Methods and compositions for genome integration

Abstract

This invention provides an effective method for the delivery and stabilization of exogenous gene materials for therapeutic purposes. [Solution] A method and composition for modulating a target genome and the stable integration of a target transgene into the cellular genome are disclosed.

Description

Related applications

[0001] cross reference

[0001] This application claims the interests of U.S. Provisional Application No. 62 / 895,441 filed on 3 September 2019, U.S. Provisional Application No. 62 / 908,800 filed on 1 October 2019, and U.S. Provisional Application No. 63 / 039,261 filed on 15 June 2020, each of which is incorporated herein by reference in whole. [Background technology]

[0002]

[0002] Cell therapy is a rapidly developing field to address diseases that are difficult to treat, such as cancer, persistent infections, and certain diseases that are resistant to other forms of treatment. Cell therapy often utilizes cells that are modified ex vivo and administered to an organism to correct defects in the body. Effective and reliable systems for manipulating the genome of cells are crucial in that, when modified cells are administered to an organism, they function optimally with long-term efficacy. Similarly, reliable mechanisms for genetic manipulation form the basis of success in gene therapy. However, there are serious shortcomings in methods for delivering nucleic acid cargoes (e.g., large cargoes) therapeutically safely and effectively. Viral delivery mechanisms are frequently used to deliver large nucleic acid cargoes to cells, but they are associated with safety issues and cannot be used to express the cargo in some cell types. In addition, subjecting cells to repeated genetic manipulation may affect the health of the cells, induce changes in the cell cycle, and render the cells unsuitable for therapeutic use. Advances in this area are constantly being explored for the effective delivery and stabilization of exogenous gene material for therapeutic purposes. [Overview of the project]

[0003]

[0003] Compositions and methods for the stable, nonviral transfer and integration of genetic material into cells are provided herein. In one embodiment, the genetic material is a self-integrating polynucleotide. The genetic material can be stably integrated into the genome of a cell. The cell may be a human cell. The method is designed for the safe and reliable integration of genetic material into the genome of a cell.

[0004]

[0004] In one embodiment, compositions and methods are provided herein that enable the integration of genetic material into the genome of a cell, wherein the genetic material that can be integrated is not specifically limited in terms of size. In some embodiments, the methods described herein provide one-step single polynucleotide-mediated delivery and integration of a gene "cargo" into the genome of a cell. The genetic material may include coding sequences, e.g., sequences encoding a transgene, peptide, recombinant protein, or antibody, or fragments thereof, wherein the methods and compositions ensure stable expression of the transcript encoded by the coding sequence. The genetic material may include, but is not limited to, non-coding sequences, e.g., regulatory RNA sequences, e.g., regulatory small interfering RNA (siRNA), microRNA (miRNA), long non-coding RNA (lncRNA), or one or more transcriptional regulators such as promoters and / or enhancers, as well as structural biomolecules, e.g., ribosomal RNA (rRNA), transfer RNA (tRNA), or fragments thereof, or combinations thereof.

[0005]

[0005] In another embodiment, methods and compositions for site-specific integration of genetic material into the cellular genome, not specifically limited in size, via nonviral delivery that ensures both the safety and effectiveness of the transfer, are provided herein. The composition may be particularly useful in developing therapeutic agents containing polynucleotides, such as polynucleotides, which include genetic material and a mechanism that enables the transfer of the polynucleotide or mRNA encoding the polynucleotide into a cell and its stable integration into the cell's genome. In some embodiments, the therapeutic agent may be a cell containing polynucleotides that has been stably integrated into the cell's genome using the methods and compositions described herein.

[0006]

[0006] In one embodiment, the present disclosure provides compositions and methods for stable gene transfer into cells. In some embodiments, the compositions and methods are for stable gene transfer into immune cells. In some cases, the immune cells are bone marrow cells. In some cases, the methods described herein relate to the development of bone marrow cells for immunotherapy.

[0007]

[0007] Immunotherapy using phagocytic cells involves creating and using modified bone marrow cells, e.g., macrophages or other phagocytic cells that attack and kill disease cells or infected cells, such as cancer cells. Modified bone marrow cells, e.g., macrophages and other phagocytic cells are prepared by incorporating into bone marrow cells, via recombinant nucleic acid technology, a modified protein containing a targeted antigen-binding extracellular domain designed to bind to a specific antigen on the surface of a target, e.g., a target cell, e.g., a cancer cell, e.g., a cancer cell, e.g., a chimeric antigen receptor. Binding of the modified chimeric receptor to the target antigen, e.g., a cancer antigen (or similarly, a disease target), initiates phagocytosis of the target. This elicits an action having two components: 1. The engulfment and lysis of the target by the phagocytic cell destroys the target and eliminates it as the front line of immune defense; 2. The antigen derived from the target is digested in phagolysosomes of bone marrow cells and presented on the surface of the bone marrow cells, which then triggers T cell activation and further activation of the immune response, as well as the development of immunological memory. Chimeric receptors are modified to enhance phagocytosis and immune activation in myeloid cells into which they are incorporated and expressed. The chimeric antigen receptors of this disclosure are variously referred to herein as chimeric fusion proteins, CFPs, phagocytic receptor (PR) fusion proteins (PFPs), or chimeric antigen receptors for phagocytosis (CAR-Ps), each term encompassing the concept of recombinant chimeric and / or fusion receptor proteins. In some embodiments, genes encoding non-receptor proteins are also co-expressed in myeloid cells, typically for enhanced chimeric antigen receptor function. In summary, methods and compositions for creating and incorporating various modified receptor and non-receptor recombinant proteins, as well as recombinant nucleic acids encoding modified receptor or non-receptor recombinant proteins, designed to enhance the phagocytic and / or immune response of myeloid cells to disease targets, are contemplated herein, such that they are consequently suitable for creating modified myeloid cells for immunotherapy.

[0008]

[0008] In one embodiment, the present disclosure provides compositions and methods for stable gene transfer into cells, wherein the cells may be any somatic cells. In some embodiments, the compositions and methods are designed for cell-specific or tissue-specific delivery. In some cases, the methods described herein relate to supplying functional proteins or fragments thereof to correct proteins that are absent or defective (mutated) in vivo, for example, for protein replacement therapy.

[0009]

[0009] The integration of recombinant nucleic acids into cells can be carried out by one or more gene transfer techniques available in the latest technologies. However, the therapeutic integration of foreign gene (e.g., nucleic acid) elements into the genome still faces several challenges. Achieving stable integration in a safe and reliable manner, and efficient and long-term expression, are some of these. Most successful gene transfer systems aimed at genomic integration of cargo nucleic acid sequences rely on viral delivery mechanisms, which have some inherent problems regarding safety and efficacy. Delivery and integration of long nucleic acid sequences are currently challenging. This cannot be achieved through the editing system.

[0010]

[0010] To date, the creation and use of modified bone marrow cells for stable, long-term gene transfer and expression of transgenes has received little attention. For example, gene transfer into differentiated mammalian cells ex vivo for cell therapy can be carried out via viral gene transfer mechanisms. However, the use of viral gene transfer vectors comes with several strategic drawbacks, including the undesirable possibility of transgene silencing over time, preferential integration into transcriptional active sites of the genome with associated undesirable activation of other genes (e.g., oncogenes), and genotoxicity. In addition to safety concerns, the increased costs and inefficient efforts involved in manufacturing, storing, and handling the integrated viruses often hinder the large-scale use of viral vector-mediated gene-modified cells in therapeutic applications. These persistent concerns regarding safety and the cost and scale of vector production associated with viral vectors necessitate alternative methods for effective therapies.

[0011]

[0011] The integration of a transgene into the genome of cells used for immunotherapy may be advantageous in that the integration is stable and fewer cells are required for delivery during therapy. On the other hand, the integration of a transgene into non-dividing cells may be challenging in that it affects the health and function of the cells, as well as the ultimate lifespan of the cells in vivo, and therefore affects the overall usefulness as a therapy. In some embodiments, the methods described herein for generating bone marrow cells for immunotherapy may be a cumulative product of several steps and compositions, including, but are not limited to, the step of selecting bone marrow cells for modification; a method and composition for incorporating recombinant nucleic acids into bone marrow cells; a method and composition for enhancing the expression of recombinant nucleic acids; a method and composition for selecting and modifying vectors; and a method for preparing recombinant nucleic acids suitable for in vivo administration for in vivo uptake and integration of recombinant nucleic acids by bone marrow cells, and therefore for generating bone marrow cells for therapy. In some embodiments, one or more embodiments of the various inventions described herein are transferable to one another, and it is expected that those skilled in the art will use these embodiments in alternative, combined, or interchangeable means without requiring excessive experimentation. All such variations of the disclosed elements are contemplated and fully encompassed herein.

[0012]

[0012] In one embodiment, transposons, or transposable elements (TEs), are considered herein as means of incorporating heterologous, synthetic, or recombinant nucleic acids encoding a desired transgene into bone marrow cells. Transposons, or transposable elements, are genetic elements that have the ability to transfer fragments of genetic material into the genome by the use of enzymes known as transposases. The mammalian genome contains a large number of transposable element (TE)-derived sequences, with up to 70% of our genome being TE-derived sequences (de Koning et al., 2011; Richardson et al., 2015). These elements can be utilized to introduce genetic material into the genome of cells. TE elements are capable of moving genetic material within the genome, often described as "jumping." TEs generally exist in the eukaryotic genome in a reversibly inactive and epigenetically silenced form. This disclosure provides methods and compositions for the efficient and stable incorporation of transgenes into macrophages and other phagocytic cells. The methods are based on the use of transposases and transposable element mRNA-coding transposases. In some embodiments, long, scattered repeat sequence 1 (L1) RNA is used for the stable integration and / or retrotransfer of the transgene into cells (e.g., macrophages or phagocytic cells).

[0013]

[0013] A method for the stable integration of foreign nucleic acid sequences into the cellular genome mediated by retrotransposons is intended herein. The method can utilize the random genomic integration mechanism of retrotransposons in cells without causing adverse effects. The methods described herein are robust and versatile methods for the incorporation of foreign nucleic acid sequences into cells, which can be used for incorporation such that the foreign nucleic acid is incorporated into a safe locus within the genome and expressed without being silenced by the cell's inherent defense mechanisms. The methods described herein can be used for the incorporation of foreign nucleic acids of approximately 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, or larger. In some embodiments, the foreign nucleic acid is not incorporated into a ribosomal locus. In some embodiments, the foreign nucleic acid is not incorporated into a ROSA26 locus or another safe harbor locus. In some embodiments, the methods and compositions described herein can incorporate the foreign nucleic acid sequence into any location within the cell's genome. Furthermore, retrotransfer systems developed for the incorporation of foreign nucleic acid sequences into predetermined specific sites within the cell's genome without causing adverse effects are contemplated herein. The disclosed methods and compositions incorporate several mechanisms for modifying retrotransposons for highly precise and specific incorporation of foreign nucleic acids into cells. The retrotransposons selected for this purpose may be human retrotransposons.

[0014]

[0014] The methods and compositions described herein represent a significant breakthrough in molecular systems and molecular mechanisms for manipulating the genome of cells. For the first time, a method is presented here that utilizes the human retrotransposon system to nonvirally deliver and stably integrate large fragments of foreign nucleic acid sequences (at least more than 100 nucleic acid bases, at least more than 1 kb, at least more than 2 kb, at least more than 3 kb, etc.) into non-conserved regions of the genome that are neither rDNA, ribosomal loci, nor designated safe harbor loci such as the ROSA26 locus.

[0015] In some embodiments, the retrotransposition system is used to stably incorporate and express non-endogenous nucleic acids into the genome, where the non-endogenous nucleic acids contain retrotransposition factors within the nucleic acid sequence. In some embodiments, the cell's endogenous retrotransposition system (e.g., proteins and enzymes) is used to stably express non-endogenous nucleic acids in the cell. In some embodiments, the cell's endogenous retrotransposition system (e.g., proteins and enzymes, e.g., the LINE-1 retrotransposition system) is used, but one or more components of the retrotransposition system may be further expressed to stably express non-endogenous nucleic acids in the cell.

[0016] In some embodiments, synthetic nucleic acids encoding a transgene and one or more components for genomic integration and / or retrotransposition are provided herein.

[0017] In one aspect, a method of integrating a nucleic acid sequence into the genome of a cell includes introducing a recombinant mRNA or a vector encoding mRNA into the cell, where the mRNA includes an insertion sequence that is a foreign sequence or a sequence that is the reverse complementary strand of a foreign sequence; a 5'UTR sequence and a 3'UTR sequence downstream of the 5'UTR sequence, where the 5'UTR sequence or the 3'UTR sequence includes a binding site for a human ORF protein, and the insertion sequence is integrated into the genome of the cell. In some embodiments, the 5'UTR sequence or the 3'UTR sequence includes a binding site for human ORF2p.

[0018]

[0018] In one aspect, a method for integrating a nucleic acid sequence into the genome of an immune cell, comprising the step of introducing a recombinant mRNA or a vector encoding mRNA, wherein the mRNA is an insert sequence comprising (i) a foreign sequence or (ii) a sequence that is the reverse complementary strand of a foreign sequence; a 5'UTR sequence and a 3'UTR sequence downstream of the 5'UTR sequence, wherein the 5'UTR sequence or the 3'UTR sequence comprises an endonuclease binding site and / or a reverse transcriptase binding site, and the introduced gene sequence is integrated into the genome of the immune cell. A method is provided herein, wherein the sequence is integrated into the genome of the immune cell.

[0019]

[0019] In one aspect, a method for integrating a nucleic acid sequence into the genome of a cell, comprising the step of introducing a recombinant mRNA or a vector encoding mRNA, wherein the mRNA is an insert sequence comprising (i) a foreign sequence or (ii) a sequence that is the reverse complementary strand of a foreign sequence; a 5'UTR sequence, a sequence of a human retrotransposon downstream of the 5'UTR sequence, and a 3'UTR sequence downstream of the sequence of the human retrotransposon, wherein the 5'UTR sequence or the 3'UTR sequence comprises an endonuclease binding site and / or a reverse transcriptase binding site, and the introduced gene sequence is integrated into the genome of the cell.

[0020]

[0020] In some embodiments, the 5'UTR sequence or the 3'UTR sequence comprises an ORF2p binding site. In some embodiments, the ORF2p binding site is a polyA sequence in the 3'UTR sequence.

[0021]

[0021] In some embodiments, the mRNA contains a human retrotransposon sequence. In some embodiments, the human retrotransposon sequence is downstream of the 5'UTR sequence. In some embodiments, the human retrotransposon sequence is upstream of the 3'UTR sequence. In some embodiments, a polynucleotide sequence (e.g., an insertion fragment) that is to be transferred into and incorporated into the cell genome is inserted at the 3' end of the sequence encoding ORF1 in the recombinant nucleic acid construct. In some embodiments, a polynucleotide sequence that is to be transferred into and incorporated into the cell genome is inserted at the 3' end of the sequence encoding ORF2 in the recombinant nucleic acid construct. In some embodiments, the sequence that is to be transferred into and incorporated into the cell genome is inserted within the 3'UTR of ORF1 or ORF2, or both. In some embodiments, a polynucleotide sequence that is to be transferred into and incorporated into the cell genome is inserted upstream of the poly-A tail of ORF2 in the recombinant nucleic acid construct.

[0022]

[0022] In some embodiments, the sequence of the human retrotransposon encodes two proteins translated from a single RNA containing two ORFs. In some embodiments, the two ORFs are non-duplication ORFs. In some embodiments, the two ORFs are ORF1 and ORF2. In some embodiments, ORF1 encodes ORF1p and ORF2 encodes ORF2p.

[0023]

[0023] In some embodiments, the sequence of the human retrotransposon includes the sequence of a non-LTR retrotransposon. In some embodiments, the sequence of the human retrotransposon includes the LINE-1 retrotransposon. In some embodiments, the LINE-1 retrotransposon is the human LINE-1 retrotransposon. In some embodiments, the sequence of the human retrotransposon includes a sequence encoding an endonuclease and / or reverse transcriptase. In some embodiments, the endonuclease and / or reverse transcriptase is ORF2p. In some embodiments, the reverse transcriptase is a group II intron reverse transcriptase domain. In some embodiments, the endonuclease and / or reverse transcriptase is the minke whale endonuclease and / or reverse transcriptase. In some embodiments, the sequence of the human retrotransposon includes a sequence encoding ORF2p. In some embodiments, the insertion sequence is integrated into the genome at a poly-T site using the specificity of the endonuclease domain of ORF2p. In some embodiments, the poly-T site includes the sequence TTTTTA.

[0024]

[0024] In some embodiments, polynucleotide constructs comprising mRNA are provided herein, wherein the mRNA comprises a sequence encoding a human retrotransposon, and (i) the sequence of the human retrotransposon comprises a sequence encoding ORF1p, (ii) the mRNA does not comprise a sequence encoding ORF1p, or (iii) the mRNA comprises a substitution sequence for the sequence encoding ORF1p having a 5'UTR sequence derived from a complementary gene. In some embodiments, the mRNA comprises a first mRNA molecule encoding ORF1p and a second mRNA molecule encoding an endonuclease and / or reverse transcriptase. In some embodiments, the mRNA is an mRNA molecule comprising a first sequence encoding ORF1p and a second sequence encoding an endonuclease and / or reverse transcriptase. In some embodiments, the first sequence encoding ORF1p and the second sequence encoding an endonuclease and / or reverse transcriptase are separated by a linker sequence.

[0025]

[0025] In some embodiments, the linker sequence includes an internal ribosome entry sequence (IRES). In some embodiments, the IRES is derived from CVB3 or EV71. In some embodiments, the linker sequence encodes a self-cleaving peptide sequence. In some embodiments, the linker sequence encodes a T2A, E2A, or P2A sequence.

[0026]

[0026] In some embodiments, the human retrotransposon sequence includes a sequence encoding ORF1p fused with an additional protein sequence and / or a sequence encoding ORF2p fused with an additional protein sequence. In some embodiments, ORF1p and / or ORF2p are fused with a nuclear retained sequence. In some embodiments, the nuclear retained sequence is an Alu sequence. In some embodiments, ORF1p and / or ORF2p are fused with an MS2 coat protein. In some embodiments, the 5'UTR sequence or 3'UTR sequence includes at least one, two, three, or more MS2 hairpin sequences. In some embodiments, the 5'UTR sequence or 3'UTR sequence includes a sequence that promotes or enhances the interaction between the polyA tail of mRNA and an endonuclease and / or reverse transcriptase. In some embodiments, the 5'UTR sequence or 3'UTR sequence includes a sequence that promotes or enhances the interaction between a polyA-binding protein (e.g., PABP) and an endonuclease and / or reverse transcriptase. In some embodiments, the 5'UTR sequence or 3'UTR sequence includes a sequence that enhances the specificity of the endonuclease and / or reverse transcriptase to the above mRNA compared to another mRNA expressed by the cell. In some embodiments, the 5'UTR sequence or 3'UTR sequence includes an Alu element sequence.

[0027]

[0027] In some embodiments, the first sequence encoding ORF1p and the second sequence encoding endonuclease and / or reverse transcriptase have the same promoter. In some embodiments, the inserted sequence has a different promoter from the promoter of the first sequence encoding ORF1p. In some embodiments, the inserted sequence has a different promoter from the promoter of the second sequence encoding endonuclease and / or reverse transcriptase. In some embodiments, the first sequence encoding ORF1p and / or the second sequence encoding endonuclease and / or reverse transcriptase have a promoter or transcription initiation site selected from the group consisting of an inducible promoter, a CMV promoter or transcription initiation site, a T7 promoter or transcription initiation site, an EF1a promoter or transcription initiation site, and combinations thereof. In some embodiments, the inserted sequence has a promoter or transcription initiation site selected from the group consisting of an inducible promoter, a CMV promoter or transcription initiation site, a T7 promoter or transcription initiation site, an EF1a promoter or transcription initiation site, and combinations thereof.

[0028]

[0028] In some embodiments, the first sequence encoding ORF1p and the second sequence encoding an endonuclease and / or reverse transcriptase are used for expression in human cells. Codon optimization is performed.

[0029]

[0029] In some embodiments, the mRNA includes a WPRE element. In some embodiments, the mRNA includes a selection marker. In some embodiments, the mRNA includes a sequence encoding an affinity tag. In some embodiments, the affinity tag is linked to a sequence encoding an endonuclease and / or reverse transcriptase.

[0030]

[0030] In some embodiments, the 3'UTR contains a polyA sequence, or the polyA sequence is added to the mRNA in vitro. In some embodiments, the polyA sequence is downstream of a sequence encoding an endonuclease and / or reverse transcriptase. In some embodiments, the insertion sequence is upstream of the polyA sequence.

[0031]

[0031] In some embodiments, the 3'UTR sequence includes an insertion sequence. In some embodiments, the insertion sequence includes a sequence that is the reverse complementary strand of the sequence encoding the foreign polypeptide. In some embodiments, the insertion sequence includes a polyadenylation site. In some embodiments, the insertion sequence includes an SV40 polyadenylation site. In some embodiments, the insertion sequence includes a polyadenylation site upstream of the sequence that is the reverse complementary strand of the sequence encoding the foreign polypeptide. In some embodiments, the insertion sequence is integrated into the genome at a locus that is not a ribosomal locus. In some embodiments, the insertion sequence is integrated into the genome at a locus that is not an rDNA locus. In some embodiments, the insertion sequence is integrated into a gene or a regulatory region of a gene, thereby disrupting the gene or downregulating gene expression. In some embodiments, the insertion sequence is integrated into a gene or a regulatory region of a gene, thereby upregulating gene expression. In some embodiments, the insertion sequence is integrated into the genome and replaces a gene. In some embodiments, the insertion sequence is stably integrated into the genome. In some embodiments, the insertion sequence retrotransfers into the genome. In some embodiments, the insertion sequence is incorporated into the genome by cleavage of the DNA strand at the target site by an endonuclease encoded by the mRNA. In some embodiments, the insertion sequence is incorporated into the genome via target-primed reverse transcription (TPRT). In some embodiments, the insertion sequence is incorporated into the genome via reverse splicing of the mRNA to a target DNA site in the genome.

[0032]

[0032] In some embodiments, the cells are immune cells. In some embodiments, the immune cells are T cells or B cells. In some embodiments, the immune cells are bone marrow cells. In some embodiments, the immune cells are selected from the group consisting of monocytes, macrophages, dendritic cells, dendritic progenitor cells, and macrophage progenitor cells.

[0033]

[0033] In some embodiments, the mRNA is self-integrated mRNA. In some embodiments, the method includes the step of introducing mRNA into cells. In some embodiments, the method includes the step of introducing a vector encoding mRNA into cells. In some embodiments, the method includes the step of introducing mRNA or an mRNA-encoding vector into cells ex vivo. In some embodiments, the method further includes the step of administering cells to a human subject. In some embodiments, the method includes the step of administering mRNA or an mRNA-encoding vector to a human subject. In some embodiments, no immune response is induced in the human subject. In some embodiments, the mRNA or vector is substantially non-immunogenic.

[0034]

[0034] In some embodiments, the vector is a plasmid or a viral vector. In some embodiments, the vector contains a non-LTR retrotransposon. In some embodiments, the vector contains a human L1 element. In some embodiments, the vector contains the L1 retrotransposon ORF1 gene. In some embodiments, the vector contains an L1 retrotransposon The vector contains the ORF2 gene of the nsposon. In some embodiments, the vector contains the L1 retrotransposon.

[0035]

[0035] In some embodiments, the mRNA is at least about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 kilobases. In some embodiments, the mRNA is at most about 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5 kilobases.

[0036]

[0036] In some embodiments, the mRNA includes a sequence that inhibits or prevents mRNA degradation. In some embodiments, the sequence that inhibits or prevents mRNA degradation inhibits or prevents mRNA degradation by exonuclease or RNAse. In some embodiments, the sequence that inhibits or prevents mRNA degradation is a G4 structure, a pseudoknot, or a triple-stranded sequence. In some embodiments, the sequence that inhibits or prevents mRNA degradation is an exoribonuclease-resistant RNA structure derived from flavivirus RNA or an ENE element derived from KSV. In some embodiments, the sequence that inhibits or prevents mRNA degradation inhibits or prevents mRNA degradation by deadenylase. In some embodiments, the sequence that inhibits or prevents mRNA degradation includes a non-adenosine nucleotide inside or at the end of the poly-A tail of the mRNA. In some embodiments, the sequence that inhibits or prevents mRNA degradation improves mRNA stability. In some embodiments, the exogenous sequence includes a sequence encoding an exogenous polypeptide. In some embodiments, the sequence encoding the foreign polypeptide is not in-frame with the sequence encoding the endonuclease and / or reverse transcriptase. In some embodiments, the foreign sequence does not contain an intron. In some embodiments, the foreign sequence includes a sequence encoding a foreign polypeptide selected from the group consisting of enzymes, receptors, transport proteins, structural proteins, hormones, antibodies, contractile proteins, and storage proteins. In some embodiments, the foreign sequence includes a sequence encoding a foreign polypeptide selected from the group consisting of chimeric antigen receptors (CARs), ligands, antibodies, receptors, and enzymes. In some embodiments, the foreign sequence includes a regulatory sequence. In some embodiments, the regulatory sequence includes a cis-acting regulatory sequence. In some embodiments, the regulatory sequence includes a cis-acting regulatory sequence selected from the group consisting of enhancers, silencers, promoters, or response elements. In some embodiments, the regulatory sequence includes a trans-acting regulatory sequence. In some embodiments, the regulatory sequence includes a trans-acting regulatory sequence encoding a transcription factor.

[0037]

[0037] In some embodiments, the insertion of the insert sequence does not adversely affect the health of the cell. In some embodiments, endonuclease, reverse transcriptase, or both are capable of site-specific insertion of the insert sequence.

[0038]

[0038] In some embodiments, the mRNA includes a sequence encoding an additional nuclease domain or a nuclease domain not derived from ORF2. In some embodiments, the mRNA includes a sequence encoding a megaTAL nuclease domain, a TALEN domain, a Cas9 domain, a zinc finger binding domain derived from the R2 retroelement, or a DNA binding domain that binds to a repeat sequence such as Rep78 derived from AAV. In some embodiments, the endonuclease includes a mutation that reduces the activity of the endonuclease compared to an endonuclea without the mutation. In some embodiments, the endonuclease is an ORF2p endonuclease, and the mutation is S228P. In some embodiments, the mRNA includes a domain that improves the accuracy and / or processing ability of the reverse transcriptase. It contains the encoding sequence. In some embodiments, the reverse transcriptase is a reverse transcriptase derived from a retroelement other than ORF2, or a reverse transcriptase that has higher accuracy and / or processing ability compared to the reverse transcriptase of ORF2p. In some embodiments, the reverse transcriptase is a group II intron reverse transcriptase. In some embodiments, the group II intron reverse transcriptase is a group IIA intron reverse transcriptase, a group IIB intron reverse transcriptase, or a group IIC intron reverse transcriptase. In some embodiments, the group II intron reverse transcriptase is TGIRT-II or TGIRT-III.

[0039]

[0039] In some embodiments, the mRNA includes a sequence comprising an Alu element and / or a ribosome-binding aptamer. In some embodiments, the mRNA includes a sequence encoding a polypeptide comprising a DNA-binding domain. In some embodiments, the 3'UTR sequence is derived from a viral 3'UTR or a beta-globin 3'UTR.

[0040]

[0040] In one embodiment, a composition comprising recombinant mRNA or an mRNA-coding vector is provided herein, wherein the mRNA comprises a human LINE-1 transposon sequence comprising a human LINE-1 transposon 5'UTR sequence, a sequence encoding ORF1p downstream of the human LINE-1 transposon 5'UTR sequence, an ORF-linker sequence downstream of the ORF1p sequence, a sequence encoding ORF2p downstream of the ORF-linker sequence, and a 3'UTR sequence derived from a human LINE-1 transposon downstream of the ORF2p sequence, wherein the 3'UTR sequence comprises an insertion sequence, and the insertion sequence is the reverse complementary strand of a sequence encoding a foreign polypeptide or the reverse complementary strand of a sequence encoding a foreign regulatory element.

[0041]

[0041] In some embodiments, the insertion sequence is integrated into the cell's genome when introduced into a cell. In some embodiments, the insertion sequence is integrated into a gene associated with a condition or disease, thereby disrupting the gene or downregulating its expression. In some embodiments, the insertion sequence is integrated into a gene, thereby upregulating its expression. In some embodiments, recombinant mRNA or a vector encoding mRNA is isolated or purified.

[0042]

[0042] In one embodiment, a composition is provided herein that comprises a nucleic acid comprising (a) a long scattered repeat sequence (LINE) polypeptide comprising human ORF1p and human ORF2p, and (b) an insertion sequence which is the reverse complementary chain of a sequence encoding a foreign polypeptide or the reverse complementary chain of a sequence encoding a foreign regulatory element, and is substantially non-immunogenic.

[0043]

[0043] In some embodiments, the composition comprises human ORF1p and human ORF2p proteins. In some embodiments, the composition comprises ribonucleoprotein (RNP) containing human ORF1p and human ORF2p in complex with nucleic acid. In some embodiments, the nucleic acid is mRNA.

[0044]

[0044] In one embodiment, a composition comprising cells comprising the composition described herein is provided herein. In some embodiments, the cells are immune cells. In some embodiments, the immune cells are T cells or B cells. In some embodiments, the immune cells are myeloid cells. In some embodiments, the immune cells are selected from the group consisting of monocytes, macrophages, dendritic cells, dendritic progenitor cells, and macrophage progenitor cells. In some embodiments, the insertion sequence is the reverse complementary chain of a sequence encoding an exogenous polypeptide, and the exogenous polypeptide is a chimeric antigen receptor (CAR).

[0045]

[0045] In one embodiment, a pharmaceutical composition comprising the composition described herein and pharmaceutically acceptable excipients is provided herein. In some embodiments, the pharmaceutical composition is for use in gene therapy. In some embodiments, the pharmaceutical composition is for use in the manufacture of a medicament for treating a disease or condition. In some embodiments, the pharmaceutical composition is for use in treating a disease or condition. In one embodiment, a method for treating a disease in a subject is provided herein, comprising the step of administering the pharmaceutical composition described herein to a subject having the disease or condition. In some embodiments, the method increases the amount or activity of a protein or functional RNA in the subject. In some embodiments, the subject has an insufficient amount or activity of a protein or functional RNA. In some embodiments, the insufficient amount or activity of a protein or functional RNA is associated with or causes a disease or condition.

[0046]

[0046] In some embodiments, the method further includes the step of administering an agent that inhibits the human silencing hub (HUSH) complex, an agent that inhibits FAM208A, or an agent that inhibits TRIM28. In some embodiments, the agent that inhibits the human silencing hub (HUSH) complex is an agent that inhibits Periphylline, TASOR, and / or MPP8. In some embodiments, the agent that inhibits the human silencing hub (HUSH) complex inhibits the assembly of the HUSH complex. In some embodiments, the agent inhibits the Fanconia anemia complex. In some embodiments, the agent inhibits FANCD2-FANC1 heterodimer monoubiquitination. In some embodiments, the agent inhibits FANCD2-FANC1 heterodimer formation. In some embodiments, the agent inhibits the Fanconia anemia (FA) core complex. The FA core complex is a component of the Fanconia anemia DNA damage repair pathway, for example, in chemotherapy-induced interstrand crosslinking. The FA core complex contains two major dimers: a FANCB subunit and a 100 kDa FA-associated protein (FAAP100) subunit, flanked by two copies of a ring finger subunit called FANCL. These two heterotrimers act as a scaffold for assembling the remaining five subunits, resulting in an elongated asymmetric structure. Destabilization of the scaffold can disrupt the entire complex, leading to a non-functional FA pathway. Examples of drugs that can inhibit the FA core complex include bortezomib, as well as the curcumin analogs EF24 and 4H-TTD.

[0047]

[0047] Accordingly, an object of the present invention is to provide novel transposon-based vectors useful for delivering gene therapy to animals. An object of the present invention is to provide novel transposon-based vectors for use in the preparation of pharmaceuticals useful for delivering gene therapy to animals or humans. Another object of the present invention is to provide novel transposon-based vectors encoding the production of a desired protein or peptide in cells. Yet another object of the present invention is to provide novel transposon-based vectors encoding the production of a desired nucleic acid in cells. A further object of the present invention is to provide a method for cell and tissue-specific incorporation of a transposon-based DNA or RNA construct, comprising the step of targeting a selected gene to a specific cell or tissue of an animal. Yet another object of the present invention is to provide a method for cell and tissue-specific expression of a transposon-based DNA or RNA construct, comprising the steps of designing a DNA or RNA construct having a cell-specific promoter that enhances the stable incorporation of a selected gene by a transposase, and expressing the selected gene in cells. An object of the present invention is to provide multi-generational gene therapy by germline administration of transposon-based vectors. Another object of the present invention is to provide gene therapy in animals by non-germ cell line administration of transposon-based vectors. Another object of the present invention is gene therapy in animals by administration of transposon-based vectors, wherein the animal produces a desired protein. One object of the present invention is to provide gene therapy that produces peptides or nucleic acids. Another object of the present invention is to provide gene therapy in animals by administration of a transposon-based vector, wherein the animal produces a desired protein or peptide that is recognized by a receptor on a target cell. Another object of the present invention is to provide gene therapy in animals by administration of a transposon-based vector, wherein the animal produces a desired fusion protein or fusion peptide, a portion of which is recognized by a receptor on a target cell in order to deliver other protein or peptide components of the fusion protein or fusion peptide to the cell and induce a biological response. Another object of the present invention is to provide a method for gene therapy in animals by administration of a transposon-based vector containing a tissue-specific promoter and the gene of interest, which facilitates tissue-specific incorporation and expression of the gene of interest that produces the desired protein, peptide, or nucleic acid. Another object of the present invention is to provide a method for gene therapy in animals by administration of a transposon-based vector containing a cell-specific promoter and the gene of interest, which facilitates cell-specific incorporation and expression of the gene of interest that produces the desired protein, peptide, or nucleic acid. A further object of the present invention is to provide a method for gene therapy in animals by administering a transposon-based vector containing a cell-specific promoter and the gene of interest, which facilitates cell-specific incorporation and expression of the gene of interest that produces a desired protein, peptide, or nucleic acid, wherein the desired protein, peptide, or nucleic acid has a desired biological effect in the animal.

[0048] Inclusion by reference

[0048] All publications, patents, and patent applications referred to herein are subject to the terms of each individual publication. To the same extent that a patent or patent application is incorporated by reference, as is specifically and individually indicated. To the extent that any publication or patent or patent application incorporated by reference conflicts with any disclosure contained herein, this Specified is intended to supersede and / or take precedence over any such conflicting material.

[0049]

[0049] Novel features of the present invention are described in detail in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description and appended drawings (further "Figures" herein) which describe exemplary embodiments in which the principles of the present invention are utilized. [Brief explanation of the drawing]

[0050] [Figure 1A]

[0050] This figure illustrates the general mechanism of action of retrotransposons. (I) is a schematic diagram representing the overall life cycle of an autonomous retrotransposon. (II) The LINE-1 retrotransposon contains a LINE-1 element that encodes two proteins, ORF1p and ORF2p, which are expressed as mRNA. The bicistronic mRNA is translated into two proteins, and ORF2p, when translated by a ribosome read-through event, binds to the 3' end of its own mRNA via a poly-A tail (III). ORF2p is cleaved at the consensus sequence TAAAA, where the poly-A at the 3' end of the mRNA hybridizes, stimulating the reverse transcriptase activity of the ORF2 protein. This protein reverse transcribes the mRNA into DNA, causing the LINE-1 sequence to be inserted into a new location in the genome (IV). [Figure 1B]

[0051] This diagram illustrates various exemplary designs for integrating mRNA encoding an introduced gene into the cell's genome. The GFP enclosed in the rectangle shown here is an exemplary introduced gene. [Figure 1C]

[0052] This diagram illustrates various exemplary designs for integrating mRNA encoding an introduced gene into the cell's genome. The GFP enclosed in the rectangle shown here is an exemplary introduced gene. [Figure 2]

[0053] Figure 2A illustrates three exemplary designs for expressing the exemplary transgene GFP by stably incorporating the GFP-coding sequence using a construct. The expected GFP expression level at 72 hours is shown on the right.

[0054] Figure 2B illustrates three exemplary designs for expressing the exemplary transgene GFP by stably incorporating sequences encoding RFP, RFP and GFP, or ORF2p and GFP using constructs. Expected GFP and RFP expression levels at 72 hours are shown on the right. [Figure 3A]

[0055] Figure 3A is an illustrative diagram of a conventional circRNA structure and formation.

[0056] Figure 3B shows two diagrams illustrating exemplary RL-GAAA tectoRNA motif designs.

[0057] Figure 3C illustrates an exemplary structure of a chip-flow fragment RNA as a platform for testing potential tectoRNAs. [Figure 3B]

[0055] Figure 3A is an exemplary diagram of a conventional circRNA structure and formation.

[0056] Figure 3B is two diagrams relating to an exemplary RL-GAAA tectoRNA motif design.

[0057] Figure 3C is a diagram illustrating an exemplary structure of a chip-flow fragment RNA as a platform for testing potential tectoRNAs. [Figure 4A]

[0058] This is a schematic diagram illustrating ORF2p binding to the poly-A region of ORF2. [Figure 4B]

[0059] This is an illustrative schematic diagram illustrating how the fusion of ORF2p and the MS2 RNA-binding domain binds to the MS2-binding RNA sequence in the 3'UTR of the mRNA encoding ORF2, thereby improving its specificity. [Figure 4C]

[0060] This figure illustrates exemplary designs of retrotransposon systems for the stable integration of nucleic acids into the cellular genome at specific sites. The top panel shows a design using an ORFp2-MegaTAL DNA-binding domain fusion in which ORF2p is mutated to inactivate its DNA-binding and endonuclease activity. The middle panel shows a chimeric ORF2p in which the endonuclease domain is replaced with a highly specific and highly accurate nuclease domain of another protein. The bottom panel shows a fusion of ORF2p and the DNA-binding domain of a heterologous protein, such that the fusion protein binds to the ORF2 binding site and further DNA sequences in the vicinity of the ORF2 site. [Figure 5-1]

[0061] This diagram illustrates an exemplary construct for incorporating mRNA encoding a transgene into the cell's genome. [Figure 5-2] Figure 5-1 continued. [Figure 5-3] Figure 5-1 continued. [Figure 6A]

[0062] Figure 6A illustrates an exemplary construct containing an ORF1p-coding sequence for integrating the mRNA encoding the transgene into the cell genome. [Figure 6B]

[0063] Figure 6B illustrates an exemplary construct that does not contain the sequence encoding ORF1p, for integrating the mRNA encoding the transgene into the cell genome. [Figure 7A]

[0064] This figure illustrates exemplary methods for improving mRNA half-life by inhibiting degradation by 5'-3' exonucleases such as XRN1 or 3'-5' exosome degradation, or by introducing a G4 structure or a structure corresponding to a pseudoknot in the 5'UTR, and / or a triple-stranded motif xrRNA and / or non-A nucleotide residues in the 3'UTR. [Figure 7B]

[0065] This is a schematic diagram illustrating bone marrow cells expressing a transgene that encodes a chimeric receptor that binds to cancer cells and induces anti-cancer activity. [Figure 7C]

[0066] Figure 7B is a graph showing the expected results regarding increased and prolonged expression of the chimeric receptor by introducing bulk or purified RNA encoding the chimeric receptor that binds to cancer cells. [Figure 8A]

[0067] Figure 8A shows an exemplary plasmid design and a LINE-1 mRNA transcript containing the expected cargo nucleic acid sequence. The plasmid has a LINE-1 sequence (containing the ORF1 and ORF2 protein coding sequences) as well as a cargo sequence, which is the nucleic acid sequence encoding GFP, where the GFP coding sequence is separated by an intron. GFP is not expressed until the sequence is integrated into the genome and the intron is spliced. [Figure 8B]

[0068] Figure 8B is a graph illustrating exemplary results showing the successful incorporation of mRNA transcripts encoded by the plasmid shown in Figure 8A, and GFP expression compared to mock-transfected cells (showing the increase in mean fluorescence intensity of GFP-positive cells). Mock-transfected cells were transfected with a vector lacking the GFP cargo sequence. [Figure 8C]

[0069] Figure 8C is a graph showing exemplary flow cytometry results from the results shown in Figure 8B. [Figure 9A]

[0070] Figure 9A shows an exemplary plasmid design and a LINE-1 mRNA transcript containing the expected cargo nucleic acid sequence. The plasmid has the LINE-1 sequence (containing the ORF1 and ORF2 protein coding sequences), as well as a cargo sequence which is a nucleic acid sequence encoding a recombinant chimeric fusion receptor protein (ATAK receptor) having an extracellular domain capable of binding to CD5 and an intracellular domain containing an FCR intracellular domain and a PI3 kinase mobilization domain. The ATAK receptor coding sequence is separated by introns. [Figure 9B]

[0071] Figure 9B is a graph illustrating exemplary results showing successful incorporation of mRNA transcripts encoded by the plasmid shown in Figure 9A, and ATAK expression compared to mock-transfected cells (showing the increase in mean fluorescence intensity of ATAK-positive cells). Mock-transfected cells were transfected with a vector lacking the ATAK cargo sequence. ATAK receptor protein expression was detected by conjugation with a labeled CD5 antibody. [Figure 9C]

[0072] Figure 9C is a graph showing exemplary flow cytometry results from the results shown in Figure 9B. [Figure 10A]

[0073] Figure 10A shows an exemplary plasmid design and the expected LINE-1 mRNA transcript containing the cargo nucleic acid sequence. The plasmid has a LINE-1 sequence (including the ORF1 and ORF2 protein coding sequences), as well as a cargo sequence which is a nucleic acid sequence encoding a recombinant chimeric fusion receptor protein (ATAK receptor), followed by a T2A autocleavage sequence, and then a fragmented GFP sequence (all reverse-oriented relative to the LINE-1 sequence). The GFP coding sequence is separated by an intron. The reverse transcription of the cargo and the expected mRNA after integration are shown. [Figure 10B]

[0074] Figure 10B is a graph illustrating exemplary results showing the successful incorporation of mRNA transcripts encoded by the plasmid shown in Figure 10A, and the expression of ATAK-T2A-GFP compared to mock-transfected cells (showing the magnification changes of GFP and ATAK double-positive cells). Mock-transfected cells were transfected with a vector lacking the ATAK cargo sequence. ATAK receptor protein expression was detected by conjugation with a labeled CD5 antibody. [Figure 10C]

[0075] Figure 10C shows representative flow cytometry data from two separate experimental trials on the expression of both GFP and CD5 conjugate (ATAK) using the experimental setup shown in Figure 10A. [Figure 10D]

[0076] Figure 10D is a graph showing representative flow cytometry data from two separate experimental trials on the expression of both GFP and CD5 conjugate (ATAK) using the experimental setup shown in Figure 10A. [Figure 11A]

[0077] Figure 11A shows an exemplary mRNA construct for retrotransfer-based gene delivery. The ORF1 and ORF2 sequences are present in two different mRNA molecules. The ORF2p (ORF2) coding mRNA contains a GFP coding sequence and is inverted. [Figure 11B]

[0078] Figure 11B is a graph showing exemplary data illustrating GFP expression when both ORF1-mRNA and ORF2-FLAG-GFPai mRNA are electroporated, normalized to the value obtained when only ORF2-FLAG-GFPai mRNA is electroporated (showing the increase in the average fluorescence intensity of GFP-positive cells). [Figure 12A]

[0079] Figure 12A is a graph showing exemplary data illustrating GFP expression when various amounts of ORF1-mRNA and ORF2-FLAG-GFPai mRNA are electroporated (showing the increase in average fluorescence intensity of GFP-positive cells). The increase ratios are for 1×ORF2-GFPao and 1×ORF1 mRNA. [Figure 12B]

[0080] Figure 12B is an exemplary fluorescence microscopy image of GFP+ cells after mRNA electroporation, as shown in Figure 11A. [Figure 13A]

[0081] Figure 13A shows exemplary mRNA constructs for gene delivery in which the ORF1 and ORF2 sequences are present in two different mRNA molecules (upper panel), as well as a LINE-1 mRNA transcript containing the ORF1 and ORF2 protein-coding sequences in a single mRNA molecule (lower panel). The mRNA contains bicistronic ORF1 and ORF2 sequences and a CMV-GFP sequence oriented from 3' to 5' in the 3' UTR. After retrotransfer of the delivered ORF2-cmv-GFP antisense (LINE-1 mRNA), cells are expected to express GFP. [Figure 13B]

[0082] Figure 13B is a graph showing exemplary data illustrating GFP expression when the construct shown in Figure 13A is electroporated (showing the increase in the average fluorescence intensity of GFP-positive cells). [Figure 14A]

[0083] Figure 14A shows an exemplary experimental design for testing whether multiple electroporation passes improve retrotransition efficiency. HEK293T cells were electroporated every 48 hours using the Maxcyte system and cultured for 24–72 hours before being evaluated for GFP-positive cells using flow. [Figure 14B]

[0084] Figure 14B is a graph showing exemplary data illustrating GFP expression at the indicated time for 1 to 5 electroporations according to Figure 14A (showing the increase in the average fluorescence intensity of GFP-positive cells). [Figure 15A]

[0085] Figure 15A shows exemplary constructs for enhancing retrotransition via mRNA delivery. In one construct, a nuclear localization signal (NLS) sequence is fused to the C-terminus of the ORF2 sequence (ORF2-NLS fusion). In another construct, a minke whale ORF2 sequence is used instead of human ORF2. In another construct, the minimal sequence of the Alu element (AJL-H33 delta) is inserted into the 3'UTR of the LINE-1 sequence. In yet another construct, an MS2 hairpin is inserted into the 3'UTR of the LINE-1 sequence, and the MS2 hairpin-binding protein (MCP) sequence is fused to the ORF2 sequence. [Figure 15B]

[0086] Figure 15B is a graph showing exemplary data illustrating GFP expression using the construct shown in Figure 15A (showing the increase in mean fluorescence intensity of GFP-positive cells). [Figure 16A]

[0087] Figure 16A shows exemplary plasmid constructs for gene delivery in which the ORF1 and ORF2 sequences are present in two different plasmid molecules (upper panel), as well as plasmids encoding LINE-1 mRNA transcripts containing the ORF1 and ORF2 protein-coding sequences of a single mRNA molecule, along with various substitution sequences for the inter-ORF sequence between ORF1 and ORF2 (lower panel). [Figure 16B]

[0088] Figure 16B is a graph showing exemplary data illustrating GFP expression using the construct shown in Figure 16A (showing the increase in mean fluorescence intensity of GFP-positive cells). [Figure 17A]

[0089] Figure 17A shows an exemplary plasmid construct (top panel) encoding a LINE-1 mRNA transcript containing the ORF1 and ORF2 protein-coding sequences and GFP sequence of a single mRNA molecule, as well as an exemplary LINE-1 mRNA transcript containing the ORF1 and ORF2 protein-coding sequences and GFP sequence of a single mRNA molecule. [Figure 17B]

[0090] Figure 17B is a graph showing exemplary data illustrating GFP expression in Jurkat cells using the constructs shown in Figure 17A (showing the increase in mean fluorescence intensity of GFP-positive cells). Plasmid constructs were transfected, and mRNA constructs were electroporated. [Figure 18A]

[0091] Figure 18A shows an exemplary plasmid design and the expected LINE-1 mRNA transcript containing the cargo nucleic acid sequence. The plasmid has a LINE-1 sequence (including the ORF1 and ORF2 protein coding sequences), as well as a cargo sequence which is a nucleic acid sequence encoding a recombinant chimeric fusion receptor protein (ATAK receptor), followed by a T2A self-cleavage sequence, and then a fragmented GFP sequence (all reverse-oriented relative to the LINE-1 sequence). The GFP coding sequence is separated by an intron. The reverse transcription of the cargo and the expected mRNA after integration are shown. [Figure 18B]

[0092] Figure 18B is a graph illustrating exemplary results showing the successful incorporation of mRNA transcripts encoded by the plasmid shown in Figure 10A, and the expression of ATAK-T2A-GFP in the bone marrow cell line (THP-1) compared to mock-transfected cells (showing the magnification changes of GFP and ATAK double-positive cells). The data are expressed 6 days after transfection, normalized to mock plasmid-transfected cells, where the mock plasmid does not contain the GFP coding sequence. [Figure 19]

[0093] This figure illustrates an exemplary experimental setup for cell synchronization. A heterogeneous cell population is sorted based on the stage of the cell cycle before delivery of an exogenous nucleic acid. Cell cycle synchronization is expected to result in higher expression and stabilization of the delivered exogenous nucleic acid. If the cells are not homogeneous after sorting, they may be further incubated with a suitable agent that halts the cell cycle at a certain stage. [Figure 20]

[0094] This figure illustrates exemplary methods for improving retrotransposon efficiency by inducing DNA double-strand breaks, with or without inhibiting the DNA repair pathway, such as by inducing the DNA ligase inhibitor SCR7 or by inhibiting host surveillance proteins using miRNA against the HUSH complex TASOR protein. [Figure 21]

[0095] This diagram illustrates an exemplary construct for incorporating mRNA encoding a transgene into the cell's genome. [Figure 22]

[0096] This diagram illustrates an exemplary construct for incorporating mRNA encoding a transgene into the cell's genome. [Figure 23]

[0097] This diagram illustrates an exemplary construct for incorporating mRNA encoding a transgene into the cell's genome. [Figure 24]

[0098] This diagram illustrates an exemplary construct for incorporating mRNA encoding a transgene into the cell's genome. [Figure 25]

[0099] This diagram illustrates an exemplary construct for incorporating mRNA encoding a transgene into the cell's genome. [Figure 26]

[0100] This diagram illustrates an exemplary construct for incorporating mRNA encoding a transgene into the cell's genome. [Figure 27]

[0101] This diagram illustrates an exemplary construct for incorporating mRNA encoding a transgene into the cell's genome. [Figure 28]

[0102] This diagram illustrates an exemplary construct for incorporating mRNA encoding a transgene into the cell's genome. [Modes for carrying out the invention]

[0051] Detailed explanation

[0103] This invention partially relates to polynucleotides being used as gene cargo (e.g., large genes). This stems from the exciting discovery that polynucleotides can be designed and developed to carry out the translocation and integration of cargo into the cellular genome. In some embodiments, polynucleotides include (i) gene material for stable expression, and (ii) a self-integrating genome integration mechanism that enables stable integration of the gene material into cells by safe and effective nonviral means. Furthermore, it is thought that the genetic material can be incorporated into loci other than ribosome loci, that the genetic material can be incorporated in a site-specific manner, and / or that the incorporated genetic material can be expressed without triggering the cell's innate silencing mechanisms.

[0052]

[0104] Clustered, regularly spaced, short palindromic repeat sequences (CRISPR) have revolutionized the field of molecular biology and led to the development of powerful gene editing. These can utilize homologous recombination repair (HDR) and be directed to genomic sites. CRISPR / Cas9 is a naturally occurring RNA-guided endonuclease. While the CRISPR / Cas9 system has demonstrated great promise for site-directed gene editing and other applications, several factors must be addressed that affect its effectiveness, particularly when used for in vivo human gene therapy. These factors include target DNA site selection, sgRNA design, off-target cleavage, the incidence / efficiency of HDRs leading to NHEJ, Cas9 activity, and delivery methods. Delivery remains a major barrier to the use of CRISPR for in vivo applications. Zinc finger nucleases (ZFNs) are fusion proteins of the Cys2-His2 zinc finger protein (ZFP) and a non-specific DNA restriction enzyme derived from the FokI endonuclease. One of the challenges with ZFPs is the design and modification of ZFPs for high-affinity binding of desired sequences, which is crucial. Furthermore, site selection is limited because not all sequences are available for ZFP binding. Another significant challenge is off-target cleavage. Activator-like effector nucleases (TALENs) are fusion proteins composed of TALE and FokI nucleases. While off-target cleavage remains a concern, TALENs have been shown to be more specific and less cytotoxic than ZFNs in some parallel comparative studies. However, TALENs are substantially larger, with cDNA encoding only 3kb. This makes the delivery of a pair of TALENs more difficult than a pair of ZFNs due to the limited size of the delivery vehicle cargo. Additionally, packaging and delivery of some TALENs into viral vectors can be problematic due to high levels of repeats in the TALEN sequence.Fusion proteins of the mutant Cas9 system, inactive dCas9, and the FokI nuclease dimer improve specificity, reduce off-target cleavage, and have fewer potential target sites due to PAM and other sgRNA design constraints.

[0053]

[0105] The present invention addresses the problems described above by providing novel effective and efficient compositions, including transposon-based vectors, for providing therapies, including gene therapy, to animals and humans. The present invention provides methods for using these compositions to provide therapies to animals and humans. These transposon-based vectors can be used in the preparation of pharmaceuticals useful for producing desired effects in recipients after administration. Gene therapy includes, but is not limited to, the introduction of genes, such as exogenous genes, into animals using transposon-based vectors. These genes can perform diverse functions in recipients, such as encoding the production of nucleic acids, e.g., RNA, or encoding the production of proteins and peptides. The present invention can facilitate the efficient incorporation of polynucleotide sequences, including the gene of interest, promoter, insertion sequence, poly(A), and any regulatory sequence. The present invention is based on the finding that human LINE-1 elements are retrotransferable in human cells and cells of other animal species and can be manipulated in various ways to achieve efficient delivery and incorporation of gene cargo into the cellular genome. Such LINE-1 elements have diverse applications in human and animal genetics, including, but not limited to, applications in the diagnosis and treatment of genetic disorders and applications in cancer. The LINE-1 element of the present invention is also useful for treating various phenotypic effects of various diseases. For example, the LINE-1 element can be used for the transfer of DNA encoding an antitumor gene product into cancer cells. Other uses of the LINE-1 element of the present invention will become apparent to those skilled in the art by reading this specification. Ro.

[0054]

[0106] Generally, the human LINE-1 element contains a 5' UTR with an internal promoter, two non-overlapping reading frames (ORF1 and ORF2), a 200 bp 3' UTR, and a 3' poly-A tail. LINE-1 retrotransposons may also contain an endonuclease domain at the N-terminus of LINE-1 ORF2. The finding that LINE-1 encodes an endonuclease demonstrates that this element is autonomously retrotransportable. LINE-1 is a modular protein containing a non-overlapping functional domain that mediates the reverse transcription and incorporation of LINE-1. In some embodiments, the sequence specificity of the LINE-1 endonuclease itself is modifiable, or the LINE-1 endonuclease can be replaced by another site-specific endonuclease.

[0055]

[0107] LINE-1 retrotransposons can be manipulated using recombinant DNA techniques to include and / or be contiguous with other DNA elements that make them suitable for insertion of heterogeneous or homogeneous DNA of substantial length (up to 1 kb, or greater than 1 kb) into the cellular genome. LINE-1 retrotransposons can also be manipulated using the same type of technique so that the insertion of DNA into the cellular genome is site-specific (the site where such DNA is inserted is known). Alternatively, LINE-1 retrotransposons may be manipulated so that the DNA insertion site is random. Retrotransposons can also be manipulated to achieve insertion of a desired DNA sequence into a region of DNA that is not normally transcribed, such that the DNA sequence is expressed in a way that does not interfere with the normal expression of the gene in the cell. In some embodiments, the integration or retrotransposition is performed in trans orientation. In some embodiments, the integration or retrotransposition is performed in cis orientation.

[0056]

[0108] Because LINE-1 is naturally present in human cells, the construct should not be rejected as non-self by the immune system when placed in human cells. In addition, the mechanism of LINE-1 retrointegration ensures that only one copy of the gene is integrated into any specific chromosomal location. Therefore, copy number control is built into the system. In contrast, gene transfer procedures using conventional plasmids offer little to no control over copy number and often result in a complex array of DNA molecules integrated in tandem at the same genomic location.

[0057]

[0109] All terms are intended to be understood in the same way as they would be understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as they would be generally understood by those skilled in the art to which this disclosure belongs.

[0058]

[0110] The section headings used in this specification are for organizational purposes only and should not be interpreted as limiting the subjects discussed.

[0111] As used herein, the singular forms "a," "an," and "the" are intended to include the plural form unless otherwise indicated by the context.

[0059]

[0112] In this application, the use of “or” means “and / or” unless otherwise stated. The terms “and / or” and “any combination thereof,” as well as their grammatical synonyms, can be used interchangeably as used herein. These terms can convey that any combination is specifically intended. For illustrative purposes only, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof” are equivalent to “A only, B only, C only, A and B, B and C, A It can mean "and C, and A and B and C." The term "or" can be used both conjunctively and disjunctively unless the context makes it clear that it is a disjunctive use.

[0060]

[0113] The terms “approximately” or “about” can mean that a particular value is within the tolerance range determined by those skilled in the art, and in part depends on the method by which that value is measured or determined, i.e., the limitations of the measurement system. For example, “approximately” may mean a standard deviation of 1 or more than 1, as is customary in the art. Alternatively, “approximately” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Or, particularly with respect to biological systems or processes, the term may mean within 10 times, up to 5 times, more preferably up to 2 times a certain value. Where a particular value is described in this application and claims, unless otherwise stated, the term “approximately” should be assumed to mean that the particular value is within the tolerance range.

[0061]

[0114] As used herein and in the claims, the words “comprising” (and any form of “comprising,” e.g., “comprise” and “comprises”), “having” (and any form of “having,” e.g., “have” and “has”), “including” (and any form of “including,” e.g., “includes” and “include”), or “containing” (and any form of “containing,” e.g., “contains” and “contain”) are non-exclusive or non-limiting and do not exclude any additional undescribed elements or method steps. Any embodiment discussed herein can be implemented with respect to any method or composition of the Disclosure, and vice versa. Furthermore, the compositions of the Disclosure can be used to achieve the methods of the Disclosure.

[0062]

[0115] References to “some embodiments,” “a certain embodiment,” “one embodiment,” or “other embodiments” in this specification mean that certain features, structures, or characteristics described with an embodiment are included in at least some embodiments of this disclosure, but not necessarily in all embodiments. To facilitate understanding of this disclosure, several terms and phrases are defined below.

[0063]

[0116] Various features of this disclosure may be described in the context of a single embodiment, but may also be described separately or in any preferred combination. Conversely, for clarity of explanation, this disclosure may be described herein in the context of separate embodiments, but may also be implemented in a single embodiment.

[0064]

[0117] This disclosure encompasses, but is not limited to, methods and compositions relating to the expression of exogenous nucleic acids in cells. In some embodiments, the exogenous nucleic acid is configured for stable integration into the genome of cells such as bone marrow cells. In some embodiments, stable integration of the exogenous nucleic acid may be achieved at a specific target within the genome. In some embodiments, the exogenous nucleic acid comprises one or more coding sequences. In some embodiments, the exogenous nucleic acid may comprise one or more coding sequences, including nucleic acid sequences encoding immune receptors. In some embodiments, this disclosure provides methods and compositions for the stable integration of nucleic acids encoding transmembrane receptors (e.g., phagocytic receptors or synthetic chimeric antigen receptors) into human macrophages or dendritic cells or preferred bone marrow cells or myeloid progenitor cells. Exogenous nucleic acid can refer to nucleic acids that are not originally present in cells and are added from outside the cell, whether or not they include sequences that may already be endogenously present in the cell. Exogenous nucleic acid may be a DNA or RNA molecule. Exogenous nucleic acid may include sequences encoding transgenes. Exogenous nucleic acid is recombinant. Genetic materials can encode recombinant proteins such as receptors or chimeric antigen receptors (CARs). Foreign nucleic acids are sometimes referred to as "gene cargo" in the context of their delivery into cells. Gene cargo can be DNA or RNA. Genetic material can generally be delivered into cells ex vivo by several different known techniques using chemical (CaCl2-mediated transfection), physical (electroporation), or biological (e.g., viral infection or transduction) means.

[0065]

[0118] In one embodiment, methods and compositions are provided herein for the delivery and stable incorporation of one or more nucleic acids, comprising nucleic acid sequences encoding one or more proteins, into cells, such as bone marrow cells, wherein stable incorporation can be achieved via a nonviral mechanism. In some embodiments, the delivery of the nucleic acid composition to bone marrow cells is achieved via a nonviral mechanism. In some embodiments, the delivery of nucleic acids can further avoid plasmid-mediated delivery. As used herein, “plasmid” means a nonviral expression vector, such as a nucleic acid molecule encoding a gene and / or a regulatory element necessary for gene expression. As used herein, “viral vector” means a virus-derived nucleic acid capable of transporting another nucleic acid into a cell. A viral vector, when present in a suitable environment, can lead to the expression of one or more proteins encoded by one or more genes harbored by the vector. Examples of viral vectors, but not limited to, include retroviruses, adenoviruses, lentiviruses, and adeno-associated virus vectors.

[0066]

[0119] In some embodiments, a method for delivering a composition into cells such as bone marrow cells is provided herein, wherein the composition comprises one or more nucleic acid sequences encoding one or more proteins, and one or more nucleic acid sequences are RNA. In some embodiments, RNA is mRNA. In some embodiments, one or more mRNAs comprising one or more nucleic acid sequences are delivered. In some embodiments, one or more mRNAs may comprise at least one modified nucleotide. As used herein, the term "nucleotide" refers to a base-sugar-phosphate combination. Nucleotides may include synthetic nucleotides. Nucleotides may include synthetic nucleotide analogs. Nucleotides can be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphates such as adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates, e.g., dATP, dCTP, dITP, dUTP, dGTP, or derivatives thereof. Examples of such derivatives include [aS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used herein, the term nucleotide may refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Exemplary examples of dideoxyribonucleoside triphosphates, but not limited to, include ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or detected by well-known techniques. Labeling can also be performed using quantum dots. Examples of detectable labels include radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzymatic labels.Fluorescent labels for nucleotides include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,NcN'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2'-aminoethyl)aminonaphthalene-1. -Sulfonic acid (EDANS) can be cited. Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAN1RA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP, available from Perkin Elmer, Foster City, Calif., FluoroLink deoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP, available from Amersham, Arlington Heights, Ill.; Boehringer Fluorescein-15-dATP, fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, TR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2'-dATP, available from Mannheim, Indianapolis, Ind., as well as Molecular Examples of chromosome-labeled nucleotides available from Probes, Eugene, and Oreg include BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, Fluorescein-12-UTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine-6-UTP, Tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP.Nucleotides can also be labeled or marked by chemical modification. Chemically modified single nucleotides can be biotin-dNTPs. Some non-limiting examples of biotinylated dNTPs include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-cICTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-1.6-dUTP, biotin-20-dUTP).

[0067]

[0120] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are interchangeable and refer to polymeric forms of nucleotides of any length, either single-stranded, double-stranded, or multi-stranded, of either deoxyribonucleotides or ribonucleotides or their analogues. Polynucleotides may be exogenous or endogenous to cells. Polynucleotides may exist in a cell-free environment. Polynucleotides may be genes or fragments thereof. Polynucleotides may be DNA. Polynucleotides may be RNA. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. Polynucleotides may contain one or more analogues (e.g., modified skeletons, sugars, or nucleic acid bases). If present, modifications to the nucleotide structure may be conjugated before or after the assembly of the polymer. Some non-limiting examples of modified nucleotides or analogs include pseudouridine, 5-bromouracil, 5-methylcytosine, peptide nucleic acids, xeno nucleic acids, morpholino, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to sugars), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, cuosin, and waiosin. Examples include coding or non-coding regions of genes or gene fragments, loci defined by ligation analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, eDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. Nucleotide sequences may be separated by non-nucleotide components.

[0068]

[0121] In some embodiments, the nucleic acid composition may comprise one or more mRNAs, each containing at least one mRNA encoding a transmembrane receptor (e.g., a phagocytic receptor or a synthetic chimeric antigen receptor) associated with immune response function, which enters human macrophages or dendritic cells or suitable myeloid cells or myeloid progenitor cells. In some embodiments, the nucleic acid composition comprises one or more mRNAs and one or more lipids for delivery of the nucleic acid to hematopoietic cells such as myeloid cells or myeloid progenitor cells. In some embodiments, one or more lipids may form liposome complexes.

[0069]

[0122] The compositions described herein, as used herein, can be used for intracellular delivery. The cells may originate from any organism having one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protist cells, plant-derived cells (e.g., cells from plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, hemp, tobacco, angiosperms, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, mosses), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens). Examples include cells derived from C. agardh (etc.), seaweed (e.g., kelp), fungal cells (e.g., yeast cells, mushroom-derived cells), animal cells, cells derived from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells derived from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), and cells derived from mammals (e.g., pigs, cattle, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.). In some cases, the cells do not have to originate from natural organisms (e.g., cells may be produced by synthesis and may be referred to as artificial cells). In some embodiments, the cells referred to herein are mammalian cells. In some embodiments, the cells are human cells. The methods and compositions described herein relate to the incorporation of genetic material into cells, more specifically into human cells, where human cells may be any human cells. In its use herein, human cells may be cells of any origin, such as somatic cells, neurons, fibroblasts, muscle cells, epithelial cells, cardiac cells, or hematopoietic cells. The methods and compositions described herein may also be applicable and useful for incorporating foreign nucleic acids into human cells that are difficult to transfect. The methods are simple and, once a suitable foreign nucleic acid construct has been designed and developed, are universally applicable.The methods and compositions described herein are applicable to the ex vivo incorporation of exogenous nucleic acids into cells. In some embodiments, the compositions may be applicable to systemic administration to an organism, where the nucleic acid material in the composition may be taken up by cells in vivo and subsequently incorporated into the cells in vivo.

[0070]

[0123] In some embodiments, the methods and compositions described herein may be intended for incorporating foreign nucleic acids into human hematopoietic cells, such as human cells of hematopoietic origin, such as human bone marrow cells or bone marrow cell precursors. However, the methods and compositions described herein may be intended for incorporating foreign nucleic acids into human hematopoietic cells, such as human bone marrow cells or bone marrow cell precursors. It can be used in any biological cell, or it can be minimally modified to be suitable for use in any biological cell. Therefore, a cell can refer to any cell that is the basic structural, functional, and / or biological unit of a living organism.

[0071]

[0124] In one embodiment, methods and compositions are provided herein for utilizing transposable elements for the stable incorporation of one or more nucleic acids into the genome of a cell, wherein the cell is a type of hematopoietic cell, such as a bone marrow cell. In some embodiments, one or more nucleic acids comprise at least one nucleic acid sequence encoding a transmembrane receptor protein having a role in the immune response. In some embodiments, the methods and compositions relate to the use of retrotransposable elements for incorporating one or more nucleic acid sequences into bone marrow cells. The nucleic acid composition may comprise one or more nuclear sequences, such as a gene, where the gene is a transgene. The term “gene,” as used herein, refers to nucleic acids (e.g., DNA such as genomic DNA and cDNA), as well as the corresponding nucleotide sequences of nucleic acids involved in encoding RNA transcripts. As used herein in relation to genomic DNA, the term includes intervening noncoding regions and regulatory regions, and may include the 5' and 3' ends. In some uses, the term encompasses transcription sequences including the 5' and 3' untranslated regions (5'UTR and 3'UTR), exons, and introns. In some genes, the transcription region may contain an “open reading frame” encoding a polypeptide. In some uses of this term, “gene” includes only the coding sequence necessary to encode a polypeptide (e.g., “open reading frame” or “coding region”). In some cases, a gene does not encode polypeptides, such as ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term “gene” includes not only the transcription sequence but also non-transcriptional regions, including upstream and downstream regulatory regions, enhancers, and promoters. A gene may refer to an “endogenous gene” or native gene that is located in its native location in the genome of an organism. A gene may refer to an “exogenous gene” or non-native gene. A non-native gene may refer to a gene that is not normally found in a host organism and is introduced into the host organism by gene transfer. A non-native gene may also refer to a gene that is not located in its native location in the genome of an organism.Non-natural genes may also refer to naturally occurring nucleic acid or polypeptide sequences (e.g., non-natural sequences) that include mutations, insertions, and / or deletions.

[0072]

[0125] The term “transgene” refers to any nucleic acid molecule introduced into a cell, which may occasionally be referred to as a recipient cell in this specification. Cells resulting from the acceptance of a transgene can be classified as transgenic cells. Transgenes may include genes that are partially or completely heterologous (i.e., non-self) to the transgenic organism or cell, or they may be genes of the same species as the endogenous genes of that organism or cell. In some cases, transgenes may include any polynucleotide, such as genes encoding polypeptides or proteins, polynucleotides transcribed into inhibitory polynucleotides, or polynucleotides that are not transcribed (e.g., lacking expression regulatory elements such as promoters that drive transcription). Transcripts and encoded polypeptides may collectively be referred to as “gene products.” If the polynucleotide originates from genomic DNA, expression may involve mRNA splicing in eukaryotic cells. In relation to expression, "upregulated" refers to an increase in the expression level of polynucleotides (e.g., RNA such as mRNA) and / or polypeptide sequences compared to the wild-type expression level, while "downregulated" refers to a decrease in the expression level of polynucleotides (e.g., RNA such as mRNA) and / or polypeptide sequences compared to the wild-type expression level. Expression of a transfected gene can occur transiently or stably in the cell. During "transient expression," the transfected gene is not transferred to daughter cells during cell division. Because this gene expression is restricted to the transfected cell, it is lost over time. In contrast, stably expressed transfected gene means the gene is not transferred to the transfected cell. This may occur when the gene is co-transfected with another gene that confers a selective advantage. Such a selective advantage may be resistance to a particular toxin presented to the cell. If the transfected gene needs to be expressed, this application assumes the use of a codon-optimized sequence. An example of a codon-optimized sequence may be one that is optimized for expression in a eukaryote, e.g., human (i.e., optimized for human expression), or one that is optimized for another eukaryote, animal, or mammal. Codon optimization for non-human host species, or for specific organs, is known. In some embodiments, a coding sequence encoding a protein may be codon-optimized for expression in a specific cell, e.g., a eukaryotic cell. The eukaryotic cell may be derived from a specific organism, e.g., a plant or mammal, e.g., but not limited to humans, or non-human eukaryotes or animals or mammals discussed herein, e.g., cells of mice, rats, rabbits, dogs, livestock, or non-human mammals or primates. Codon optimization refers to the process of modifying nucleic acid sequences while maintaining the natural amino acid sequence by replacing at least one codon in the natural sequence (e.g., approximately 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more than approximately 1, 2, 3, 4, 5, 10, 15, 20, 25, 50) with a codon that is more or most frequently used in the host cell's gene, in order to enhance expression in the target host cell. Different species exhibit a particular bias towards certain codons for specific amino acids. Codon bias (differences in codon usage frequency among organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which is thought to depend, among other things, on the characteristics of the codon being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of selected tRNAs in cells can generally reflect the codons most frequently used in peptide synthesis. Therefore, genes can be tuned for optimal gene expression in a given organism based on codon optimization.Codon frequency tables are readily available, for example, in the "Codon Frequency Database" available at www.kazusa.orjp / codon / , and these tables may be modified in some respects. Computer algorithms for codon-optimizing specific sequences for expression in specific host cells are also available, such as Gene Forge (Aptagen, Jacobus, PA).

[0073]

[0126] As used herein, a "multicistronic transcript" refers to an mRNA molecule containing two or more protein-coding regions or cistrons. mRNA containing two coding regions is referred to as a "bisistronic transcript." A "5' proximal" coding region or cistron is the coding region in which the translation start codon (usually AUG) is closest to the 5' end of the multicistronic mRNA molecule. A "5' distal" coding region or cistron is a coding region or cistron in which the translation start codon (usually AUG) is closest to the 5' end of the mRNA, but is not the start codon.

[0074]

[0127] The terms "transfection" or "transfected" refer to the introduction of nucleic acids into cells by non-viral or virus-based methods. Nucleic acid molecules can be complete proteins or gene sequences encoding functional portions thereof. See, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1–18.88.

[0075]

[0128] The term “promoter,” as used herein, refers to a polynucleotide sequence capable of driving the transcription of a coding sequence in a cell. Therefore, promoters used in the polynucleotide constructs of this disclosure include cis-acting transcriptional regulatory elements and regulatory sequences involved in regulating or modulating the timing and / or rate of gene transcription. For example, promoters include enhancers, promoters, transcription termination factors, origins of replication, chromosomal integration sequences, 5' and These can be cis-acting transcriptional regulatory elements, including the 3' untranslated region or intron sequences. These cis-acting sequences typically interact with proteins or other biomolecules that perform gene transcription (switching them on / off, regulating them, modulating them, etc.). Constitutive promoters are promoters that can initiate transcription in almost all tissue types, while tissue-specific promoters initiate transcription in only one or a few specific tissue types. Inducible promoters are promoters that initiate transcription only under specific environmental, developmental, or drug or chemical conditions. Exemplary inducible promoters may be doxycycline or tetracycline-inducible promoters. Tetracycline-regulating promoters may be either tetracycline-inducible or tetracycline-repressive, known as tet-on systems and tet-off systems. The tet-regulating system is tetracycline-dependently dependent on two components: a tetracycline-regulating regulator (also called a transactivator) (tTA or rtTA) and a tTA / rtTA-dependent promoter that controls the expression of downstream cDNA. tTA is a fusion protein containing the repressor of the Tn10 tetracycline-resistant operon of Escherichia coli and the carboxyl terminus of protein 16 (VP16) of herpes simplex virus. The tTA-dependent promoter consists of a minimal RNA polymerase II promoter fused with a tet operator (tetO) sequence (a series of seven homogeneous operator sequences). This fusion converts the tet repressor into a potent transcriptional activator in eukaryotic cells. In the absence of tetracycline or its derivatives (e.g., doxycycline), tTA binds to the tetO sequence, enabling transcriptional activation of the tTA-dependent promoter. However, in the presence of doxycycline, tTA cannot interact with its target, and transcription does not occur. The tet system using tTA is called tet-OFF because tetracycline or doxycycline allows for downregulation of transcription. In contrast, in the tet-ON system, a variant of tTA, referred to as rtTA, has been isolated using random mutagenesis.In contrast to tTA, rtTA is not functional in the absence of doxycycline and requires the presence of a ligand for transactivation. The term "exon" refers to nucleic acid sequences found in genomic DNA that are predicted by bioinformatics and / or experimentally confirmed to result in a continuous sequence in a mature mRNA transcript. The term "intron" refers to sequences present in genomic DNA that, under endogenous conditions, are transcribed into RNA (e.g., mRNA precursor) molecules but are spliced ​​from endogenous RNA (e.g., mRNA precursor) before the RNA is translated into protein, and are predicted by bioinformatics and / or experimentally confirmed not to encode some or all of the expressed protein.

[0076]

[0129] The term “splice receptor site” refers to a sequence present in genomic DNA that is predicted by bioinformatics and / or experimentally confirmed to be a receptor site during the splicing of mRNA precursors, and may include identified and unidentified, naturally occurring and artificially obtained or obtainable splice receptor sites.

[0077]

[0130] An "internal ribosome entry site" or "IRES" refers to a nucleotide sequence that enables 5' end / cap-independent translation initiation, thereby increasing the likelihood of expressing two proteins from a single messenger RNA (mRNA) molecule. IRESs are typically located in the 5' UTR of positive-strand RNA viruses with a capless genome. Another means of expressing two proteins from a single mRNA molecule is by inserting a 2A peptide(like) sequence between the coding sequences of those proteins. The 2A peptide(like) sequence mediates the self-processing of the primary translation product through a process variously referred to as "ribosome skipping," "stop-go" translation, and "stop carry-on" translation. The sequence is present in various groups of positive-sense and double-stranded RNA viruses, including those belonging to the families Picornaviridae, Flaviviridae, Tetraviridae, Disistroviridae, Reoviridae, and Totiviridae.

[0078]

[0131] The term "2A peptide" refers to a class of 18-22 amino acid (AA) long viral oligopeptides that mediate the "cleavage" of polypeptides during translation in eukaryotic cells. The "2A" designation refers to a specific region of the viral genome, and generally, various viral 2A peptides are named after the viruses from which they were derived. The first 2A peptide discovered was F2A (foot-and-mouth disease virus), followed by the identification of E2A (equine rhinitis A virus), P2A (porcine tesiovirus-1 2A), and T2A (thosea asigna virus 2A). The mechanism of 2A-mediated "self-cleavage" is thought to involve ribosome skipping of the formation of a glycyl-prolyl peptide bond at the C-terminus of the 2A sequence. 2A peptide(s) sequences mediate the self-processing of primary translation products through a process variously referred to as "ribosome skipping," "stop-go" translation, and "stop-carry-on" translation. 2A peptide(like) sequences are present in various groups of positive-sense and double-stranded RNA viruses, including those belonging to the families Picornaviridae, Flaviviridae, Tetraviridae, Disistroviridae, Reoviridae, and Totiviridae.

[0079]

[0132] As used herein, the term “operably linked” refers to a functional relationship between two or more segments, such as nucleic acid segments or polypeptide segments. Typically, this term refers to the functional relationship of a transcriptional regulatory sequence to a transcriptional sequence.

[0080]

[0133] The term "termination sequence" refers to a nucleic acid sequence that is recognized by polymerase in a host cell and results in the termination of transcription. A termination sequence is a DNA sequence at the 3' end of a native or synthetic gene that enables the termination of mRNA transcription of an upstream open reading frame or both mRNA transcription and ribosome translation. Prokaryotic termination sequences generally consist of a GC-rich region with 2 rotational symmetry followed by an AT-rich sequence. A commonly used termination sequence is the T7 termination sequence. A variety of termination sequences are known in the art, including TINT3, TL13, TL2, TR1, TR2, and T6S termination signals from bacteriophage lambda, as well as termination signals from bacterial genes such as the trp gene of E. coli, and can be used in the nucleic acid constructs of the present invention.

[0081]

[0134] The term "polyadenylation sequence" (also called "poly-A site" or "poly-A sequence") refers to a DNA sequence that leads to both termination and polyadenylation of a nascent RNA transcript. Since transcripts lacking a poly-A tail are typically unstable and rapidly degraded, efficient polyadenylation of recombinant transcripts is desirable. The poly-A signal used in expression vectors can be either "exotic" or "endogenous." An endogenous poly-A signal is a signal naturally found at the 3' end of the coding region of a given gene in the genome. An heterologous poly-A signal is a signal isolated from one gene and positioned at the 3' end of the coding sequence of another gene, such as a protein. A commonly used heterologous poly-A signal is the SV40 poly-A signal. The SV40 poly-A signal is contained in a 237 bp BamHI / BclI restriction fragment and leads to both termination and polyadenylation; numerous vectors contain the SV40 poly-A signal. Another commonly used heterologous polyA signal is derived from the bovine growth hormone (BGH) gene, and the BGH polyA signal is also available in several commercially available vectors. The polyA signal derived from the herpes simplex virus thymidine kinase (HSV tk) gene is also used as a polyA signal in several commercially available expression vectors. Polyadenylation signals facilitate the transport of RNA from the cell nucleus to the cytoplasm and increase the cellular half-life of such RNA. Polyadenylation signals are located at the 3' end of mRNA.

[0082]

[0135] The terms “complement,” “complements,” “complementary,” and “complementarity,” as used herein, refer to sequences that are complementary to and hybridizable to a given sequence. In some cases, a sequence that hybridizes with a given nucleic acid is referred to as the “complementary” or “countercomplementary” of a given molecule if the sequence of bases across a given region can bind complementaryly to the sequence of a binding partner, for example, to form AT, AU, GC, and GU base pairs. Generally, a first sequence that is hybridizable to a second sequence is specifically or selectively hybridizable to the second sequence, and as a result, hybridization to the second sequence or set of second sequences is preferred over hybridization to non-target sequences during the hybridization reaction (for example, being thermodynamically more stable under a given set of conditions, such as stringent conditions commonly used in the art). Typically, hybridizable sequences share some degree of sequence complementarity over all or part of their respective lengths, including, for example, 25% to 100% complementarity, such as at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence complementarity.For example, sequence identity for the purpose of evaluating complementarity percentage can be measured by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needle aligner available at www.ebi.ac.uk / Tools / psa / embossneedle / nucleotide.html), the BLAST algorithm (see, e.g., the optional, pre-configured BLAST alignment tool available at blast.ncbi.nlm.nih.gov / Blast.cgi), or the Smith-Waterman algorithm (see, e.g., the optional, pre-configured EMBOSS Water aligner available at www.ebi.ac.ukaools / psa / emboss_water / nucleotide.html). Optimal alignment can be evaluated using any suitable parameters of the selected algorithm, including initial parameters.

[0083]

[0136] Complementarity can be perfect or substantial / sufficient. Perfect complementarity between two nucleic acids can mean that the two nucleic acids can form a double helix in which each base is bonded to a complementary base by Watson-Crick pairing. Substantial or sufficient complementarity can mean that the sequence in one strand is not completely and / or perfectly complementary to the sequence in the opposing strand, but that sufficient bonding occurs between the bases of the two strands to form a stable hybrid complex under a set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be determined using sequences and standard mathematical calculations to find the melting temperature (T) of the hybridized strand. m By predicting T) or by using conventional methods m This can be predicted by empirical decisions.

[0084]

[0137] A "transposon," also known herein as a "jumping gene," is a segment within a chromosome that can change position within the genome. There are two distinct classes of transposons: Class I or retrotransposons, which move via RNA intermediates and "copy-and-paste" mechanisms, and Class II or DNA transposons, which move via excision-integration or "cut-and-paste" mechanisms (Ivics Nat Methods 2009). Bacterial, lower eukaryote (e.g., yeast), and invertebrate transposons are largely species-specific and are not considered usable for efficient DNA transfer in vertebrate cells. "Sleeping Beauty" (Ivics Cell 1997) is a transposon artificially reconstructed by sequence shuffling of an inactive TE derived from fish. It was the first active transposon. This enabled successful DNA integration by transposition into vertebrate cells, including human cells. Sleeping Beauty is a class II DNA transposon belonging to the Tcl / Mariner family of transposons (Ni Genomics Proteomics 2008). On the other hand, further functional transposons have been identified or reconstructed from different species, including Drosophila, frogs, and even the human genome, all of which have been shown to enable DNA transposition into vertebrate and even human host cell genomes. Each of these transposons has advantages and disadvantages in terms of transposition efficiency, expression stability, gene payload capacity, etc. Exemplary class II transposases that have been created include Sleeping Beauty, PiggyBac, Frog Prince, Himarl, Passport, Minos, hAT, Toll, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR, and Hsmarl.

[0085]

[0138] As used herein, "heterogeneous" includes molecules such as DNA and RNA that are not naturally found in the cell into which it is inserted. For example, when mouse or bacterial DNA is inserted into the genome of a human cell, such DNA is referred to herein as heterogeneous DNA. In contrast, as used herein, the term "homogeneous" refers to molecules such as DNA and RNA that are naturally found in the cell into which it is inserted. For example, the insertion of mouse DNA into the genome of a mouse cell constitutes the insertion of homogeneous DNA into a cell. In the latter case, it is not necessary for the homogeneous DNA to be inserted into a site in the cell genome where it is naturally found; rather, the homogeneous DNA may be inserted into a site other than where it is naturally found, thereby causing a genetic alteration (mutation) at the insertion site.

[0086]

[0139] A "transposase" is an enzyme that can form a functional complex with a transposon end-containing composition (e.g., a transposon, a transposon end) and catalyzes the insertion or transfer of the transposon end-containing composition to double-stranded DNA incubated for in vitro transposon reactions. The term "transposon end" refers to double-stranded DNA containing the nucleotide sequence ("transposon end sequence") necessary to form a complex with a functional transposase or integrase enzyme in an in vitro transfer reaction.

[0087]

[0140] The transposon end forms a complex, synaptic complex, transposon complex, or transposon composition with a transposase or integrase that recognizes and binds to the transposon end. These complexes can insert or transfer the transposon end to target DNA that is incubated together in an in vitro transposition reaction. The transposon end exhibits two complementary sequences, consisting of an importing transposon end sequence or importing strand and a non-importing transposon end sequence or non-importing strand. For example, a transposon terminal that forms a complex with a highly active Tn5 transposase active in an in vitro transposition reaction includes an importing strand exhibiting the following importing transposon terminal sequence: 5'AGATGTGTATAAGAGACAG3' and a non-importing strand exhibiting the following "non-importing transposon terminal sequence": (5'CTGTCTCTTATACACATCT3'). The 3' end of the importing strand either binds to or is transferred to the target DNA in the in vitro transposition reaction. The non-importing strand, exhibiting a transposon terminal sequence complementary to the importing transposon terminal sequence, neither binds to nor is transferred to the target DNA in the in vitro transposition reaction.

[0088]

[0141] In some embodiments, the importing and non-importing strands are covalently bonded. For example, in some embodiments, the importing and non-importing strand sequences are provided, for example, in a single oligonucleotide in a hairpin arrangement. Thus, the free end of the non-importing strand is directly bonded to the target DNA by a transfer reaction. Although direct binding does not occur, the non-imported strand becomes indirectly bound to the DNA fragment by linking with the imported strand via a hairpin-like loop. As used herein, "cleavage domain" refers to a sensitive nucleic acid sequence.

[0089]

[0142] A “restriction site domain” refers to a tag domain that exhibits a sequence intended to facilitate cleavage using restriction endonucleases. For example, in some embodiments, the restriction site domain is used to generate a linear ssDNA fragment with two tags. In some embodiments, the restriction site domain is used to generate a compatible double-stranded 5' end in the tag domain so that the compatible double-stranded 5' end can be ligated to another DNA molecule using a template-dependent DNA ligase. In some embodiments, the restriction site domain in the tag exhibits a sequence of a restriction site that is rarely present, if at all, in the target DNA (e.g., a restriction site for low-frequency cleavage restriction endonucleases such as NotI or AscI).

[0090]

[0143] As used herein, the term “recombinant nucleic acid molecule” refers to a recombinant DNA molecule or a recombinant RNA molecule. A recombinant nucleic acid molecule is any nucleic acid molecule that contains a conjugate of nucleic acid molecules of different origins that would not naturally bind together. Recombinant RNA molecules include RNA molecules transcribed from recombinant DNA molecules. Recombinant nucleic acids can be synthesized in the laboratory. Recombinant nucleic acids can be prepared by using recombinant DNA techniques that utilize enzymatic modification of DNA, such as enzyme restriction digestion, ligation, and DNA cloning. Recombinant DNA can be transcribed in vitro to produce messenger RNA (mRNA), which can then be isolated, purified, and used for transfecting cells. Recombinant nucleic acids may encode proteins or polypeptides. Under suitable conditions, recombinant nucleic acids can be incorporated into living cells and expressed within living cells. As used herein, “expression” of nucleic acids usually refers to the transcription and / or translation of nucleic acids. The product of nucleic acid expression is usually a protein, but may be mRNA. The detection of mRNA encoded by recombinant nucleic acids in cells into which recombinant nucleic acids have been incorporated is considered clear evidence that the nucleic acid is “expressed” in the cell. The process of inserting or incorporating nucleic acids into cells can be done through transformation, transfection, or transduction. Transformation is the process by which bacterial cells take up non-self nucleic acids. This process is modified for the amplification, protein production, and other applications of plasmid DNA. Transformation introduces recombinant plasmid DNA into competent bacterial cells that take up extracellular DNA from the environment. Some bacterial species are naturally competent under certain environmental conditions, but competence is artificially induced in a laboratory setting. Transfection is the forced introduction of small molecules such as DNA, RNA, or antibodies into eukaryotic cells. To further complicate matters, “transfection” also refers to the introduction of bacteriophages into bacterial cells.While "infection" refers to the natural infection of humans or animals with wild-type viruses, "transduction" is mostly used to describe the introduction of recombinant viral vector particles into target cells.

[0091]

[0144] A "stem-loop" sequence refers to a nucleic acid sequence (e.g., an RNA sequence) that is sufficiently self-complementary to hybridize and form a stem, and a non-complementary region that bulges out in a loop. The stem may contain mismatches or bulges.

[0092]

[0145] The term "vector" refers to a nucleic acid molecule capable of transporting or mediating the expression of heterologous nucleic acids. "Vector sequence," as used herein, refers to a nucleic acid sequence comprising at least one origin of replication and at least one select marker gene. A vector capable of operatively ligating the expression of a gene and / or nucleic acid sequence is referred herein to as an "expression vector."

[0093]

[0146] A plasmid is a species of the genus encompassed by the term “vector.” Generally, useful expression vectors often exist in the form of a “plasmid,” which refers to a circular double-stranded DNA molecule that, in vector form, does not bind to a chromosome and typically contains components for the stable or transient expression of encoded DNA. Other expression vectors that can be used in the methods disclosed herein include, but are not limited to, plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, or viral vectors, such vectors that can be integrated into the host genome or autonomously replicate in cells. Vectors can be DNA or RNA vectors. Other forms of expression vectors known to those skilled in the art that perform equivalent functions, such as self-replicating extrachromosomal vectors or vectors that can be integrated into the host genome, can also be used. Exemplary vectors are those capable of autonomous replication and / or expression of ligated nucleic acids. A safe harbor locus is a region within the genome to which additional foreign or heterologous nucleic acid sequences can be inserted, and which the host genome can accommodate the inserted genetic material. Examples of safe harbor sites include, but are not limited to, the AAVS1 site, GGTA1 site, CMAH site, B4GALNT2 site, B2M site, ROSA26 site, COLA1 site, and TIGRE site. For example, the heterologous nucleic acids described herein can be incorporated into one or more sites in the cellular genome, where one or more sites are selected from the group consisting of the AAVS1 site, GGTA1 site, CMAH site, B4GALNT2 site, B2M site, ROSA26 site, COLA1 site, and TIGRE site. In some embodiments, the nucleic acid cargo containing the transgene may be delivered to the R2D locus.

[0094]

[0147] In some embodiments, the nucleic acid cargo containing the transgene can be delivered to the genome in intergeneric or intragenetic regions. In some embodiments, the nucleic acid cargo containing the transgene is integrated into the genome within 0.1kb, 0.25kb, 0.5kb, 0.75kb, 1kb, 2kb, 3kb, 4kb, 5kb, 7.5kb, 10kb, 15kb, 20kb, 25kb, 50kb, 75kb, or 100kb 5' or 3' from the endogenous active gene. In some embodiments, the nucleic acid cargo containing the transgene is integrated into the genome within 0.1kb, 0.25kb, 0.5kb, 0.75kb, 1kb, 2kb, 3kb, 4kb, 5kb, 7.5kb, 10kb, 15kb, 20kb, 25kb, 50, 75kb, or 100kb 5' or 3' from the endogenous promoter or enhancer. In some embodiments, the nucleic acid cargo containing the transgene is between 50 and 50,000 base pairs, for example, between 50 and 40,000 bp, between 500 and 30,000 bp, between 500 and 20,000 bp, between 100 and 15,000 bp, between 500 and 10,000 bp, between 50 and 10,000 bp, or between 50 and 5,000 bp. In some embodiments, the nucleic acid cargo containing the transgene is less than 1,000, 1,300, 1,500, 2,000, 3,000, 4,000, 5,000, or 7,500 nucleotides in length.

[0095] L1 and non-L1 retrotransposon systems

[0148] Retrotransposons are actively involved in the reorganization of unique genomes. Retrotransposons can contain transposition factors. Broadly speaking, retrotransposons are DNA sequences that can be transcribed into RNA, translated into proteins, and have the ability to reverse transcribe themselves into DNA. Approximately 45% of the human genome consists of sequences resulting from transposition events. Retrotransposition can result in target site deletions or the addition of non-retrotransposon DNA to the genome through a process called 5' and 3' transduction. Recombination between non-homologous retrotransposons causes deletions, duplications, or rearrangements of gene sequences. Ongoing retrotransposition can generate novel splice sites, polyadenylation signals, and promoters, thus constructing new transcription modules.

[0096]

[0149] Generally, retrotransposons can be classified into two classes: retrovirus-like LTR retrotransposons and non-LTR elements such as the human L1 element, Neurospora TAD element (Kinsey, 1990, Genetics 126:317-326), Drosophila I element (Bucheton et al., 1984, Cell 38:153-163), and Bombyx mori R2Bm (Luan et al., 1993, Cell 72:595-605). These two types of retrotransposons are structurally different and use fundamentally different mechanisms for retrotransposition. Exemplary and non-exclusive examples of LINE code polypeptides can be found in GenBank commission numbers AAC51261, AAC51262, AAC51263, AAC51264, AAC51265, AAC51266, AAC51267, AAC51268, AAC51269, AAC51270, AAC51271, AAC51272, AAC51273, AAC51274, AAC51275, AAC51276, AAC51277, AAC51278, and AAC51279.

[0097]

[0150] The decision to focus on LINE-1 and develop it into the system described herein is for several reasons, at least in part, as illustrated below: (a) the LINE-1 (or L1) element is autonomous, as it single-handedly encodes all the mechanisms necessary to complete this reverse transcription and integration process; (b) the L1 element is so abundant in the human genome that it can be considered a naturalized element; and (c) the L1 retrotransposon retrotransposes its own mRNA with a high degree of specificity compared to other mRNAs floating in the cell.

[0098]

[0151] L1 expresses a 6kb bicistronic RNA encoding a 40kDa open reading frame-1 RNA-binding protein (ORF1p) with essential but uncertain function, and a 150kDa ORF2 protein with endonuclease and reverse transcriptase (RT) activity. L1 retrotransition is a complex process involving L1 transcription, transport of its RNA to the cytoplasm, translation of the bicistronic RNA, formation of ribonucleoprotein (RNP) particles, retransition to the nucleus, and reverse transcription with target-primer reverse transcription at the integration site. Several transcription factors that interact with L1 have been identified. Transcribed L1 RNA forms RNPs in cis with the protein translated from the transcript. L1 is integrated into genomic DNA by targeted-site primer reverse transcription (TPRT) via ORF2p cleavage at 5'-TTTT-3', where the poly(A) sequence of L1 RNA anneals, stimulating reverse transcriptase (RT) activity to construct L1 cDNA.

[0099]

[0152] Other mobile elements of the genome can "hijack" L1 ORFs for retrotransposition. For example, Alu elements are mobile DNA elements that belong to a class of short scattered repeat sequences (SINEs) that are non-autonomous retrotransposons and acquire trans factors for incorporation. Alu elements and SINE-1 elements can also retrotranspose by ORF1p and ORF2p by trans-associating with L1 ribonucleoprotein. Similar to L1 RNA to some extent, Alu elements often terminate with a long A chain, often referred to as an A tail, and also have a smaller A-rich region (indicated by AA) that bisects a branched dimeric structure. Alu elements are likely to contain internal components of the RNA polymerase III promoter (e.g., commonly referred to as A-box and B-box promoters), but do not encode the termination factor of RNA polymerase III. Alu elements can terminate transcription by utilizing a T-nucleotide segment located at various distances downstream from the Alu element. A typical Alu transcript encompasses the entire Alu molecule, including the A tail, and has a 3' region specific to each locus. Alu RNA folds into distinct structures for each monomeric unit. RNA is 7SL RNA S It has been shown to bind to RP9 and RP14 heterodimers, as well as to poly(A)-binding protein (PABP). The poly(A) tail of Alu primes the T-rich (TTTT) region of the genome, attracting ORF2p to bind to the primer region, and cleaving the T-rich region via the endonuclease activity of ORF2p. The T-rich region is then primed for reverse transcription by ORF2p bound to the 3'A tail region of the Alu element. This creates a cDNA copy of the Alu element's body. Nicks are generated in the second strand by an unknown mechanism, priming the second strand synthesis. The new Alu element then mates with a short direct repeat, which is a replication of the DNA sequence between the first and second nicknames. Due to their preference for gene-rich regions, Alu elements are very widely distributed within RNA molecules. The full-length Alu (approximately 300 bp) originates from the signal-recognizing particle RNA 7SL and consists of two similar monomers with an A-rich linker in between, A and B boxes present in the 5' monomer, and a poly-A tail lacking the aforementioned polyadenylation signal, resulting in an extended tail (up to 100 bp in length). Alu can be transcribed by RNA polymerase III using the internal promoters within the A and B boxes, but it does not contain an ORF and therefore does not encode a protein product.

[0100]

[0153] Other non-L1 transposons include SVA and HERV-K. The full-length SVA (SINE-VNTR-Alu) element (approximately 2-3 kb) is a synthetic unit containing a CCCTCT repeat sequence, two Alu-like sequences, a VNTR, a SINE-R region with an env (envelope) gene, the 3'LTR of HERV-K10, and a polyadenylation signal followed by a poly-A tail. It is unknown whether the SVA element possesses an internal promoter, but SVA is most likely transcribed by RNA polymerase II.

[0101]

[0154] The full-length HERV-K element (approximately 9–10 kb) consists of an ancient artifact of an endogenous retroviral sequence and includes two adjacent LTR regions flanking three retroviral ORFs: (1) gag encoding the structural protein of the retroviral capsid, (2) pol-pro encoding enzymes, namely proteases, RT, and integrases, and (3) env encoding proteins that enable horizontal transfer. The LTR of HERV-K contains an internal bidirectional promoter thought to be under the transcriptional control of RNA polymerase II.

[0102]

[0155] L1 retrotransposition and RNA binding can occur at or near the poly-A tail. The 3'UTR plays a role in the recognition of stringent LINE RNA by the ORF1 protein (ORF1p). The stringent LINE RNA may contain a stem-loop structure located at the end of the 3'UTR. A branched molecule consisting of the junction of the transposon 3' terminal cDNA with the target DNA and the specific positioning of L1 RNA within the ORF2 protein (ORF2p) was detected during the initial stages of L1 retrotransposition in vitro. Secondary or tertiary RNA structures shared by L1 and Alu, sometimes together with the poly-A tail, are likely responsible for recognition and binding by ORF2. In some embodiments, the stem-loop structure located downstream of the poly-A sequence correlates with cleavage strength.

[0103]

[0156] Mechanisms that restrict or resolve L1 integration have also evolved to maintain genomic gene integrity and stability. Non-homologous end repair proteins, such as XRCC1, Ku70, and DNA-PK, are involved in resolving L1 integration during insertion. In addition, cells have evolved several proteins to counteract unrestricted retrotransitions, including the APOBEC3 family of cytosine deaminases, the adenosine deaminase ADAR1, chromatin remodeling factors, and members of the piRNA pathway for post-transcriptional gene silencing that function in male germline cells.

[0104] I. Compositions and methods comprising nucleic acid constructs involved in the stable expression of coding proteins

[0157] Encodes one or more proteins for expression in cells such as bone marrow cells. Recombinant nucleic acids are provided herein. In one embodiment, the recombinant nucleic acid is designed for the stable expression of one or more proteins or polypeptides encoded by the recombinant nucleic acid. In some embodiments, stable expression is achieved by incorporating the recombinant nucleic acid into the cellular genome.

[0105]

[0158] Those skilled in the art will readily understand that the compositions and methods described herein can be used to design products in which recombinant nucleic acids may contain one or more sequences capable of encoding oligonucleotides that can become regulatory nucleic acids, such as oligonucleotide products as inhibitors, or oligonucleotides as activators, rather than being translated as protein or polypeptide components.

[0106]

[0159] In one embodiment, a composition is provided herein comprising a synthetic nucleic acid comprising a nucleic acid sequence encoding a gene of interest and one or more retrotransfer factors for stably incorporating a non-endogenous nucleic acid into a cell. In some embodiments, the cells are hematopoietic cells. In some embodiments, the cells are myeloid cells. In some embodiments, the cells are progenitor cells. In some embodiments, the cells are undifferentiated. In some embodiments, the cells have further differentiation potential. In some embodiments, the cells are not stem cells.

[0107] A.LINE / Alu Retrotransposon Structure

[0160] In some embodiments, the present disclosure stably incorporates non-endogenous nucleic acids into the genome and expresses them. A retrotransferable system can be used to express non-endogenous nucleic acids, wherein the retrotransferable system includes retrotransfer factors within the nucleic acid sequence. In some embodiments, the disclosure can utilize an endogenous retrotransferable system (e.g., proteins and enzymes) within the cell to stably express non-endogenous nucleic acids in the cell. In some embodiments, the disclosure can utilize an endogenous retrotransferable system (e.g., proteins and enzymes, e.g., the LINE1 retrotransferable system), but one or more components of the retrotransferable system may be further expressed to stably express non-endogenous nucleic acids in the cell.

[0108]

[0161] In some embodiments, synthetic nucleic acids are provided herein that encode a transgene and encode one or more components for retrotransfer. The synthetic nucleic acids described herein are interchangeably referred to as nucleic acid constructs, transgenes, or exogenous nucleic acids.

[0109]

[0162] In one embodiment, a method for incorporating a nucleic acid sequence into the genome of a cell is provided herein, comprising the steps of introducing recombinant mRNA or an mRNA-encoding vector into a cell, wherein the mRNA comprises an insertion sequence comprising a foreign sequence or a sequence that is the reverse complementary strand of a foreign sequence; a 5'UTR sequence and a 3'UTR sequence downstream of the 5'UTR sequence, wherein the 5'UTR sequence or the 3'UTR sequence comprises a binding site for a human ORF protein, and the insertion sequence is incorporated into the genome of the cell.

[0110]

[0163] In some embodiments, the 5'UTR sequence or 3'UTR sequence contains a binding site for human ORF2p.

[0164] In one embodiment, a method for incorporating a nucleic acid sequence into the genome of an immune cell, comprising the step of introducing recombinant mRNA or an mRNA-encoding vector, wherein the mRNA is an insertion sequence comprising (i) a foreign sequence or (ii) the reverse complementary strand of a foreign sequence; a 5'UTR sequence and a 3'UTR sequence downstream of the 5'UTR sequence, wherein the 5'UTR sequence or the 3'UTR sequence is an endonuclease-binding site and / or A method is provided herein for incorporating a transgene sequence into the genome of an immune cell, comprising a 5'UTR sequence and a 3'UTR sequence, which include a reverse transcriptase binding site.

[0111]

[0165] In one embodiment, a method for incorporating a nucleic acid sequence into the genome of a cell is provided, comprising the step of introducing recombinant mRNA or an mRNA-coding vector, wherein the mRNA comprises an insertion sequence comprising (i) a foreign sequence or (ii) the reverse complementary strand of a foreign sequence; a 5'UTR sequence, a sequence of a human retrotransposon downstream of the 5'UTR sequence, and a 3'UTR sequence downstream of the human retrotransposon sequence, wherein the 5'UTR sequence or the 3'UTR sequence comprises an endonuclease-binding site and / or a reverse transcriptase-binding site, the human retrotransposon sequence encoding two proteins translated from a single RNA containing two ORFs, and the insertion sequence is incorporated into the genome of a cell.

[0112]

[0166] In some embodiments, the 5'UTR sequence or the 3'UTR sequence includes an ORF2p binding site. In some embodiments, the ORF2p binding site is a poly(A) sequence in the 3'UTR sequence.

[0113]

[0167] In some embodiments, the mRNA contains a human retrotransposon sequence. In some embodiments, the human retrotransposon sequence is downstream of the 5'UTR sequence. In some embodiments, the human retrotransposon sequence is upstream of the 3'UTR sequence.

[0114]

[0168] In some embodiments, the sequence of the human retrotransposon encodes two proteins translated from a single RNA containing two ORFs. In some embodiments, the two ORFs are non-duplication ORFs. In some embodiments, the two ORFs are ORF1 and ORF2. In some embodiments, ORF1 encodes ORF1p and ORF2 encodes ORF2p.

[0115]

[0169] In some embodiments, the human retrotransposon sequence includes the sequence of a non-LTR retrotransposon. In some embodiments, the human retrotransposon sequence includes the LINE-1 retrotransposon. In some embodiments, the LINE-1 retrotransposon is the human LINE-1 retrotransposon. In some embodiments, the human retrotransposon sequence includes a sequence encoding an endonuclease and / or reverse transcriptase. In some embodiments, the endonuclease and / or reverse transcriptase is ORF2p. In some embodiments, the reverse transcriptase is a group II intron reverse transcriptase domain. In some embodiments, the endonuclease and / or reverse transcriptase is the minke whale endonuclease and / or reverse transcriptase. In some embodiments, the human retrotransposon sequence includes a sequence encoding ORF2p. In some embodiments, the insertion sequence is integrated into the genome at a poly-T site using the specificity of the endonuclease domain of ORF2p. In some embodiments, the poly-T site includes the sequence TTTTTA.

[0116]

[0170] In some embodiments, (i) the sequence of the human retrotransposon includes a sequence encoding ORF1p, (ii) the mRNA does not include a sequence encoding ORF1p, or (iii) the mRNA includes a substitution sequence for the ORF1p sequence having a 5'UTR sequence derived from a complementary gene. In some embodiments, the mRNA includes a first mRNA molecule encoding ORF1p and a second mRNA molecule encoding an endonuclease and / or reverse transcriptase. In some embodiments, the mRNA is an mRNA molecule containing a first sequence encoding ORF1p and a second sequence encoding an endonuclease and / or reverse transcriptase. In some embodiments, the first sequence encoding ORF1p and The second sequence encoding the endonuclease and / or reverse transcriptase is separated by a linker sequence.

[0117]

[0171] In some embodiments, the linker sequence includes an internal ribosome entry sequence (IRES). In some embodiments, the IRES is derived from CVB3 or EV71. In some embodiments, the linker sequence encodes a self-cleaving peptide sequence. In some embodiments, the linker sequence encodes a T2A, E2A, or P2A sequence.

[0118]

[0172] In some embodiments, the human retrotransposon sequence includes a sequence encoding ORF1p fused with an additional protein sequence and / or a sequence encoding ORF2p fused with an additional protein sequence. In some embodiments, ORF1p and / or ORF2p are fused with a nuclear retained sequence. In some embodiments, the nuclear retained sequence is an Alu sequence. In some embodiments, ORF1p and / or ORF2p are fused with an MS2 coat protein. In some embodiments, the 5'UTR sequence or 3'UTR sequence includes at least one, two, three, or more MS2 hairpin sequences. In some embodiments, the 5'UTR sequence or 3'UTR sequence includes a sequence that promotes or enhances the interaction between the polyA tail of mRNA and endonucleases and / or reverse transcriptases. In some embodiments, the 5'UTR sequence or 3'UTR sequence includes a sequence that promotes or enhances the interaction between polyA-binding protein (PABP) and endonucleases and / or reverse transcriptases. In some embodiments, the 5'UTR sequence or 3'UTR sequence includes a sequence that enhances the specificity of the endonuclease and / or reverse transcriptase to the above mRNA compared to another mRNA expressed by the cell. In some embodiments, the 5'UTR sequence or 3'UTR sequence includes an Alu element sequence.

[0119]

[0173] In some embodiments, the first sequence encoding ORF1p and the second sequence encoding endonuclease and / or reverse transcriptase have the same promoter. In some embodiments, the inserted sequence has a different promoter from the promoter of the first sequence encoding ORF1p. In some embodiments, the inserted sequence has a different promoter from the promoter of the second sequence encoding endonuclease and / or reverse transcriptase. In some embodiments, the first sequence encoding ORF1p and / or the second sequence encoding endonuclease and / or reverse transcriptase have a promoter or transcription initiation site selected from the group consisting of an inducible promoter, a CMV promoter or transcription initiation site, a T7 promoter or transcription initiation site, an EF1a promoter or transcription initiation site, and combinations thereof. In some embodiments, the inserted sequence has a promoter or transcription initiation site selected from the group consisting of an inducible promoter, a CMV promoter or transcription initiation site, a T7 promoter or transcription initiation site, an EF1a promoter or transcription initiation site, and combinations thereof.

[0120]

[0174] In some embodiments, the first sequence encoding ORF1p and the second sequence encoding an endonuclease and / or reverse transcriptase are codon-optimized for expression in human cells.

[0121]

[0175] In some embodiments, the mRNA includes a WPRE element. In some embodiments, the mRNA includes a selection marker. In some embodiments, the mRNA includes a sequence encoding an affinity tag. In some embodiments, the affinity tag is ligated to a sequence encoding an endonuclease and / or reverse transcriptase.

[0122]

[0176] In some embodiments, the 3'UTR contains a polyA sequence, or the polyA sequence is added to the mRNA in vitro. In some embodiments, the polyA sequence is downstream of a sequence encoding an endonuclease and / or reverse transcriptase. In this state, the inserted sequence is upstream of the polyA sequence.

[0123]

[0177] In some embodiments, the 3'UTR sequence includes an insertion sequence. In some embodiments, the insertion sequence includes a sequence that is the reverse complementary strand of the sequence encoding the foreign polypeptide. In some embodiments, the insertion sequence includes a polyadenylation site. In some embodiments, the insertion sequence includes an SV40 polyadenylation site. In some embodiments, the insertion sequence includes a polyadenylation site upstream of the sequence that is the reverse complementary strand of the sequence encoding the foreign polypeptide. In some embodiments, the insertion sequence is integrated into the genome at a locus that is not a ribosomal locus. In some embodiments, the insertion sequence is integrated into a gene or a regulatory region of a gene, thereby disrupting the gene or downregulating gene expression. In some embodiments, the insertion sequence is integrated into a gene or a regulatory region of a gene, thereby upregulating gene expression. In some embodiments, the insertion sequence is integrated into the genome and replaces a gene. In some embodiments, the insertion sequence is stably integrated into the genome. In some embodiments, the insertion sequence retrotransfers into the genome. In some embodiments, the insertion sequence is integrated into the genome by cleavage of the DNA strand at the target site by an mRNA-encoded endonuclease. In some embodiments, the insertion sequence is incorporated into the genome via reverse transcription (TPRT) using the target as a primer. In some embodiments, the insertion sequence is incorporated into the genome via reverse splicing of mRNA to a target DNA site in the genome.

[0124]

[0178] In some embodiments, the cells are immune cells. In some embodiments, the immune cells are T cells or B cells. In some embodiments, the immune cells are bone marrow cells. In some embodiments, the immune cells are selected from the group consisting of monocytes, macrophages, dendritic cells, dendritic progenitor cells, and macrophage progenitor cells.

[0125]

[0179] In some embodiments, the mRNA is self-integrated mRNA. In some embodiments, the method includes the step of introducing mRNA into cells. In some embodiments, the method includes the step of introducing a vector encoding mRNA into cells. In some embodiments, the method includes the step of introducing mRNA or an mRNA-encoding vector into cells ex vivo. In some embodiments, the method further includes the step of administering cells to a human subject. In some embodiments, the method includes the step of administering mRNA or an mRNA-encoding vector to a human subject. In some embodiments, no immune response is induced in the human subject. In some embodiments, the mRNA or vector is substantially non-immunogenic.

[0126]

[0180] In some embodiments, the vector is a plasmid or a viral vector. In some embodiments, the vector contains a non-LTR retrotransposon. In some embodiments, the vector contains a human L1 element. In some embodiments, the vector contains the L1 retrotransposon ORF1 gene. In some embodiments, the vector contains the L1 retrotransposon ORF2 gene. In some embodiments, the vector contains an L1 retrotransposon.

[0127]

[0181] In some embodiments, the mRNA is at least about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 kilobases. In some embodiments, the mRNA is at most about 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5 kilobases.

[0128]

[0182] In some embodiments, the mRNA includes sequences that inhibit or prevent mRNA degradation. In some embodiments, the sequence that inhibits or prevents mRNA degradation inhibits or prevents mRNA degradation by exonuclease or RNAse. In some embodiments, the sequence that inhibits or prevents mRNA degradation is a G4 structure, a pseudoknot, or a triple-stranded sequence. In some embodiments, the sequence that inhibits or prevents mRNA degradation is an exoribonuclease-resistant RNA structure derived from flavivirus RNA or an ENE element derived from KSV. In some embodiments, the sequence that inhibits or prevents mRNA degradation inhibits or prevents mRNA degradation by deadenylase. In some embodiments, the sequence that inhibits or prevents mRNA degradation contains a non-adenosine nucleotide inside or at the end of the poly-A tail of the mRNA. In some embodiments, the sequence that inhibits or prevents mRNA degradation improves mRNA stability. In some embodiments, the exogenous sequence includes a sequence encoding an exogenous polypeptide. In some embodiments, the sequence encoding the exogenous polypeptide is not in-frame with a sequence encoding an endonuclease and / or reverse transcriptase. In some embodiments, the sequence encoding the foreign polypeptide is not in-frame with the sequence encoding the endonuclease and / or reverse transcriptase. In some embodiments, the foreign sequence does not contain an intron. In some embodiments, the foreign sequence includes a sequence encoding a foreign polypeptide selected from the group consisting of enzymes, receptors, transport proteins, structural proteins, hormones, antibodies, contractile proteins, and storage proteins. In some embodiments, the foreign sequence includes a sequence encoding a foreign polypeptide selected from the group consisting of chimeric antigen receptors (CARs), ligands, antibodies, receptors, and enzymes. In some embodiments, the foreign sequence includes a regulatory sequence. In some embodiments, the regulatory sequence includes a cis-acting regulatory sequence. In some embodiments, the regulatory sequence includes a cis-acting regulatory sequence selected from the group consisting of enhancers, silencers, promoters, or response elements. In some embodiments, the regulatory sequence includes a trans-acting regulatory sequence. In some embodiments, the regulatory sequence includes a trans-acting regulatory sequence encoding a transcription factor.

[0129]

[0183] In some embodiments, the insertion of the insert sequence does not adversely affect the health of the cell. In some embodiments, endonucleases, reverse transcriptases, or both are capable of site-specific insertion of the insert sequence.

[0130]

[0184] In some embodiments, the mRNA includes a sequence encoding an additional nuclease domain or a nuclease domain not derived from ORF2. In some embodiments, the mRNA includes a sequence encoding a megaTAL nuclease domain, a TALEN domain, a Cas9 domain, a zinc finger binding domain derived from the R2 retroelement, or a DNA binding domain that binds to a repetitive sequence such as Rep78 derived from AAV. In some embodiments, the endonuclease includes a mutation that reduces the activity of the endonuclease compared to an endonuclea without the mutation. In some embodiments, the endonuclease is an ORF2p endonuclease, and the mutation is S228P. In some embodiments, the mRNA includes a sequence encoding a domain that improves the accuracy and / or processing ability of the reverse transcriptase. In some embodiments, the reverse transcriptase is a reverse transcriptase derived from a retroelement other than ORF2, or a reverse transcriptase with higher accuracy and / or processing ability compared to an ORF2p reverse transcriptase. In some embodiments, the reverse transcriptase is a group II intron reverse transcriptase. In some embodiments, the group II intron reverse transcriptase is the group IIA intron reverse transcriptase, the group IIB intron reverse transcriptase, or the group IIC intron reverse transcriptase. In some embodiments, the group II intron reverse transcriptase is TGIRT-II or TGIRT-III.

[0131]

[0185] In some embodiments, the mRNA includes a sequence containing an Alu element and / or a ribosome-binding aptamer. In some embodiments, the mRNA includes a sequence encoding a polypeptide containing a DNA-binding domain. In some embodiments, the 3'UTR sequence is It originates from the virus 3'UTR or beta-globin 3'UTR.

[0132]

[0186] In one embodiment, the present invention provides a composition comprising recombinant mRNA or an mRNA-encoding vector, wherein the mRNA comprises a human LINE-1 transposon sequence including a human LINE-1 transposon 5'UTR sequence, a sequence encoding ORF1p downstream of the human LINE-1 transposon 5'UTR sequence, an ORF-linker sequence downstream of the ORF1p sequence, a sequence encoding ORF2p downstream of the ORF-linker sequence, and a 3'UTR sequence derived from a human LINE-1 transposon downstream of the ORF2p sequence, wherein the 3'UTR sequence comprises an insertion sequence, and the insertion sequence is the reverse complementary strand of a sequence encoding a foreign polypeptide or the reverse complementary strand of a sequence encoding a foreign regulatory element.

[0133]

[0187] In some embodiments, the insertion sequence, when introduced into a cell, is integrated into the cell's genome. In some embodiments, the insertion sequence is integrated into a gene associated with a condition or disease, thereby disrupting the gene or downregulating its expression. In some embodiments, the insertion sequence is integrated into a gene, thereby upregulating its expression. In some embodiments, recombinant mRNA or a vector encoding mRNA is isolated or purified.

[0134]

[0188] In one embodiment, a composition is provided herein that comprises a nucleic acid comprising (a) a long scattered repeat sequence (LINE) polypeptide comprising human ORF1p and human ORF2p, and (b) an insertion sequence which is the reverse complementary chain of a sequence encoding a foreign polypeptide or the reverse complementary chain of a sequence encoding a foreign regulatory element, and is substantially non-immunogenic.

[0135]

[0189] In some embodiments, the composition comprises human ORF1p and human ORF2p proteins. In some embodiments, the composition comprises ribonucleoprotein (RNP) containing human ORF1p and human ORF2p in complex with a nucleic acid. In some embodiments, the nucleic acid is mRNA.

[0136]

[0190] In one embodiment, a composition comprising cells containing the composition described herein is provided herein. In some embodiments, the cells are immune cells. In some embodiments, the immune cells are T cells or B cells. In some embodiments, the immune cells are myeloid cells. In some embodiments, the immune cells are selected from the group consisting of monocytes, macrophages, dendritic cells, dendritic progenitor cells, and macrophage progenitor cells. In some embodiments, the insertion sequence is the reverse complementary chain of a sequence encoding an exogenous polypeptide, and the exogenous polypeptide is a chimeric antigen receptor (CAR).

[0137]

[0191] In one embodiment, a pharmaceutical composition comprising the composition described herein and pharmaceutically acceptable excipients is provided herein. In some embodiments, the pharmaceutical composition is for use in gene therapy. In some embodiments, the pharmaceutical composition is for use in the manufacture of a medicament for treating a disease or condition. In some embodiments, the pharmaceutical composition is for use in treating a disease or condition. In one embodiment, a method for treating a disease in a subject is provided herein, comprising the step of administering the pharmaceutical composition described herein to a subject having the disease or condition. In some embodiments, the method increases the amount or activity of a protein or functional RNA in the subject. In some embodiments, the subject has an insufficient amount or activity of a protein or functional RNA. In some embodiments, the insufficient amount or activity of a protein or functional RNA is associated with or causes a disease or condition.

[0138]

[0192] In some embodiments, the method further includes the step of administering a human silencing hub (HUSH) complex inhibitor, a FAM208A inhibitor, or a TRIM28 inhibitor. In some embodiments, the human silencing hub (HUSH) complex inhibitor is a periphylline, TASOR, and / or MPP8 inhibitor. In some embodiments, the human silencing hub (HUSH) complex inhibitor inhibits the assembly of the HUSH complex.

[0139]

[0193] In some embodiments, the drug inhibits the Fanconia anemia complex. In some embodiments, the drug inhibits FANCD2-FANC1 heterodimer monoubiquitination. In some embodiments, the drug inhibits FANCD2-FANC1 heterodimer formation. In some embodiments, the drug inhibits the Fanconia anemia (FA) core complex. The FA core complex is a component of the Fanconia anemia DNA damage repair pathway, for example, in chemotherapy-induced interstrand crosslinking. The FA core complex contains two major dimers: a FANCB subunit and a 100 kDa FA-related protein (FAAP100) subunit, flanked by two copies of a ring finger subunit called FANCL. These two heterotrimers act as a scaffold for assembling the remaining five subunits, resulting in an elongated asymmetric structure. Destabilization of the scaffold can disrupt the entire complex, leading to a non-functional FA pathway. Examples of drugs that can inhibit the FA core complex include bortezomib, as well as the curcumin analogs EF24 and 4H-TTD.

[0140]

[0194] In some embodiments, the inserted sequence can be placed under the control of tissue-specific elements, and as a result, the entire inserted DNA becomes functional only in cells where the tissue-specific elements are active.

[0141]

[0195] In one embodiment, a method and composition for stable gene transfer into cells are provided herein by introducing a target heteronucleotide or gene (e.g., a transgene, regulatory sequence, e.g., a sequence relating to interfering nucleic acids, siRNA, miRNA) adjacent to a sequence that causes retrotransfer of a heteronucleotide sequence into the cell's genome. In some embodiments, the heteronucleotide is referred to herein for illustrative purposes as an insertion fragment, which is a nucleic acid sequence that can be reverse transcribed and inserted into the cell's genome according to the intended design of the construct described herein. In some embodiments, the heteronucleotide is also referred to herein for illustrative purposes as a cargo or cargo sequence. The cargo may include a sequence of heteronucleotide to be inserted into the genome. In some embodiments, the cells may be mammalian cells. Mammalian cells may be of epithelial, mesothelial, or endothelial origin. In some embodiments, the cells may be stem cells. In some embodiments, the cells may be progenitor cells. In some embodiments, the cells may be terminally differentiated cells. In some embodiments, the cells may be muscle cells, cardiac cells, epithelial cells, hematopoietic cells, mucosal cells, epidermal cells, squamous cells, chondrocytes, osteocytes, or any cells of mammalian origin. In some embodiments, the cells are hematopoietic lineage cells. In some embodiments, the cells are myeloid lineage cells, or phagocytic cells, such as monocytes, macrophages, dendritic cells, or myeloid progenitor cells. In some embodiments, the nucleic acid encoding the transgene is mRNA.

[0142]

[0196] In some embodiments, the retrotransposable element may be derived from a non-LTR retrotransposon.

[0197] A method for incorporating a nucleic acid sequence into the genome of a cell is provided herein, comprising the step of introducing recombinant mRNA or an mRNA-encoding vector into a cell, wherein the mRNA comprises an insertion sequence, and the insertion sequence is incorporated into the genome of the cell. In some embodiments, the insertion sequence is (i) a foreign sequence, or (ii) a sequence that is the reverse complementary strand of a foreign sequence; a 5'UTR sequence and a 3'UTR sequence downstream of the 5'UTR sequence, and 5'U The ORF protein comprises a 5'UTR sequence and a 3'UTR sequence, the TR sequence or 3'UTR sequence containing a binding site for a human ORF protein. In some embodiments, the ORF protein is a human LINE1 ORF2 protein. In some embodiments, the ORF protein is a non-human ORF protein. In some embodiments, the ORF protein is a chimeric protein, a recombinant protein, or a modified protein.

[0143]

[0198] A method for incorporating a nucleic acid sequence into the genome of an immune cell is provided herein, comprising the step of introducing recombinant mRNA or an mRNA-encoding vector, wherein the mRNA comprises (a) an insertion sequence, which is (i) a foreign sequence or (ii) the reverse complementary strand of a foreign sequence; and (b) a 5'UTR sequence and a 3'UTR sequence downstream of the 5'UTR sequence, wherein the 5'UTR sequence or the 3'UTR sequence comprises an endonuclease-binding site and a reverse transcriptase-binding site, and the method for incorporating the transgene sequence into the genome of an immune cell is provided herein.

[0144]

[0199] In some embodiments, structural elements mediating RNA integration or transposition may be encoded in a synthetic construct that is expected to deliver the heterologous gene of interest to a cell. In some embodiments, the synthetic construct may include a nucleic acid encoding the heterologous gene of interest and a structural element that induces integration or retrotransposition of the heterologous gene of interest into the genome. In some embodiments, the structural element that induces integration or retrotransposition may include a 5'L1 RNA region and a 3'L1 region, the latter including a poly-A3' region for priming. In some embodiments, the 5'L1 RNA region may include one or more stem-loop regions. In some embodiments, the L1 3' region may include one or more stem-loop regions. In some embodiments, the 5' and 3'L1 regions are constructed to be adjacent to the nucleic acid sequence encoding the heterologous gene of interest (transgene). In some embodiments, the structural element may include a region derived from L1 or Alu RNA that includes a hairpin-loop structure containing A-box and B-box elements that are ribosome binding sites. In some embodiments, the synthetic nucleic acid may include an L1-Ta promoter.

[0145]

[0200] Two types of LINE RNA recognition by ORF2p—strict and sluggish—may exist. In the strict type, the RT recognizes its own 3'UTR tail, while in the sluggish type, the RT does not require any specific recognition other than the polyA tail. The classification into strict and sluggish types arose from the observation that some LINE / SINE pairs share the same 3' end. Regarding the strict type, experimental studies have shown that the 3'UTR stem-loop facilitates retrotransition. The 5'UTR of the LINE retrotransition sequence has been shown to contain three conserved stem-loop regions.

[0146]

[0201] In some embodiments, the transgene or transcript of interest may be flanked at the 5' and 3' ends by transposers derived from L1 or Alu sequences. In some embodiments, the 5' region of the retrotransposon contains an Alu sequence. In some embodiments, the 3' region of the retrotransposon contains an Alu sequence. In some embodiments, the 5' region of the retrotransposon contains an L1 sequence. In some embodiments, the 3' region of the retrotransposon contains an L1 sequence. In some embodiments, the transgene or transcript of interest is flanked by an SVA transposon sequence.

[0147]

[0202] In some embodiments, the transcript of interest may include an L1 or Alu sequence encoding a binding region for ORF2p and a 3' poly-A priming region. In some embodiments, the heterologous nucleic acid encoding the transgene of interest may be adjacent to the L1 or Alu sequence encoding a binding region for ORF1p and a 3' poly-A priming region. The 3' region may include one or more stem-loop structures. In some embodiments, the transcript of interest is structured for cis-integration or retro-transfer. In some embodiments, the transcript of interest is structured for trans-integration or retro-transfer.

[0148]

[0203] In some embodiments, the retrotransposon is a human retrotransposon. The sequence of the human retrotransposon may include a sequence encoding an endonuclease and / or reverse transcriptase. The sequence of the human retrotransposon may encode two proteins translated from a single RNA containing two non-duplication ORFs. In some embodiments, the two ORFs are ORF1 and ORF2.

[0149]

[0204] Accordingly, a method for stably incorporating a heterologous nucleic acid encoding a transgene into the genome of cells such as bone marrow cells is provided herein, comprising the steps of introducing a nucleic acid into the cell that encodes a transgene; one or more 5' nucleic acid sequences adjacent to the region encoding the transgene, including the 5' region of a retrotransposon; and one or more 3' nucleic acid sequences adjacent to the region encoding the transgene, including the 3' region of a retrotransposon, wherein the 3' region of the retrotransposon comprises a genomic DNA priming sequence and a LINE transposase binding sequence, and has endonuclease and reverse transcriptase (RT) activity, respectively.

[0150]

[0205] A method for incorporating a nucleic acid sequence into the genome of a cell is provided herein, comprising the step of introducing recombinant mRNA or an mRNA-coding vector, wherein the mRNA comprises an insertion sequence comprising (i) a foreign sequence or (ii) a reverse complementary strand of a foreign sequence; (b) a 5'UTR sequence, a sequence of a human retrotransposon downstream of the 5'UTR sequence, and a 3'UTR sequence downstream of the human retrotransposon sequence, wherein the 5'UTR sequence or the 3'UTR sequence comprises an endonuclease-binding site and a reverse transcriptase-binding site, the human retrotransposon sequence encoding two proteins translated from a single RNA containing two ORFs, and the insertion sequence is incorporated into the genome of a cell.

[0151]

[0206] In some embodiments, the method includes the step of using a single nucleic acid molecule to deliver and incorporate an insertion sequence into the cell's genome. The single nucleic acid molecule may be a plasmid vector. The single nucleic acid may be a DNA or RNA molecule. The single nucleic acid may be mRNA.

[0152]

[0207] In some embodiments, the method includes the step of introducing one or more polynucleotides comprising a human retrotransposon and a heterologous nucleic acid sequence into a cell. In some embodiments, the one or more polynucleotides comprise (i) a first nucleic acid molecule encoding ORF1p, and (ii) a second nucleic acid molecule encoding ORF2p and a cargo sequence. In some embodiments, the first and second nucleic acids are mRNA. In some embodiments, the first and second nucleic acids are DNA encoded, for example, in a separate plasmid vector.

[0153]

[0208] Self-integrating polynucleotides containing sequences to be inserted into the cellular genome are provided herein, the insertion fragments being stably integrated into the genome by the naked self-integrating polynucleotide. In some embodiments, the polynucleotide is RNA. In some embodiments, the polynucleotide is mRNA. In some embodiments, the polynucleotide is modified mRNA. In some embodiments, the modification ensures protection from RNAse in the intracellular environment. In some embodiments, the modification includes substitutional modified nucleotides, such as 5-methylcytidine, pseudouridine, or 2-thiouridine.

[0154]

[0209] In some embodiments, a single polynucleotide is used for the delivery and genomic integration of the insertion (or cargo) nucleic acid. In some embodiments, the single polynucleotide is bicistronic. In some embodiments, the single polynucleotide is tricistronic. It is nic. In some embodiments, a single polynucleotide is multicistronic. In some embodiments, two or more polynucleotide molecules are used for the delivery and genomic integration of insertion (or cargo) nucleic acids.

[0155]

[0210] In some embodiments, a retrotransferable gene can be generated which comprises (i) a heterologous nucleic acid (insertion fragment) encoding a transgene or non-coding sequence to be inserted into the cell's genome, (ii) a nuclear sequence encoding one or more retrotransposon ORF coding sequences, and (iii) one or more UTR regions of an ORF coding sequence such that the heterologous nucleic acid encoding the transgene or non-coding sequence to be inserted is contained within the UTR sequence, and the 3' region of the retrotransposon ORF coding sequence comprises a genomic DNA priming sequence.

[0156]

[0211] In some embodiments, retrotransposable elements can be introduced into cells to stably integrate a transgene into genomic DNA. In some embodiments, the retrotransposable element comprises (a) a retrotransposon protein-coding sequence and 3'UTR, and (b) a sequence containing a heterogeneous nucleic acid to be inserted (e.g., integrated) into the cell's genome. The retrotransposon protein-coding sequence and 3'UTR are complete and sufficient units for delivering the heterogeneous nucleic acid sequence into the cell's genome and may include a sequence in the 3'UTR for binding to genomic DNA at a region cleaved by the retrotransposable element, such as an endonuclease, reverse transcriptase, or a sequence in the 3'UTR to prime it and initiate reverse transcription and integration of the heterogeneous nucleic acid.

[0157]

[0212] In some embodiments, the code sequence of the insert is oriented forward with respect to the code sequence of one or more ORFs. In some embodiments, the code sequence of the insert is oriented backward with respect to the code sequence of one or more ORFs. The code sequence of the insert and the code sequence of one or more ORFs may include separate modulating elements, such as a 5'UTR, a 3'UTR, a promoter, an enhancer, etc. In some embodiments, the 3'UTR or 5'UTR of the insert may include the code sequence of one or more ORFs, and similarly, the code sequence of the insert may be located within the 3'UTR of the code sequence of one or more ORFs.

[0158]

[0213] In some embodiments, a retrotransferable genetic factor can be generated comprising a retrotransferable genetic factor comprising (a) an insertion sequence, which comprises (i) a foreign sequence and a sequence that is the reverse complementary strand of the foreign sequence; a 5'UTR sequence and a 3'UTR sequence downstream of the 5'UTR sequence, wherein the 5'UTR sequence or the 3'UTR sequence contains a binding site for a human ORF protein.

[0159]

[0214] In some embodiments, the retrotransposon may include a sine or line element. In some embodiments, the retrotransposon includes a sine or line stem-loop structure such as an Alu element.

[0160]

[0215] In some embodiments, the retrotransposon is a LINE-1 (L1) retrotransposon. In some embodiments, the retrotransposon is human LINE-1. Human LINE-1 sequences are abundant in the human genome. There are approximately 13,224 human L1 sequences in total, of which 480 are active, accounting for about 3.6%. Therefore, human L1 proteins are well-tolerated in humans and are non-immunogenic. Furthermore, strict regulation of random transposition in humans ensures that random transposase activity is not induced by the introduction of the L1 system described herein. In addition, the retrotransposable constructs designed herein may include targeting and specific insertion of the insertion sequence. In some embodiments, the retrotransposable gene is designed to prevent random insertion that would result in genomic instability. This may include designs that take care to prevent the process from starting while simultaneously overcoming the silencing mechanisms that are actively and widely present in human cells.

[0161]

[0216] It contains nucleic acid sequences.

[0162]

[0217] catcgcggccttgtttacgatagctaagacgtggaatcagcctaagtgccccacaatgatcgattggatcaagaaaatgtggcatatttataccatggagtattacgcagcaattaagaatgacgaatttatttccttcgttgggacctggatgaagctggagactattattctgagcaagctgtctcaggagcaaaagacaaagcatagaatcttctctctcattggtggtaactaa and a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0163]

[0218] In some embodiments, the structure is MVIGTYISIITLNVNGLNAPTKRHRLAEWIQKQDPYICCLQETHFRPRDTYRLKVRGWKKIFHANGNQKKAGVAILISDKIDFKIKNVTRDKEGHYIMIQGSIQEEDITIINIYAPNIGAPQYIRQLLTAIKEEIDSNTIIVGDFNTSLTPMDRSSKMKINKETEALNDTIDQIDLIDIYR aatatggtactggcacaaaaacagaaacatagatcaatggaacaagatagaaagcccagagattaacccacgcacctatggtcaactaatctatgacaaaggaggcaaagatatacaatggagaaaagacagtctcttcaataagtggtgctgggaaaactggacagccacatgtaaaagatgaaattatactccctaaccacatacacaaaaataaactcaaaatggattagagacctaaataagaactggacactataaaactttagagaaaacataggagaacactctttgacataaatcacagcaagagatcttttcgatccacctcctaggtaatggaaataaaaacaaaaataaacaagtgggacctaatgaacttcaaagcttttgcacagcaaaggaaaccataaacaagacgaaaagacaaccctcagaatgggagaaata tttgcaaatgaatcaacggagaaaggattaatctccaaaatatataaaacagctcattcagctcaatatcaaagaaacaaacaccccaatccaaaaatgggcagaagacctaaatagacatttctccaaaagaagacatacagacggccacgaagcacatgaaaagatgctcaacatcactaattattagagaaatgcaaatcaaaactacaatgaggtatcacctcactcc tgttagaatgggcatcatcagaaaatctacaaacaacaaatgctggagagggtgtggagaaaagggaaccctcttgcactgttggtgggaatgtaaattgatacagccactatgggagaacaatatggaggttccttaaaaaactaaaaatagaattaccatatgacccagcaatcccactactgggcatatacccagagaaaacgtaattcaaaaaagacacatgcacccg aatgttcattgcagcactatttacaatagccaggtcatggaagcaacctaaatgcccatcgacagacgaatggataaagaagatgtggtacatatatacaatggaatattactcagccataaaaag gaacgaaattgggtcattttagagacgtggatggatctagagactgtcatacagagtgaagtaagtcagaaagagaaaaacaaatatcgtatattaacgcatatatgtggaacctggaaaaatgg The nucleic acid sequence comprises tacagatgaaccggtctgcaggacagaaattgagacacaaatgtaa and having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0164]

[0219] In some embodiments, the construct includes a nucleic acid sequence encoding a nuclear localization sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with PAAKRVKLD. In some embodiments, the nuclear localization sequence fuses with the ORF2p sequence. In some embodiments, the construct includes a nucleic acid sequence encoding a flag tag having the sequence DYKDDDDK. In some embodiments, the flag tag fuses with the ORF2p sequence. In some embodiments, the flag tag fuses with the nuclear localization sequence.

[0165]

[0220] In some embodiments, the structure is ASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYAMASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGN The molecule contains a nucleic acid sequence encoding an MS2 coat protein having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with PIPSAIAANSGIY. In some embodiments, the MS2 coat protein sequence is fused with the ORF2p sequence.

[0166]

[0221] In some embodiments, the transgene may include an adjacent sequence containing an Alu ORF2p recognition sequence.

[0222] In some embodiments, additional elements may be introduced into the mRNA. In some embodiments, the additional elements may be IRES elements or T2A elements. In some embodiments, the mRNA transcript contains one, two, three, or more stop codons at the 3' end.

[0167]

[0223] In some embodiments, one, two, three, or more stop codons are designed to be in tandem. In some embodiments, one, two, three, or more stop codons are designed to be present in all three reading frames. In some embodiments, one, two, three, or more stop codons may be present in multiple reading frames and designed to be in tandem.

[0168]

[0224] In some embodiments, one or more target-specific nucleotides may be added to the priming end of the L1 or Alu RNA priming region.

[0225] In some embodiments, the 5'UTR sequence or 3'UTR sequence may be able to bind to one or more endogenous proteins that regulate gene retrotransfer and / or stable integration, in addition to being able to bind to the ORF protein. In some embodiments, the adjacent sequence may be able to bind to the PABP protein.

[0169]

[0226] In some embodiments, the 5' region adjacent to the transcript may contain a strong promoter. In some embodiments, the promoter is a CMV promoter.

[0227] In some embodiments, an additional nucleus encoding L1 ORF2p is introduced into the cell. In some embodiments, the sequence encoding L1 ORF1 is removed, leaving only L1-ORF2. In some embodiments, the nucleic acid encoding the transgene with flanking elements is mRNA. In some embodiments, endogenous L1-ORF1p function may be suppressed or inhibited.

[0170]

[0228] In some embodiments, the nucleic acid encoding the transgene having a retrotransitional neighbor element includes one or more nucleic acid modifications. In some embodiments, the nucleic acid encoding the transgene having a retrotransitional neighbor element includes one or more nucleic acid modifications to the transgene. In some embodiments, the modification includes codon optimization of the transgene sequence. In some embodiments, codon optimization is for more efficient recognition by the human translation mechanism, resulting in more efficient expression in human cells. In some embodiments, one or more nucleic acid modifications are carried out in a 5' neighbor sequence or a 3' neighbor sequence containing one or more stem-loop regions. The nucleic acid encoding the transgene having a retrotransitional neighbor element is It contains one, two, three, four, five, six, seven, eight, nine, ten, or more nucleic acid modifications.

[0171]

[0229] In some embodiments, the retrotransferred transgene is stably expressed throughout the lifespan of the cell. In some embodiments, the cells are myeloid cells. In some embodiments, the myeloid cells are monocyte progenitor cells. In some embodiments, the myeloid cells are immature monocytes. In some embodiments, the monocytes are undifferentiated monocytes. In some embodiments, the myeloid cells are CD14+ cells. In some embodiments, the myeloid cells do not express the CD16 marker. In some embodiments, the myeloid cells can remain functionally active for a desired period of more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or longer under preferred conditions. Preferred conditions may represent in vitro conditions, in vivo conditions, or a combination of both.

[0172]

[0230] In some embodiments, the retrotransposed transgene can be stably expressed in cells for approximately 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, the retrotransposed transgene is stably expressed in cells for more than 10 days. In some embodiments, the retrotransposed transgene is stably expressed in cells for more than 2 weeks. In some embodiments, the retrotransposed transgene is stably expressed in cells for approximately 1 month.

[0173]

[0231] In some embodiments, the retrotransposed transgene may be modified for stable expression. In some embodiments, the retrotransposed transgene may be modified for resistance to in vivo silencing.

[0174]

[0232] In some embodiments, the expression of the retrotransposed transgene can be controlled by a strong promoter. In some embodiments, the expression of the retrotransposed transgene can be controlled by a moderately strong promoter. In some embodiments, the expression of the retrotransposed transgene can be controlled by a strong promoter that can be regulated in an in vivo environment. In some embodiments, the promoter is the CMV promoter. In some embodiments, the promoter is the L1-Ta promoter.

[0175]

[0233] In some embodiments, ORF1p may be overexpressed. In some embodiments, ORF2 may be overexpressed. In some embodiments, ORF1p or ORF2p, or both, are overexpressed. In some embodiments, in the case of ORF1 overexpression, ORF1p is at least 1.1 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 12 times, 14 times, 16 times, 18 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or at least 100 times compared to cells that do not overexpress ORF1.

[0176]

[0234] In some embodiments, in the case of overexpression of the ORF2 sequence, ORF2p is at least 1.1 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 12 times, 14 times, 16 times, 18 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or at least 100 times compared to cells that do not overexpress ORF2p.

[0177] Retrotransfer accuracy and target specificity

[0235] The LINE-1 element binds to the poly(A) tail of its own mRNA and performs retrotransition. It can be initiated. The LINE-1 element prefers to retrotranspose itself onto its own mRNA rather than random mRNA (Dewannieux et al., 2013, 3,000 times more LINE-1 retrotransposition compared to random mRNA). In addition, the LINE-1 element It is also possible to incorporate nonspecific polyA sequences into the genome.

[0178]

[0236] In one embodiment, retrotransposition compositions and methods for using them with enhanced retrotransposition specificity are provided herein. For example, a highly specific retrotransposition composition can be used for highly specific and efficient reverse transcription and subsequent integration into the genome of target cells, such as myeloid cells. In some embodiments, the retrotransposition compositions provided herein include a retrotransposition cassette comprising one or more additional components that enhance integration or retrotransposition specificity. For example, a retrotransposon cassette may encode one or more additional elements that enable high-affinity RNA-protein interactions that compete for and eliminate nonspecific binding between a polyA sequence and ORF2.

[0179]

[0237] Therefore, several means for enhancing integration or retrotransition efficiency are disclosed herein.

[0238] One exemplary means of enhancing integration or retrotransfer efficiency is cellular external manipulation. The endonuclease function of retrotransfer mechanisms delivered to cells is likely to be inhibited by cellular translocation silencing mechanisms, such as DNA repair pathways. For example, small molecules can be used to modulate or inhibit DNA repair pathways in cells before introducing nucleic acids. For example, cell sorting and / or synchronization can be used before introducing nucleic acids by electroporation, etc., as cell cycle-synchronized cell populations have been shown to increase gene transfer into cells. Cell sorting can be used to synchronize or homogenize cell types to increase uniform transfer and expression of foreign nucleic acids. Homogeneity can be achieved by sorting stem cells from non-stem cells. Another exemplary means of enhancing integration or retrotransfer efficiency is to enhance biochemical activity. For example, this can be achieved by improving reverse transcriptase processing ability or DNA cleavage (endonuclease) activity. Another exemplary means of enhancing integration or retrotransfer efficiency is to disrupt endogenous silencing mechanisms. For example, this can be achieved by replacing the entire LINE-1 sequence with LINE-1 from a different organism. Another exemplary means of enhancing integration or retrotransition efficiency is to enhance translation and ribosome binding. For example, this can be achieved by increasing the expression of the LINE-1 protein, increasing the LINE protein that binds to LINE-1 mRNA, or increasing the LINE-1 complex that binds to ribosomes. Another exemplary means of enhancing integration or retrotransition efficiency is to increase nuclear translocation or retention. For example, this can be achieved by fusing the LINE-1 sequence with a nuclear retention signal sequence. Another exemplary means of enhancing integration or retrotransition efficiency is to enhance sequence-specific insertion. For example, this can be achieved by fusing the target domain with ORF2 to increase sequence-specific retrotransition.

[0180]

[0239] In one embodiment, the method comprises the step of enhancing the retrotransposon by modifying the UTR sequence of the LINE-1 ORF to improve the specificity and robustness of cargo expression. In some embodiments, the 5'UTR upstream of the ORF1 or ORF2 coding sequence may be further modified to include a sequence complementary to the sequence of a target region in the genome, which facilitates homologous recombination at a specific site where the ORF nuclease can act and retrotransposition can occur. In some embodiments, the sequence that can bind to the target sequence by homology is 2 to 15 nucleotides long. In some embodiments, the sequence homologous to the genomic target contained in the 5'UTR of ORF1 mRNA may be about 3 nucleotides, about 4 nucleotides, about 5 nucleotides, about 6 nucleotides, about 7 nucleotides, about 8 nucleotides, about 9 nucleotides, or about 10 nucleotides long. In some embodiments, the sequence homologous to the genomic target is about 12 or about 15 nucleotides long. In some embodiments, the sequence homologous to the genomic target comprises approximately 2-5, 2-6, 2-8, 2-10, or 2-12 consecutive nucleotides that share complementarity with each target region within the genome.

[0181]

[0240] In some embodiments, ORF2 associates with or fuses with an additional protein domain containing RNA-binding activity. In some embodiments, the retrotransposon cassette includes an homogeneous RNA sequence having affinity for the additional protein domain that associates with or fuses with ORF2. In some embodiments, ORF2 associates with or fuses with the MS2-MCP coat protein. In some embodiments, the retrotransposon cassette further includes an MS2 hairpin RNA sequence in a 3' or 5' UTR sequence that interacts with the MS2-MCP coat protein. In some embodiments, ORF2 associates with or fuses with the PP7 coat protein. In some embodiments, the retrotransposon cassette further includes a PP7 hairpin RNA sequence in a 3' or 5' UTR sequence that interacts with the MS2-MCP coat protein. In some embodiments, one or more additional elements improve the retrotransposition specificity by at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 100 times, at least 200 times, at least 300 times, at least 500 times, at least 1000 times, at least 1500 times, at least 2000 times, at least 3000 times, at least 5000 times, or more, compared to a retrotransposon cassette without one or more additional elements.

[0182]

[0241] The DNA endonuclease domain consists of a series of purines at the 3' end of the target site and a series of pyrimidines (Py) following the target site. n ↓(Pu) n It is thought to have specificity for (adenosine). An example sequence is (adenosine) n ↓(Chimijin) n It is possible.

[0183]

[0242] In one embodiment, a method using retrotransposition with high target specificity is provided herein. Accordingly, a method and composition for the stable incorporation of a transgene into the genome of myeloid cells such as monocytes or macrophages is provided herein, wherein the method comprises the step of incorporating the transgene using a non-LTR retrotransposon system, and retrotransposition with target specificity, high precision, and accuracy is carried out at a specific genomic locus. Accordingly, in some embodiments, the method comprises the step of administering to cells a composition comprising a system having one or more retrotransposable elements and at least one transgene adjacent to it, and one or more nucleic acids encoding one or more proteins to improve transposition specificity, and / or further comprising the step of modifying one or more genes associated with retrotransposition.

[0184]

[0243] The nucleic acid containing the transgene located in the UTR region of a retrotransferant is often referred to as the retrotransfer cassette. Therefore, in some embodiments, the retrotransfer cassette includes a nucleic acid encoding the transgene and adjacent to the Alu transferant. The retrotransferant includes sequences for binding to retrotransposons, e.g., L1-transposons, e.g., L1-ORF proteins, i.e., ORF1p and ORF2p. It is known that ORF proteins bind to their own mRNA sequences for retrotransposition. Therefore, the retrotransfer cassette includes the nucleic acid encoding the transgene; adjacent L1-ORF2p binding sequences; and / or L1-ORF1p binding sequences, and outside the transgene sequence, sequences encoding the L1-ORF1p coding sequences and L1-ORF2p coding sequences. In some embodiments, a spacer region, also referred to as the ORF1-ORF2 interregion, is located between L1-ORF1 and L1-ORF2. In some embodiments, the L1-ORF1 and L1-ORF2 coding sequences are oriented opposite to the coding region of the transgene. The retrotransfer cassette may include a poly-A region downstream of the L1-ORF2 coding sequence, with the transgene sequence positioned downstream of the poly-A sequence. L1-ORF2 is an endonuclease (EN The L1-ORF2 sequence in the retrotransfer cassette described herein is a complete (intact) sequence, i.e., it encodes a full-length natural (WT) L1-ORF2 sequence. In some embodiments, the L1-ORF2 sequence in the retrotransfer cassette described herein includes a partial or modified sequence.

[0185]

[0244] The systems described herein may include promoters for expressing L1-ORF1p and L1-ORF2p. In some embodiments, transgene expression is driven by separate promoters. In some embodiments, the transgene and ORF are tandem-oriented. In some embodiments, the transgene and ORF are oppositely oriented.

[0186]

[0245] In some embodiments, the method includes the step of incorporating one or more elements in addition to the retrotransposon cassette. In some embodiments, the one or more additional elements include nucleic acid sequences encoding one or more domains of a heterologous protein. The heterologous protein may be a sequence-specific nucleic acid-binding protein, such as a sequence-specific DNA-binding protein domain (DBD). In some embodiments, the heterologous protein is a nuclease or a fragment thereof. In some embodiments, the additional elements include nucleic acid sequences encoding one or more nuclease domains or fragments thereof derived from the heterologous protein. In some embodiments, the heterologous nuclease domains have reduced nuclease activity. In some embodiments, the heterologous nuclease domains are inactive. In some embodiments, the ORF2 nuclease is inactive, but one or more nuclease domains derived from the heterologous protein are configured to confer specificity to the retrotransposition. In some embodiments, one or more nuclease domains or fragments thereof derived from the heterologous protein target a specific desired polynucleotide in the genome to which the retrotransposition and incorporation of the polynucleotide of interest should be incorporated. In some embodiments, one or more nuclease domains derived from heterologous proteins include megaTAL, TALEN, or zinc finger nuclease domains, e.g., megaTAL, TALE, or zinc finger domains fused to or associated with a nuclease domain, e.g., a FokI nuclease domain. In some embodiments, one or more nuclease domains derived from heterologous proteins include a CRISPR-Cas protein domain accompanied by a specific guide nucleic acid, e.g., a guide RNA (gRNA) to a specific target locus. In some embodiments, the CRISPR-Cas protein is a Cas9, Cas12a, Cas12b, Cas13, CasX, or CasY protein domain. In some embodiments, one or more nuclease domains derived from heterologous proteins have target specificity.

[0187]

[0246] In some embodiments, an additional nuclease domain may be incorporated into the ORF2 domain. In some embodiments, the additional nuclease may fused with the ORF2p domain. In some embodiments, the additional nuclease domain may fused with ORF2p, which is ORF2p containing a mutation in the ORF2p endonuclease domain. In some embodiments, the mutation inactivates the ORF2p endonuclease domain. In some embodiments, the mutation is a point mutation. In some embodiments, the mutation is a deletion. In some embodiments, the mutation is an insertion. In some embodiments, the mutation suppresses ORF2 endonuclease (nickase) activity. In some embodiments, the mutation inactivates DNA target recognition by the ORF2p endonuclease. In some embodiments, the mutation extends to a region associated with DNA recognition by the ORF2p nuclease. In some embodiments, the mutation reduces DNA target recognition by the ORF2p endonuclease. In some embodiments, the ORF2p endonuclease domain mutation is located in the N-terminal region of the protein. In some embodiments, the ORF2p endonuclease domain mutation is located in the protein's conserved region. In some embodiments, the ORF2p endonuclease domain mutation is located in the protein's conserved N-terminal region. In some embodiments, the mutation is present in the L1 endonuclease domain. In some embodiments, the mutation is present in the L1 endonuclease domain and includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more consecutive amino acids, including the 2nd In some embodiments, the mutation includes D145 of the L1 endonuclease domain. In some embodiments, the mutation may be D145A. In some embodiments, the mutation may include D205 of the L1 endonuclease domain. In some embodiments, the mutation may be D205G. In some embodiments, the mutation may include H230 of the L1 endonuclease domain. In some embodiments, the mutation may include S228 of the L1 endonuclease domain. In some embodiments, the mutation may be S228P.

[0188]

[0247] In some embodiments, the mutation reduces the recognition of the DNA target by ORF2p endonuclease by at least 50%. In some embodiments, the mutation reduces the recognition of the DNA target by ORF2p endonuclease by at least 60%. In some embodiments, the mutation reduces the recognition of the DNA target by ORF2p endonuclease by at least 70%. In some embodiments, the mutation reduces the recognition of the DNA target by ORF2p endonuclease by at least 80%. In some embodiments, the mutation reduces the recognition of the DNA target by ORF2p endonuclease by at least 90%. In some embodiments, the mutation reduces the recognition of the DNA target by ORF2p by at least 95%. In some embodiments, the mutation reduces the recognition of the DNA target by ORF2p by at least 100%.

[0189]

[0248] In some embodiments, the mutation is a deletion. In some embodiments, the deletion is complete, i.e., 100% of the L1 endonuclease domain is deleted. In some embodiments, the deletion is partial. In some embodiments, approximately 98%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, or 50% of the ORF2 endonuclease domain is deleted.

[0190]

[0249] In some embodiments, an additional nuclease domain is inserted into the ORF2 protein sequence. In some embodiments, the ORF2 endonuclease domain is deleted and replaced with an endonuclease domain from a heterologous protein. In some embodiments, the ORF2 endonuclease is partially deleted and replaced with an endonuclease domain from a heterologous protein. The endonuclease domain from a heterologous protein may be a megaTAL nuclease domain. The endonuclease domain from a heterologous protein may be a TALEN. The endonuclease domain from a heterologous protein may be Cas9 accompanied by a gRNA specific to a particular locus.

[0191]

[0250] In some embodiments, the endonuclease is (i) an endonuclease having a specific target of the genome, and (ii) producing 5'-P and 3'-OH terminals at the cleavage site.

[0192]

[0251] In some embodiments, the additional endonuclease domain derived from a heterologous protein is an endonuclease domain derived from the relevant retrotransposon.

[0252] In some embodiments, the endonuclease domain derived from a heterologous protein may include a bacterial endonuclease modified to target a specific site. In some embodiments, the endonuclease domain derived from a heterologous protein may include a homing endonuclease. It may contain a domain of the rarease or a fragment thereof. In some embodiments, the endonuclease is a homing endonuclease. In some embodiments, the homing endonuclease is a modified LAGLIDADG homing endonuclease (LHE) or a fragment thereof. In some embodiments, the additional endonuclease can be a restriction endonuclease, Cre, Cas TAL, or a fragment thereof. In some embodiments, the endonuclease may contain a group II intron-encoded protein (ribozyme) or a fragment thereof.

[0193]

[0253] The modified or engineered L1-ORF2p that confers specific DNA targeting ability for the additional / heterologous endonuclease discussed in the preceding paragraphs is expected to be highly advantageous in driving stable integration of the transgene of interest into the genome. The modified L1-ORF2p, when expressed in cells, can achieve a much lower off-target effect than when using the native, i.e., unmodified, L1-ORF2p. In some embodiments, the modified L1-ORF2p does not produce an off-target effect.

[0194]

[0254] In some embodiments, the modified or engineered L1-ORF2p targets recognition sites other than the normal (Py) n ↓(Pu) n sites. In some embodiments, the modified L1-ORF2p targets recognition sites that include (Py) n ↓(Pu) n sites, such as recognition sites that include the TTTT / AA site, such as hybrid target sites. In some embodiments, the modified L1-ORF2p targets recognition sites that have at least one nucleotide in addition to the conventional L1-ORF2 (Py) n ↓(Pu) n sites, such as TTTT / AAG, or TTTT / AAC, or TTTT / AAT, TTTT / AAA, GTTTT / AA, CTTTT / AA, ATTTT / AA, or TTTTT / AA. In some embodiments, the modified L1-ORF2p targets recognition sites that have at least one nucleotide in addition to the conventional L1-ORF2p (Py) n ↓(Pu)n In addition to the target site, other recognition sites are also targeted. In some embodiments, the modified L1-ORF2p is different from the conventional L1-ORF2p(Py) n ↓(Pu) n The recognition site targets a site other than the specified site. In some embodiments, the modified L1-ORF2p targets a recognition site that is 4, 5, 6, 7, 8, 9, 10 nucleotides long or longer. In some embodiments, the modified or altered L1-ORF2p recognition site may be 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides, or more.

[0195]

[0255] Modified L1-ORF2p can be modified to retain the ability to bind to its own mRNA after translation and reverse transcribe it with high efficiency. In some embodiments, modified L1-ORF2p has enhanced reverse transcription efficiency compared to natural (WT) L1-ORF2p.

[0196]

[0256] In some embodiments, the retrotransferant-containing system further includes gene modifications that reduce nonspecific retrotransfer. In some embodiments, the gene modification may include a sequence encoding L1-ORF2p. In some embodiments, the modification may include mutations in one or more amino acids essential for ORF2p to bind to a protein that assists ORF2p in binding to target genomic DNA. The protein that assists ORF2p in binding to target genomic DNA may be part of the chromatin-ORF interactome. In some embodiments, the modification may include one or more amino acids essential for binding to a protein that assists ORF2p DNA endonuclease activity. In some embodiments, the modification may include one or more amino acids essential for binding to a protein that assists ORF2p RT activity. In some embodiments, the modification may be contained in a protein-binding site of ORF2p such that the association between the protein and ORF2p is altered, where the binding of the protein to ORF2p is required for binding to chromatin. In some embodiments, the modification may be contained in a protein-binding site of ORF2p such that the association between the protein and ORF2p is stringent and / or specific than in the absence of the modification. In some embodiments, modifications to the protein-binding site of the ORF2p coding sequence result in changes to ORF2p and tan. As a result of the altered association with proteins, the binding of ORF2p to target DNA exhibits improved specificity. In some embodiments, the modification may reduce the binding of ORF2 to one or more proteins that are part of the ORF2p chromatin interactome.

[0197]

[0257] In some embodiments, the gene modification may be located in the PIP domain of ORF2p.

[0258] In some embodiments, gene modifications may be present in one or more genes encoding proteins that bind to ORF2p and assist in ORF2p recognition, binding, endonuclease, or RT activity. In some embodiments, gene modifications may be present in the ORF2p interaction site of each protein, or in a site that affects the protein's interaction with ORF2p or the interaction between ORF2p and target DNA, in one or more genes encoding the PCNA, PARP1, PABP, MCM, TOP1, RPA, PURA, PURB, RUVBL2, NAP1, ZCCHC3, UPF1, or MOV10 proteins. In some embodiments, modifications may be present in the ORF2p interaction site of the ORF2p-binding domain of PCNA, or in a site that affects the protein's interaction with ORF2p or the interaction between ORF2p and target DNA. In some embodiments, modifications may be present in the ORF2p-binding domain of TOP1. In some embodiments, modifications may be present in the ORF2p-binding domain of RPA. In some embodiments, modifications may be located in the ORF2p interaction site of the ORF2p-binding domain of PARP1, or in a site that affects the interaction of the protein with ORF2p or the interaction between ORF2p and target DNA. In some embodiments, modifications may be located in the ORF2p interaction site of the ORF2p-binding domain of PABP (e.g., PABPC1), or in a site that affects the interaction of the protein with ORF2p or the interaction between ORF2p and target DNA. In some embodiments, gene modifications may be located in the MCM gene. In some embodiments, gene modifications may be located in the ORF2p interaction site of the gene encoding the MCM3 protein, or in a site that affects the interaction of the protein with ORF2p or the interaction between ORF2p and target DNA. In some embodiments, gene modifications may be located in the ORF2p interaction site of the gene encoding the MCM5 protein, or in a site that affects the interaction of the protein with ORF2p or the interaction between ORF2p and target DNA.In some embodiments, gene modifications may be located in the ORF2p interaction site or a site that affects the interaction of the protein with ORF2p or the interaction between ORF2p and target DNA in the gene encoding the MCM6 protein. In some embodiments, gene modifications may be located in the ORF2p interaction site or a site that affects the interaction of the protein with ORF2p or the interaction between ORF2p and target DNA in the gene encoding the MEPCE protein. In some embodiments, gene modifications may be located in the ORF2p interaction site or a site that affects the interaction of the protein with ORF2p or the interaction between ORF2p and target DNA in the gene encoding the RUVBL1 or RUVBL2 protein. In some embodiments, gene modifications may be located in the ORF2p interaction site or a site that affects the interaction of the protein with ORF2p or the interaction between ORF2p and target DNA in the gene encoding the TROVE protein.

[0198]

[0259] In some embodiments, the retrotransition systems disclosed herein include one or more elements that improve the accuracy of reverse transcription.

[0260] In some embodiments, the L1-ORF2 RT domain is modified. In some embodiments, the modification may include one or more of the following: improving accuracy, improving processing ability, improving DNA-RNA substrate affinity, or inactivating RNase H activity.

[0199]

[0261] In some embodiments, the modification involves introducing one or more mutations into the RT domain of L1-ORF2 such that the accuracy of RT is improved. This includes point mutations. In some embodiments, mutations include modifications such as substitutions of one, two, three, four, five, six, or more amino acids in the L1-ORF2p RT domain. In some embodiments, mutations include deletions of one or more amino acids in the L1-ORF2p RT domain, for example, one, two, three, four, five, six, seven, eight, nine, ten, or more amino acids. In some embodiments, mutations may include insertion-deletion mutations. In some embodiments, mutations may include frameshift mutations.

[0200]

[0262] In some embodiments, the modification may include the inclusion of an additional RT domain or fragment thereof derived from the second protein. In some embodiments, the second protein is a viral reverse transcriptase. In some embodiments, the second protein is a nonviral reverse transcriptase. In some embodiments, the second protein is a retrotransferase. In some embodiments, the second protein is a non-LTR retrotransferase. In some embodiments, the second protein is a group II intron protein. In some embodiments, the group II intron is as TGIRTII. In some embodiments, the second protein is a Cas nickase, where the retrotransferase system further includes introducing guide RNA. In some embodiments, the second protein is a Cas9 endonuclease, where the retrotransferase system further includes introducing guide RNA. In some embodiments, the second protein or fragment thereof fuses with the N-terminus of an L1-ORF2 RT domain or a modified L1-ORF2 RT domain. In some embodiments, the second protein or fragment thereof fuses with the C-terminus of the L1-ORF2 RT domain or a modified L1-ORF2 RT domain.

[0201]

[0263] In some embodiments, an additional RT domain or fragment derived from a second protein is incorporated into the retrotransfer system in addition to the full-length WT L1-ORF2p RT domain. In some embodiments, the additional RT domain or fragment derived from a second protein is incorporated in the presence of a modified (or altered) L1-ORF2p RT domain or fragment, where the modification (or alteration) may include mutations for enhancement of the L1-ORF2p RT processing ability, stability, and / or accuracy of the modified L1-ORF2p RT compared to the natural or WT ORF2p.

[0202]

[0264] In some embodiments, the reverse transcriptase domain may be replaced with other retroelements or group II introns, such as other RT domains derived from TGIRTII that have higher processing capacity and accuracy.

[0203]

[0265] In some embodiments, the modification may involve fusion with an additional RT domain or fragment thereof derived from a second protein. In some embodiments, the second protein may contain a retroelement. The additional RT domain or fragment thereof derived from the second protein is configured to improve the accuracy of reverse transcription of the fused L1-ORF2p RT domain. In some embodiments, the nucleic acid encoding the additional RT domain or fragment thereof fuses with a natural or WT L1-ORF2 coding sequence. In some embodiments, the nucleic acid encoding the additional RT domain or fragment thereof derived from a second protein fuses with a modified L1-ORF2 coding sequence. In some embodiments, the modification involves introducing one or more mutations into the L1-ORF2 RT domain or fragment thereof such that the accuracy of the fused RT is improved. In some embodiments, the mutations in the L1-ORF2 RT domain or fragment thereof include point mutations. In some embodiments, the mutations include modifications such as the substitution of one, two, three, four, five, six, or more amino acids in the L1-ORF2p RT domain. In some embodiments, the mutation includes the deletion of one or more amino acids in the L1-ORF2p RT domain, for example, one, two, three, four, five, six, seven, eight, nine, ten, or more amino acids. In some embodiments, the mutation may include insertion-deletion mutations. In some embodiments, the mutation may include frameshift mutations.

[0204]

[0266] In some embodiments, the modified L1-ORF2p RT domain has improved processing ability compared to the WT L1-ORF2p RT domain.

[0267] In some embodiments, the modified L1-ORF2p RT domain has a processing ability and / or accuracy that is at least 10% higher than that of the WT L1-ORF2p RT domain. In some embodiments, the modified L1-ORF2p RT domain has a processing ability and / or accuracy that is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 150%, 200%, 300%, 400%, 500%, 1000%, or higher than that of the WT L1-ORF2p RT domain. In some embodiments, the modified RT can process nucleic acid segments greater than 6 kb. In some embodiments, the modified RT can process nucleic acid segments greater than 7 kb. In some embodiments, the modified RT can process nucleic acid segments greater than 8 kb. In some embodiments, the modified RT can process nucleic acid segments greater than 9 kb. In some embodiments, the modified RT can process nucleic acid segments greater than 10 kb.

[0205] B. Group II Introns and Ribozymes

[0268] Group II enzymes are mobile ribozymes that self-splice RNA precursors and generate excised intron lariat RNAs. The intron encodes a reverse transcriptase. The reverse transcriptase can stabilize RNA with respect to both forward and reverse splicing and then convert the incorporated intron RNA into DNA.

[0206]

[0269] Group II RNAs are characterized by a conserved secondary structure spanning 400 - 800 b nucleotides. The secondary structure is formed by six domains, DI - VI, which are organized into a structure resembling a wheel with the domains radiating out from a central point. The domains interact to form a conserved tertiary structure, which brings separate sequences together to form an active site. The active site binds to splice site and branch point residue nucleotides and activates the catalytic action of splicing in the association of Mg2+ cations. The DV domain is within the active site and has conserved catalytic AGC and AY bulges, both of these regions binding to the Mg2+ ions required for catalysis. DI is the largest domain, with its upper and lower halves separated by kappa and zeta motifs. The lower half contains an epsilon' motif that associates with the active site. The upper half contains sequence elements that bind to the 5' and 3' exons at the active site. DIV encodes an intron - encoded protein (IEP), and the sub - domain IVa near the 5' end contains a high - affinity binding site for the IEP. Group II introns have conserved 5' and 3' end sequences, GUGYG and AY respectively.

[0207]

[0270] Group II RNA introns can be utilized to retrotransfer a target sequence into DNA via reverse transcription with the target as a primer. This process of transfer by Group II RNA introns is often referred to as retrohoming. Group II introns recognize a DNA target site by base - pairing of the intron RNA with the DNA target sequence and can be modified to change the target of a specific sequence carried within the intron to a desired DNA site.

[0208]

[0271] In some embodiments, the methods and compositions for retrotransition described herein may include group II intron sequences, modified group II intron sequences, or fragments thereof. Exemplary group II IEPs (maturases) include, but are not limited to, bacterial, fungal, and yeast IEPs that are functional in human cells. In particular, nucleases leave a 3'-OH group at the DNA cleavage site that can be utilized by another RT for priming and reverse transcription. Exemplary group II maturases are TGIRT (thermal It may be a stable group II intron maturase.

[0209]

[0272] In one or more embodiments of several aspects described herein, the nucleic acid construct comprises RNA. In one or more embodiments of several aspects of this disclosure, the nucleic acid construct is RNA. In one or more embodiments of several aspects of this disclosure, the nucleic acid construct is mRNA. In one embodiment, the mRNA comprises a sequence of a heterogene or a portion thereof, wherein the heterogene or the portion thereof encodes a polypeptide or a protein. In some embodiments, the mRNA comprises a sequence encoding a fusion protein. In some embodiments, the mRNA comprises a sequence encoding a recombinant protein. In some embodiments, the mRNA comprises a sequence encoding a synthetic protein. In some embodiments, the nucleic acid comprises one or more sequences, wherein one or more sequences encode one or more heterogeneous proteins, one or more recombinant proteins, or one or more synthetic proteins, or a combination thereof. In some embodiments, the nucleic acid comprises one or more sequences, wherein one or more sequences encode one or more heterogeneous proteins, including a synthetic protein or a recombinant protein. In some embodiments, the synthetic or recombinant protein is a recombinant fusion protein.

[0210]

[0273] In one or more embodiments of several aspects of this disclosure, the nucleic acid construct is developed for expression in eukaryotic cells. In some embodiments, the nucleic acid construct is developed for expression in human cells. In some embodiments, the nucleic acid construct is developed for expression in hematopoietic cells. In some embodiments, the nucleic acid construct is developed for expression in bone marrow cells. In some embodiments, bone marrow cells are human cells.

[0211] II. Modifications in nucleic acid constructs for enhancing the expression of coding proteins

[0274] In certain aspects of this disclosure, recombinant nucleic acids are tangents encoded by the nucleic acid sequence. Modifications are made to enhance protein expression. Enhanced expression of the encoded protein can have functions related to nucleic acid stability, translation efficiency, and the stability of the translated protein. Several modifications for incorporation into the design of nucleic acid constructs are intended herein, which can confer nucleic acid stability, for example, the stability of messenger RNA encoding a foreign or heterologous protein, which may be a synthetic recombinant protein or a fragment thereof.

[0212]

[0275] In some embodiments, the nucleic acid is mRNA containing one or more sequences, where one or more sequences encode one or more heterologous proteins, including synthetic or recombinant fusion proteins.

[0213]

[0276] In some embodiments, one or more modifications are made in mRNA containing a sequence encoding a recombinant or fusion protein to increase the mRNA half-life.

[0214]

[0277] Structural elements to inhibit 5'-3' degradation by exonucleases: 5' cap and 3' UTR modification

[0278] A suitable 5' cap structure is crucial in the synthesis of functional messenger RNA. In some embodiments, the 5' cap contains guanosine triphosphate organized as GpppG at the 5' end of the nucleic acid. In some embodiments, the mRNA contains m7-GpppG, which is a 5'7-methylguanosine cap. The 5'7-methylguanosine cap improves mRNA translation efficiency and prevents degradation by mRNA 5'-3' exonuclease. In some embodiments, the mRNA contains an "anti-reverse" cap analog (ARCA, m 7,3’-O It includes GpppG). However, translation efficiency can be significantly improved by using the ARCA method. In some embodiments, the guanosine cap is a cap 0 structure. In some embodiments, the guanosine cap is a cap 1 structure. In addition to its essential role in cap-dependent initiation of protein synthesis, the mRNA cap is an exit from 5' to 3'. The mRNA cap functions as a protective group against sonuclease cleavage, as well as a unique identifier for recruiting protein factors for mRNA precursor splicing, polyadenylation, and nuclear export. The mRNA cap acts as an anchor for recruiting initiation factors that initiate 5'-to-3' loop formation of mRNA during protein synthesis and translation. Three enzymatic activities, namely RNA triphosphatase (TPase), RNA guanylyltransferase (GTase), and guanine-N7 methyltransferase (guanine-N7MTase), are required to generate the cap 0 structure. Each of these enzymatic activities performs a step essential to the conversion of the 5' triphosphate of nascent RNA into the cap 0 structure. RNA TPase removes the gamma phosphate from the 5' triphosphate to produce 5' diphosphate RNA. GTase transfers the GMP group from GTP to the 5' diphosphate via a lysine-GMP covalent intermediate. Guanine-N7MTase then adds a methyl group to the N7 amine of the guanine cap to form the cap 0 structure. Regarding the cap 1 structure, m7G-specific 2'O methyltransferase (2'OMTase) methylates the +1 ribonucleotide at the 2'O position of ribose to generate the cap 1 structure. The nuclear RNA capping enzyme interacts with the polymerase subunit of the RNA polymerase II complex at the phosphorylated Ser5 of the C-terminal heptad repeat. RNA guanine-N7 methyltransferase also interacts with the RNA polymerase II phosphorylated heptad repeat. In some embodiments, the cap is a G4 structure cap.

[0215]

[0279] In some embodiments, mRNA is synthesized by in vitro transcription (IVT). In some embodiments, mRNA synthesis and capping may be carried out in a single step. Capping may be carried out in the same reaction mixture as IVT. In some embodiments, mRNA synthesis and capping may be carried out in separate steps. The mRNA thus formed by IVT is purified and then capped.

[0216]

[0280] In some embodiments, a nucleic acid construct, such as an mRNA construct, may contain one or more sequences encoding a protein, or the polypeptide of interest may be designed to contain elements that protect, prevent, inhibit, or reduce mRNA degradation by an endogenous 5'-3' exoribonuclease, such as Xrn1. Xrn1 is a cellular enzyme in the normal RNA degradation pathway that degrades 5' monophosphorylated RNA. However, some viral RNA structural elements have been found to be particularly resistant to such RNAse, such as the Xrn1-resistant structure in flavivirus sfRNA, called "xrRNA." For example, the mosquito-borne flavivirus (MBFV) genome contains separate RNA structures in the 3' untranslated region (UTR) that block the progression of Xrn1. These RNA elements are sufficient to block Xrn1 without the use of accessory proteins. The xrRNA stops the enzyme at a predetermined location, and as a result, the viral RNA located downstream of the xrRNA is protected from degradation. For example, xrRNAs derived from Zika virus or Murray Valley encephalitis virus contain a three-way junction and multiple pseudoknot interactions that result in an unusual and complex folding pattern requiring a set of nucleotides conserved across the MBFV structure. The xrRNA stops the enzyme at a predetermined location, thereby protecting the viral RNA downstream of the xrRNA from degradation. The 5' end of the RNA is thought to remain protected from Xrn1-like exonucleases by traversing the ring-like structure of the folding pattern.

[0217]

[0281] In some embodiments, a nucleic acid construct containing one or more sequences encoding a protein of interest may contain one or more xrRNA structures incorporated therein. In some embodiments, the xrRNA is a segment of nucleotides having a conserved region at the 3'UTR of one or more viral xrRNA sequences. In some embodiments, 1, 2, 3, 4 5, 6, 7, 8, 9, 10, or more xrRNA elements are incorporated into the nucleic acid construct. In some embodiments, two or more xrRNA elements are incorporated in tandem into the nucleic acid construct. In some embodiments, the xrRNA includes one or more regions containing a conserved sequence or a fragment thereof or a modification thereof. In some embodiments, the xrRNA is positioned at the 3'UTR of a retrotransposon element. In some embodiments, the xrRNA is positioned upstream of a sequence encoding one or more proteins or polypeptides. In some embodiments, the xrRNA is positioned at the 3'UTR of a retrotransposon element, such as an ORF2 sequence, and upstream of a sequence encoding one or more proteins or polypeptides.

[0218]

[0282] In some embodiments, the xrRNA structure includes an MBFV xrRNA sequence or a sequence that is at least 90% identical thereto. In some embodiments, the xrRNA structure includes a tick-borne flavivirus (TBFV) xrRNA sequence or a sequence that is at least 90% identical thereto. In some embodiments, the xrRNA structure includes a tick-borne flavivirus (TBFV) xrRNA sequence or a sequence that is at least 90% identical thereto. In some embodiments, the xrRNA structure includes a tick-borne flavivirus (TBFV) xrRNA sequence or a sequence that is at least 90% identical thereto. In some embodiments, the xrRNA structure includes an xrRNA sequence derived from a member of an unknown vector arthropod flavivirus (NKVFV) or a sequence that is at least 90% identical thereto. In some embodiments, the xrRNA structure includes an xrRNA sequence derived from a member of an insect-specific flavivirus (ISFV) or a sequence that is at least 90% identical thereto. In some embodiments, the xrRNA structure includes a Zika virus xrRNA sequence or a sequence that is at least 90% identical thereto. Any known xrRNA structural element or any non-obvious, conceivable variation thereof may be used for the purposes described herein.

[0219]

[0283] Several messenger RNAs from various organisms exhibit one or more pseudoknot structures that provide resistance to 5'-3' exonucleases. A pseudoknot is an RNA structure consisting of two helical segments connected by a single-stranded region or loop at its smallest, although several distinct folding topologies of pseudoknots exist.

[0220] PolyA tail modification

[0284] The poly(A) structure in the 3'UTR of mRNA is an important regulator of mRNA half-life. Exosome deadenylation at the poly-A tail initiates degradation of the mRNA body. In some embodiments, the length of the poly-A tail of the mRNA construct is carefully considered and designed to maximize the expression of the protein encoded by the mRNA coding region and mRNA stability. In some embodiments, the nucleic acid construct includes one or more poly-A sequences. In some embodiments, the poly-A sequence at the 3'UTR of the sequence encoding one or more proteins or polypeptides contains 20 to 200 adenosine nucleic acid bases. In some embodiments, the poly-A sequence contains 30 to 200 adenosine nucleic acid bases. In some embodiments, the poly-A sequence contains 50 to 200 adenosine nucleic acid bases. In some embodiments, the poly-A sequence contains 80 to 200 adenosine nucleic acid bases. In some embodiments, the mRNA segment containing the sequence encoding one or more proteins or polypeptides includes a 3'UTR having a poly-A tail containing about 180 adenosine nucleic acid bases, or about 140 adenosine nucleic acid bases, or about 120 adenosine nucleic acid bases. In some embodiments, the polyA tail contains approximately 122 adenosine nucleic acid bases. In some embodiments, the polyA sequence contains 50 adenosine nucleic acid bases. In some embodiments, the polyA sequence contains 30 adenosine nucleic acid bases. In some embodiments, the adenosine nucleic acid bases in the polyA tail are arranged in tandem with or without intervening non-adenosine bases. In some embodiments, one or Multiple non-adenosine nucleic acid bases are incorporated into the poly-A tail, conferring further resistance to certain exonucleases.

[0221]

[0285] In some embodiments, the adenosine segment in the poly-A tail of the construct contains one or more non-adenosine (A) nucleic acid bases. In some embodiments, the non-A nucleic acid bases are located at positions -3, -2, -1, and / or +1 in the poly-A 3' terminal region. In some embodiments, the non-A bases include guanosine (G), cytosine (C), or uracil (U). In some embodiments, the non-A base is G. In some embodiments, there are two or more non-A bases in tandem, e.g., GG. In some embodiments, modifications at the 3' end of the poly-A tail having one or more non-A bases are targeted to disrupt A base stacking in the poly-A tail. Poly-A base stacking is effective against deadenylation by various deadenylationases, and therefore, the 3' end of poly ending in -AAAG, -AAAGA, or -AAAGGA is effective in conferring stability against deadenylation. In some organisms, GC sequences interposed in poly-A sequences have been shown to effectively attenuate 3'-5' exonuclease-mediated degradation. The modifications intended herein include interposed non-A residues or non-A residue double chains interposed in the 3'-terminal poly-A segment.

[0222]

[0286] In some embodiments, a triple-chain structure is introduced to the 3'UTR that effectively inhibits or slows down the exonuclease activity associated with the 3' end.

[0287] In some embodiments, mRNA with the modifications described above has an extended half-life and exhibits stable expression for a longer period than unmodified mRNA. In some embodiments, mRNA is stably expressed for 2, 3, 4, 5, 6, 7, 8, 9, or more than 10 days, or longer, and the mRNA or its protein product is in It can be detected in vivo in vivo. In some embodiments, the mRNA is detected in vivo for up to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days. In some embodiments, the protein product of the mRNA is detected in vivo for up to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or 30 days.

[0223] CircRNA and tectoRNA

[0288] Circular RNAs are useful for the design and production of stable RNAs that are used as messenger RNAs, such as long multi-repeat protein chains. There are few methods for making circular RNAs (circRNAs). As a method, when a bisected self-splicing intron is located at both ends of the transcribed mRNA, an RNA ligase is used to splice itself and leave a product that is ligated, and protein-mediated ligation of the RNA ends using a bisected self-splicing intron can be mentioned (Figure 3A). Another technique is T4 that acts as an RNA ligase when both ends of the ligated RNA are held together by oligonucleotides. It is useful for the design and production of stable RNAs used as messenger RNAs, such as long multi-repeat protein chains. There are few methods for making circular RNAs (circRNAs). As a method, when a bisected self-splicing intron is located at both ends of the transcribed mRNA, an RNA ligase is used to splice itself and leave a product that is ligated, and protein-mediated ligation of the RNA ends using a bisected self-splicing intron can be mentioned (Figure 3A). Another technique is T4 that acts as an RNA ligase when both ends of the ligated RNA are held together by oligonucleotides. It depends on the ability of the DNA ligase. All of these techniques have the disadvantage of being inefficient and requiring large amounts of enzymes. A third technique uses the cyclization or circularization activity of group I introns, where it is assumed that the majority of the intron sequence performing the reaction will reliably remain as part of the ring. Group I introns share a set of complex secondary and tertiary structures containing a series of conserved RNA stem loops that form a catalytic core. Many of these introns have self-splicing activity in vitro and can splice to form two ligated exons as RNA without accessory protein factors. The products produced by group I autocatalytic reactions are (1) an upstream exon ligated with the 3' splice site of a downstream exon at the 5' splice site, and (2) a linear intron that can undergo further reversible autocatalysis to form a circular intron. Such highly structured large nucleic acids The presence of sequences severely limits the types of RNA sequences that can be made circular using this technique. In addition, the catalytic activity of introns remains and can interfere with the structure and function of circular RNA.

[0224]

[0289] It is useful to improve the reaction rate, and therefore the overall efficiency, by bringing the two ends of the RNA closer together. Previous studies have achieved this by including complementary RNA sequences at the 3' and 5' ends of the mRNA, so that the two ends of the mRNA are closer together during hybridization of these sequences, and as a result, the mRNA can undergo ligation or auto-splicing reactions at a faster overall rate compared to when there are no complementary sequences. These sequences are called homology arms of auto-splicing cyclization reactions (Figure 3A). A major problem with such hybridization strategies is that if a sequence complementary to either of the homology arms is present in the coding region, the hybridization may actually inhibit the splicing reaction, and the arms may have to be optimized for each new coding region. An alternative to this strategy described herein is the use of RNA sequences that fold into a three-dimensional structure to form sequence-independent, stable binding interactions.

[0225]

[0290] Non-Watson-Crick RNA tertiary interactions can be utilized to construct "tectoRNA" molecular units, which are defined as self-assembling RNA molecules. The use of such types of tertiary interactions is linked to cation concentration (e.g., Mg 2+ This allows the assembly process to be controlled and modulated by manipulating the temperature and / or a suitable temperature, as well as by using modularly designed "selective" RNA molecules. For the self-assembly of the one-dimensional array, a basic modular unit was designed that includes a four-directional junction with interaction modules for each helical arm. In some embodiments, the interaction modules are GAAA loops or specific GAAA loop receptors. Each tectoRNA can interact with two other tectoRNAs, i.e., two with each partner molecule, through the formation of four loop-receptor interactions.

[0226]

[0291] In some embodiments, the tectoRNA structure is suitably selected and incorporated into RNA containing exons and introns to form circRNA. In some embodiments, the incorporation is carried out by well-known molecular biological techniques such as ligation. In some embodiments, the tectoRNA forms a structure that is stable at high temperatures. The tectoRNA structure does not compete with the internal RNA sequence, thereby resulting in highly efficient circulation and splicing.

[0227]

[0292] CircRNAs may contain coding sequences described in any of the preceding sections. For example, a circRNA may contain a sequence encoding a fusion protein that includes a ligator or receptor molecule. The receptor may be a phagocytic receptor fusion protein.

[0228]

[0293] In some embodiments, the intron is a self-splicing intron.

[0294] In some embodiments, the terminal region having a tertiary structure, also referred to as the scaffold region for circRNA, is approximately 30 to 100 nucleotides long. In some embodiments, the tertiary structure motif is approximately 45, 50, 55, 60, 65, 70, or 75 nucleotides long. In some embodiments, the tertiary motif is formed at high temperatures. In some embodiments, the tertiary motif is stable.

[0229]

[0295] In some embodiments, nucleic acid constructs comprising one or more sequences having one or more modifications described herein and encoding one or more proteins or polypeptides are stable when administered in vivo. The nucleic acid is mRNA. In some embodiments, mRNA containing one or more sequences encoding one or more proteins or polypeptides is stable in vivo for more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the protein encoded by the sequence in mRNA can be detected in vivo for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the protein encoded by the sequence in mRNA can be detected in vivo for about 7 days after mRNA administration. In some embodiments, the protein encoded by the mRNA sequence can be detected in vivo for approximately 14 days after mRNA administration. In some embodiments, the protein encoded by the mRNA sequence can be detected in vivo for approximately 21 days after mRNA administration. In some embodiments, the protein encoded by the mRNA sequence can be detected in vivo for approximately 30 days after mRNA administration. In some embodiments, the protein encoded by the mRNA sequence can be detected in vivo for more than approximately 30 days after mRNA administration.

[0230]

[0296] In some embodiments, enhancing nucleic acid uptake or integration into cells is intended to enhance retrotransfer expression. One method involves obtaining a homogeneous cell population and initiating nucleic acid integration, for example, via transfection in the case of plasmid vector constructs, or via electroporation or any other means that can be suitably used to deliver nucleic acid molecules to cells. In some embodiments, cell cycle synchronization may be explored. Cell cycle synchronization can be performed by selecting cells with respect to a particular common phenotype. In some embodiments, the cell population may be subjected to treatment with reagents that can halt the cell cycle progression of all cells at a particular stage. Exemplary reagents can be found in commercial databases, e.g., www.tocris.com / cell-biology / cell-cycle-inhibitors or www.scbt.com / browse / chemicals-Other-Chemicals-cell-cycle-arresting-compounds. For example, itraconazole or nocodazole are reagents that inhibit the cell cycle in the G1 phase or arrest the cell cycle in the G0 / G1 phase, such as 5-[(4-ethylphenyl)methylene]-2-thioxo-4-thiazolidinone (compound 10058-F4) (Tocris Bioscience), or G2M cell cycle inhibitors such as AZD5438 (chemical name, 4-[2-methyl-1-(1-methylethyl)-1H-imidazole-5-yl]-N-[4-(methylsulfonyl)phenyl]-2-pyrimidineamine) that block the cell cycle in the G2M, G1, or S phase. Cyclosporine, hydroxyurea, and thymidine are well-known reagents that can induce cell cycle arrest. Some reagents can irreversibly alter the cell state or be toxic to cells. Depending on the cell type, serum removal from cells for approximately 2 to 16 hours prior to electroporation or transfection can also be an easy and reversible strategy for cell synchronization.

[0231]

[0297] In some embodiments, retrotransfer efficiency may be improved by inducing the generation of DNA double-strand breaks in cells transfected or electroporated with the retrotransfer constructs described herein, and / or by modulating DNA repair mechanisms. The application of these techniques may be limited depending on the end use of the cells that may undergo ex vivo genetic engineering for the stable incorporation of nucleic acid sequences by this method. In some cases, the use of such techniques may be contemplated when robust expression of the protein or transcript encoded by the incorporated nucleic acid is expected as a result of a determined period. A method for introducing double-strand breaks into cells involves the step of exposing the cells to controlled ionizing radiation of approximately 0.1 Gy or less for a short period of time.

[0232]

[0298] In some embodiments, the efficiency of LINE-1-mediated retrotransfer can be improved by treating cells with small molecule inhibitors of DNA repair proteins to increase the opportunities for reverse transcriptase to act. Exemplary small molecule inhibitors of DNA repair proteins include benzamide (CAS 55-21-0), olaparib (Lynparza) (CAS 763113-22-0), rucaparib (Clovis-AG014699, PF-01367338 Pfizer), niraparib (MK-827 Tesaro) (CAS 1038915-60-4); and veliparib (ABT-888 Abbvie) (CAS912444-00-9); Camptothecin (CPT) (CAS7689-03-4); Irinotecan (CAS100286-90-6); Topotecan (Hycamtin® GlaxoSmithKline) (CAS123948-87-8); NSC19630 (CAS72835-26-8); NSC617145 (CAS203115-63-3); ML Possible candidates include 216 (CAS1430213-30-1); 6-hydroxy DL-DOPA (CAS21373-30-8); D-103; D-G23; DIDS (CAS67483-13-0); B02 (CAS1290541-46-6); RI-1 (CAS415713-60-9); RI-2 (CAS1417162-36-7); and streptonigrin (SN) (CAS3930-19-6).

[0233] III. Nucleic Acid Cargo: A. Introduced gene

[0299] In one embodiment, a transgene or a transgene is a heterologous nucleic acid sequence inserted into the genome of a cell. Non-coding sequences are delivered as mRNA. mRNA may contain approximately 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or more than 10,000 bases. In some embodiments, mRNA may exceed 10,000 bases in length. In some embodiments, mRNA may be approximately 11,000 bases in length. In some embodiments, mRNA may be approximately 12,000 bases in length. In some embodiments, mRNA contains a transgene sequence encoding a fusion protein. In some embodiments, nucleic acids are delivered as plasmids.

[0234]

[0300] In some embodiments, nucleic acids are delivered to cells by transfection. In some embodiments, nucleic acids are delivered to cells by electroporation. In some embodiments, transfection or electroporation is repeated two or more times to enhance the integration of nucleic acids into cells.

[0235]

[0301] The stable, retrotransposon-mediated incorporation of recombinant nucleic acids encoding phagocytic or ligation receptor (PR) fusion proteins (CFPs) is intended herein. In some embodiments, the CFP comprises a PR subunit comprising a transmembrane domain, an intracellular domain including an intracellular signaling domain, and an extracellular domain including an antigen-binding domain specific to an antigen of a target cell, wherein the transmembrane domain and the extracellular domain are operatively ligated.

[0236]

[0302] In some embodiments, the nucleic acid comprises a sequence encoding a chimeric fusion protein (CFP), the CFP comprising an extracellular domain containing a CD5-binding domain and a transmembrane domain operatively linked to the extracellular domain. In some embodiments, the CD5-binding domain is a CD5-binding protein, e.g., an antigen-binding fragment, Fab fragment, scFv domain, or sdAb domain of an antibody. In some embodiments, the CD5-binding domain is (i)EI(ii) The scFv comprises a variable heavy chain (VH) sequence having at least 90% sequence identity with QLVQSGGGLVKPGGSVRISCAASGYTFTNYGMNWVRQAPGKGLEWMGWINTHTGEPTYADSFKGRFTFSLDDSKNTAYLQINSLRAEDTAVYFCTRRGYDWYFDVWGQGTTVTV, and (ii) a variable light chain (VL) sequence having at least 90% sequence identity with DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRANRLESGVPSRFSGSGSGTDYTLTISSLQYEDFGIYYCQQYDESPWTFGGGTKLEIK. In some embodiments, the CFP further comprises an intracellular domain, where the intracellular domain comprises one or more intracellular signaling domains, and the wild-type protein comprising the intracellular domain does not contain an extracellular domain. In some embodiments, one or more intracellular signaling domains comprises a phagocytic signaling domain. In some embodiments, the phagocytic signaling domain includes intracellular signaling domains derived from receptors other than Megf10, MerTk, FcαR, and Bai1. In some embodiments, the phagocytic signaling domain includes intracellular signaling domains derived from FcγR, FcαR, or FcεR. In some embodiments, the phagocytic signaling domain includes an intracellular signaling domain having at least 90% sequence identity with LYCRRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPP. In some embodiments, one or more intracellular signaling domains further include a pro-inflammatory signaling domain. In some embodiments, the pro-inflammatory signaling domain includes a PI3 kinase (PI3K) mobilization domain. In some embodiments, the pro-inflammatory signaling domain includes a sequence having at least 90% sequence identity with YEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENM. In some embodiments, the pro-inflammatory signaling domain is derived from an intracellular signaling domain of CD40.In some embodiments, the pro-inflammatory signaling domain includes a sequence having at least 90% sequence identity with KVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ. In some embodiments, the transmembrane domain includes a CD8 transmembrane domain. In some embodiments, the transmembrane domain includes a sequence having at least 90% sequence identity with IYIWAPLAGTCGVLLLSLVIT. In some embodiments, the extracellular domain further includes a hinge domain derived from CD8, which operatively ligates to the transmembrane domain and the CD5 binding domain. In some embodiments, the extracellular domain includes a sequence having at least 90% sequence identity with ALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD. In some embodiments, the CFP comprises an scFv that specifically binds to CD5, an extracellular domain comprising a hinge domain derived from CD8; a hinge domain derived from CD28; or at least a portion of an extracellular domain derived from CD68; a CD8 transmembrane domain, a CD28 transmembrane domain; or a CD68 transmembrane domain; and an intracellular domain comprising at least two intracellular signaling domains, wherein the at least two intracellular signaling domains comprise a first intracellular signaling domain derived from FcγR or FcεR and a second intracellular signaling domain comprising a PI3K mobilization domain or a second intracellular signaling domain derived from CD40. In some embodiments, the recombinant polynucleic acid is mRNA or circRNA. In some embodiments, the nucleic acid is delivered to myeloid cells. In some embodiments, the nucleic acid is delivered to CD14+ cells, CD14+CD16- cells, M0 macrophages, M2 macrophages, M1 macrophages, or mosaic myeloid cells / macrophages.In some embodiments, the fusion protein is EIQLVQSGGGLVKPGGSVRISCAASGYTFTNYGMNWVRQAPGKGLEWMGWINTHTGEPTYADSFKGRFTFSLDDSKNTAYLQINSLRAEDTAVYFCTRRGYDWYFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRANRLESGVPSRFSGSGSGTDYTLTI. It contains a sequence that has at least 90% sequence identity with SSLQYEDFGIYYCQQYDESPWTFGGGTKLEIKSGGGGSGALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDIYIWAPLAGTCGVLLLSLVITLYCRRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQGSGSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENM.In some embodiments, the fusion protein is EIQLVQSGGGLVKPGGSVRISCAASGYTFTNYGMNWVRQAPGKGLEWMGWINTHTGEPTYADSFKGRFTFSLDDSKNTAYLQINSLRAEDTAVYFCTRRGYDWYFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRANRLESGVPSRFSGSGSGTDYTLTISSL QYEDFGIYYCQQYDESPWTFGGGTKLEIKSGGGGSGALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDIYIWAPLAGTCGVLLLS LVITLYCRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ or EI QLVQSGGGLVKPGGSVRISCAASGYTFTNYGMNWVRQAPGKGLEWMGWINTHTGEPTYADSFKGRFTFSLDDSKNTAYLQINSLRAEDTAVYFCTRRGYDWYFDVWGQGTTVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRANRLESGVPSRFSGSGSGTDYTLTISSLQYEDFGIYYCQQYDESPWTFG It contains a sequence that has at least 90% sequence identity with GGTKLEIKSGGGGSGALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDIYIWAPLAGTCGVLLLSLVITLYCRRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ.

[0237]

[0303] In some embodiments, the fusion protein is a transmembrane protein, an intracellular protein, or an intracellular protein. In one embodiment, the fusion protein is intended to enhance the function of immune cells selected from monocytes, macrophages, dendritic cells, or their precursors, such as myeloid cells. In one embodiment, the fusion protein increases the cellular function of immune cells, such as phagocytosis. This disclosure is not limited to the transgenes that can be expressed using the methods and compositions described. The transgenes shown in this section are illustrative.

[0238]

[0304] Exemplary candidate transgenes for stable integration into the genome of phagocytic cells are provided herein. In one embodiment, the transgene is a recombinant nucleic acid encoding a phagocytic receptor (PR) fusion protein (CFP). The recombinant nucleic acid has a PR subunit comprising an intracellular domain including (i) a transmembrane domain and (ii) a phagocytic receptor intracellular signaling domain; and an extracellular antigen-binding domain specific to the antigen of a target cell, wherein the transmembrane domain and the extracellular antigen-binding domain are operatively linked, resulting in the activation of antigen binding to the target by the extracellular antigen-binding domain of the fusion receptor in the intracellular signaling domain of the phagocytic receptor. In some embodiments, the recombinant nucleic acid encodes a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor is a chimeric antigen receptor (phagocytosis) (CAR-P). In some embodiments, the fusion protein is a recombinant protein for immobilizing antiphagocytic signals. In some embodiments, the fusion protein is a phagocytic-enhancing chimeric protein. In some embodiments, the chimeric protein is an active phagocytic It has an intracellular domain that includes a Gunal signaling domain. In some embodiments, the chimeric protein enhances phagocytic activity by enhancing the inflammatory activity of the phagocytic cells that express the chimeric protein. In some embodiments, the transgene is designed to express a contact-activated chimeric protein that is activated by contact with an antigen on a target cell, causing the phagocytic cell to phagocytose and kill the target cell.

[0239]

[0305] The terms “spacer” or “linker,” when used in relation to a fusion protein, refer to a peptide sequence that binds to the protein domain of the fusion protein. Generally, spacers have no specific biological activity other than binding or preserving a certain minimum distance or other spatial relationship between proteins or RNA sequences. However, in some embodiments, the constituent amino acids of a spacer can be selected to affect certain properties of the molecule, such as molecular folding, net charge, or hydrophobicity. Suitable linkers for use in some embodiments of this disclosure are, but are not limited to, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers, which are well known to those skilled in the art. Linkers are used to separate two antigenic peptides by a distance sufficient to ensure that each antigenic peptide folds properly, in some embodiments. Exemplary peptide linker sequences adopt a mobile, elongated three-dimensional structure and do not tend to generate a regular secondary structure. Typical amino acids in the mobile protein region include Gly, Asn, and Ser. Substantially any permutation of amino acid sequences containing Gly, Asn, and Ser can be expected to satisfy the above criteria for linker sequences. Other nearly neutral amino acids, such as Thr and Ala, can also be used in linker sequences.

[0240]

[0306] The following are some exemplary proteins encoded by transgenes that can be expressed to enhance the immune function of phagocytic cells. This is not an exhaustive list, but serves as an exemplary list of transgene designs within the scope of this disclosure.

[0241]

[0307] In some embodiments, the PSP subunit includes the transmembrane (TM) domain of the phagocytic receptor.

[0308] In some embodiments, the PSP subunit includes the ICD domain of the phagocytic receptor.

[0242]

[0309] In some embodiments, the ICD encoded by recombinant nucleic acid includes a domain selected from the group consisting of lectin, dectin 1, mannose receptor (CD206), scavenger receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), and CD169 receptor.

[0243]

[0310] In some embodiments, ICD is a lectin, dectin 1, mannose receptor (CD206), scavenger receptor A1 (SRA1), MARCO (macrophage receptor with collagen structure, also known as SRA6, SCARA2), CD36 (thrombospondin receptor, also known as scavenger receptor class B, member 3), CD163 (scavenger receptor, cysteine-rich type 1), MSR1, SCARA3, COLEC12 (scavenger receptor with type C lectin, also known as SCARA4, or collectin 12), SCARA5, SCARB1, SCARB2, CD68 (SCARD, microsialin), OLR1 (oxidized low-density lipoprotein receptor 1, LOX1, or type C lectin domain family 8 member A), SCARF1, SCARF2, SRCRB4D, It contains signaling domains derived from one or more of the following: SSC5D and CD169 (also known as sialoadhesin receptor, SIGLEC1).

[0244]

[0311] In some embodiments, the recombinant nucleic acid encodes, for example, the intracellular domain of human MARCO. The PSR subunit includes the intracellular domain of human MARCO having a 44-amino acid ICD with the amino acid sequence:MRNKKILKEDELLSETQQAAFHQIAMEPFEINVPKPKRRNGVNF. In some embodiments, the PSR subunit includes variants that are at least 70%, 75%, 80%, 85%, 90%, or 95% identical to the intracellular domain of MARCO.

[0245]

[0312] In some embodiments, for example, the PSR (phagocytic scavenger receptor) includes the transmembrane region of human MARCO.

[0313] In some embodiments, the recombinant nucleic acid encodes the intracellular domain of human SRA1. The PSR subunit includes the intracellular domain of human SRA1 having a 50-amino acid ICD with the amino acid sequence:MEQWDHFHNQQEDTDSCSESVKFDARSMTALLPPNPKNSPSLQEKLKSFK. In some embodiments, the PSR subunit includes variants that are at least 70%, 75%, 80%, 85%, 90%, or 95% identical to the intracellular domain of human SRA1. The intracellular domain of SRA has a phosphorylation site.

[0246]

[0314] In some embodiments, the PSR includes the transmembrane region of human SRA1.

[0315] In some embodiments, for example, recombinant nucleic acids include the intracellular domain of CD36. In some embodiments, recombinant nucleic acids include the TM domain of CD36. Naturally occurring full-length CD36 has two TM domains and two short intracellular domains, and the extracellular domain of CD36 binds to oxidized LDL. Both intracellular domains contain a pair of cysteine, which is an acylated fatty acid. It lacks known signaling domains (e.g., kinase, phosphatase, g protein binding, or scaffold domains). The N-terminal cytoplasmic domain is very short (5-7 amino acid residues) and closely associated with the inner lobe of the plasma membrane. The carboxyl-terminal domain contains 13 amino acids and contains a CXCX5K motif homologous to a region in the intracellular domains of CD4 and CD8, which is known to interact with signaling molecules. The intracellular domain of CD36 is capable of assembling signaling complexes that activate lyn kinase, MAP kinase, and focal adhesion kinase (FAK), as well as inactivating src homolog 2-containing phosphotyrosine phosphatase (SHP-2). Some guanine nucleotide exchange factors (GEFs) have been identified as potentially important signaling intermediates.

[0247]

[0316] In some embodiments, the recombinant nucleic acid encodes, for example, the intracellular domain of human SCARA3. In some embodiments, the PSR subunit includes a variant that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to the intracellular domain of human SCARA3. In some embodiments, the PSR includes the TM domain of SCARA3. In some embodiments, the TM domain is approximately 20–30 amino acids long.

[0248]

[0317] Scavenger receptors can arise as homodimers or heterodimers. For example, MARCO arises as a homotrimer.

[0318] In some embodiments, the TM domain or ICD domain of the PSP does not originate from FcR, Megf10, Bai1, or MerTK. In some embodiments, the ICD of the PSR does not contain the intracellular domain of CD3 zeta.

[0249]

[0319] In some embodiments, the intracellular domain and transmembrane domain are derived from FcR beta.

[0320] In one embodiment, the recombinant nucleic acid encodes a chimeric antigenic receptor (CAR-P) for enhancing phagocytosis, which is a phagocytic scavenger receptor (PSR) fusion protein (CFP) comprising a recombinant PSR intracellular signaling domain, the recombinant PSR intracellular signaling domain comprising (a) an extracellular domain including an extracellular antigen-binding domain specific to the antigen of a target cell, (b) a transmembrane domain, and (c) a recombinant PSR intracellular signaling domain comprising a first portion derived from a phagocytic receptor and a second portion derived from a non-phagocytic receptor.

[0250]

[0321] In some embodiments, the second portion is not a PI3K mobilization domain. In some embodiments, the second portion is a PI3K mobilization domain.

[0322] The second portion derived from the nonphagocytic receptor may include an intracellular signaling domain that enhances the phagocytic and / or inflammatory capacity of modified phagocytic cells expressing recombinant nucleic acid. In some embodiments, the second portion derived from the nonphagocytic receptor includes two or more intracellular domains (ICDs). In some embodiments, the second portion derived from the nonphagocytic receptor includes a second ICD. In some embodiments, the second portion derived from the nonphagocytic receptor includes second and third ICDs. In some embodiments, the second portion derived from the nonphagocytic receptor includes second, third, and fourth ICDs, where the second portion is encoded by recombinant nucleic acid. Each second portion derived from the nonphagocytic receptor, including a second, third, or fourth ICD, is described below.

[0251] Chimeric antigen receptors for enhancing intracellular signaling and inflammation activation

[0323] In one embodiment, recombinant nucleic acids, in addition to phagocytic ICDs, can infect, for example, macrophages. It encodes a second intracellular domain that confers the ability to evoke potent pro-inflammatory immune activation when engaged in combating disease. The second intracellular domain (second ICD) fuses with the cytoplasmic terminal of the first phagocytic ICD. The second intracellular domain provides the second signal necessary to trigger the inflammasome and pro-inflammatory signals. Nod-like receptors (NLRs) are a subset of receptors that are activated in the innate immune response and oligomerize to form multiprotein complexes that serve as a platform to recruit pro-inflammatory caspases and induce their cleavage and activation. This leads to direct activation of ROS, often resulting in severe cell death known as pyroptosis. There are four inflammasome complexes, namely NLRP1m, NLRP3, IPAF, and AIM2.

[0252]

[0324] The tumor microenvironment (TME) constitutes an immunosuppressive environment. The effects of IL-10, glucocorticoid hormones, apoptotic cells, and immune complexes can interfere with innate immune cell function. Immune cells, including phagocytic cells, become tolerance-genic. In macrophages, this phenotype, commonly known as the M2 phenotype, differs from the M1 phenotype, in which macrophages are potent and capable of killing pathogens. For example, macrophages exposed to LPS or IFN-gamma may polarize into the M1 phenotype, while macrophages exposed to IL-4 or IL-13 may polarize into the M2 phenotype. LPS or IFN-gamma can interact with Toll-like receptor 4 (TLR4) on the surface of macrophages, inducing the Trif and MyD88 pathways, and inducing the activation of transcription factors IRF3, AP-1, and NFKB, and thus activating TNF genes, interferon genes, CXCL10, NOS2, IL-12, etc., which are necessary in the pro-inflammatory M1 macrophage response. Similarly, IL-4 and IL-13 activate the Jak / Stat6 pathway, which binds to IL-4R and regulates the expression of genes associated with the anti-inflammatory response (M2 response), such as CCL17, ARG1, IRF4, IL-10, and SOCS3. The expression of CD14, CD80, D206 and the low expression of CD163 are indicators of macrophage polarization to the M1 phenotype.

[0253]

[0325] In some embodiments, recombinant nucleic acids include cytoplasmic domains related to inflammatory responses. It encodes one or more additional intracellular domains. In some embodiments, expression of recombinant nucleic acids encoding phagocytic receptor (PR) fusion proteins (CFPs) containing cytoplasmic domains related to the inflammatory response in modified macrophages confers a potent pro-inflammatory response similar to the M1 phenotype.

[0254]

[0326] In some embodiments, the cytoplasmic domains related to the inflammatory response may be the signaling domains or regions of TLR3, 4, 9, MYD88, TRIF, RIG-1, MDA5, CD40, IFN receptor, NLRP-1~14, NOD1, NOD2, Pyrin, AIM2, NLRC4, and CD40.

[0255]

[0327] In some embodiments, the expression of recombinant nucleic acid encoding a phagocytic scavenger receptor (PSR) fusion protein (CFP) includes a pro-inflammatory cytoplasmic domain related to the activation of the IL-1 signaling cascade.

[0256]

[0328] In some embodiments, the cytoplasmic portion of the chimeric receptor (e.g., phagocytic receptor (PR) fusion protein (CFP)) includes a cytoplasmic domain derived from a Toll-like receptor, such as the intracellular signaling domain of Toll-like receptor 3 (TLR3), Toll-like receptor 4 (TLR4), Toll-like receptor 7 (TLR7), Toll-like receptor 8 (TLR8), or Toll-like receptor 9 (TLR9). In some embodiments, the cytoplasmic portion of the chimeric receptor includes a preferred region derived from interleukin-1 receptor-associated kinase 1 (IRAK1). In some embodiments, the cytoplasmic portion of the chimeric receptor includes a preferred region derived from differentiation primary response protein (MYD88). In some embodiments, the cytoplasmic portion of the chimeric receptor includes preferred regions derived from myelin and lymphocyte proteins (MAL). In some embodiments, the cytoplasmic portion of the chimeric receptor includes a preferred region derived from retinoic acid-inducible gene (RIG-1).

[0257]

[0329] In some embodiments, the transmembrane domain of the PSR includes one transmembrane domain from among the MYD88, TLR3, TLR4, TLR7, TLR8, TLR9, MAL, and IRAK1 proteins.

[0258]

[0330] In some embodiments, the recombinant PSR intracellular signaling domain comprises a first portion derived from a phagocytic receptor and a second portion derived from a non-phagocytic receptor, which includes a phosphorylation site. In some embodiments, the phosphorylation site includes an amino acid sequence suitable for autophosphorylation. In some embodiments, the phosphorylation site includes an amino acid sequence suitable for phosphorylation by Src family kinases. In some embodiments, the phosphorylation site includes an amino acid sequence that can bind to the SH2 domain in the kinase upon phosphorylation. In some embodiments, the receptor tyrosine kinase domain fuses to the cytoplasmic terminal of the CFP in addition to the first cytoplasmic portion. In some embodiments, phosphorylation is tyrosine phosphorylation.

[0259]

[0331] In some embodiments, the second intracellular domain is an immunoreceptor tyrosine activation motif (ITAM). ITAM motifs are found in mammalian α and β immunoglobulin proteins, TCRγ receptors, FCRγ receptor subunits, CD3 chain receptors, and NFAT activating molecules.

[0260]

[0332] In some embodiments, the CFP intracellular domain contains one ITAM motif. In some embodiments, the CFP intracellular domain contains more than one ITAM motif. In some embodiments, the CFP intracellular domain contains two or more ITAM motifs. In some embodiments, the CFP intracellular domain contains three or more ITAM motifs. In some embodiments, the CFP intracellular domain contains four or more ITAM motifs. Some embodiments In some embodiments, the CFP intracellular domain contains five or more ITAM motifs. In some embodiments, the CFP intracellular domain contains six or more ITAM motifs. In some embodiments, the CFP intracellular domain contains seven or more ITAM motifs. In some embodiments, the CFP intracellular domain contains eight or more ITAM motifs. In some embodiments, the CFP intracellular domain contains nine or more ITAM motifs. In some embodiments, the CFP intracellular domain contains ten or more ITAM motifs.

[0261]

[0333] In some embodiments, one or more domains in the first phagocytic ICD contain mutations.

[0334] In some embodiments, one or more domains in the second ICD include mutations that enhance the kinase-binding domain, mutations that generate a phosphorylation site, mutations that generate an SH2-binding site, or a combination thereof.

[0262] Co-expression of inflammatory genes

[0335] In one embodiment, recombinant nucleic acids co-express with CFP in modified cells, which are pro-inflammatory precursors. This includes the gene coding sequence. In some embodiments, the pro-inflammatory gene is a cytokine. Examples include, but are not limited to, TNF-α, IL-1α, IL-1β, IL-6, CSF, GMCSF, or IL-12, or interferon.

[0263]

[0336] The recombinant nucleic acid encoding the pro-inflammatory gene may be monocistronic, where the two coding sequences of (a) PSP and (b) the pro-inflammatory gene are post-transcribed or post-translational cleaved for independent expression.

[0264]

[0337] In some embodiments, the two coding sequences include self-cleaving domains that code for, for example, the P2A sequence.

[0338] In some embodiments, the two code regions are separated by an IRES region.

[0265]

[0339] In some embodiments, the two coding sequences are encoded by a bicistronic genetic factor. The coding regions of (a) PSP and (b) pro-inflammatory genes may be unidirectional, where each is under separate regulatory control. In some embodiments, both coding regions are bidirectional and drive in opposite directions. Each coding sequence is under separate regulatory control.

[0266]

[0340] The co-expression of pro-inflammatory genes is designed to confer potent inflammatory stimulation to macrophages and activate surrounding tissues in relation to inflammation.

[0267] Integrin activation domain

[0341] Cell-cell and cell-substrate adhesion is facilitated by the extracellular domains of integrins and various tannins. While mediated by binding to protein ligands, these adhesion interactions and their translation into dynamic cellular responses, such as cell extension or migration, require integrin cytoplasmic tails. These short tails bind to intracellular ligands that connect the receptor to signaling pathways and cytoskeletal networks (Calderwood DA, 2004, Integrin Activation, Journal of Cell Science 117, 657-666). Integrins are heterodimeric adhesion receptors formed by the non-covalent bonding of α and β subunits. Each subunit is a type I transmembrane glycoprotein with a relatively large extracellular domain and, with the exception of the β4 subunit, a short cytoplasmic tail. Individual integrin family members have the ability to recognize multiple ligands. Integrins reflect the major function of integrins in cell adhesion to the extracellular matrix, and are linked to numerous extracellular matrix proteins (bone matrix proteins, collagen, fibronectin, fibrinogen, laminin, thoron). They can bind to integrins (bospondin, vitronectin, and von Willebrand factor). Many "counterreceptors" are ligands that reflect the role of integrins in mediating cell-cell interactions. Integrins undergo conformational changes to improve ligand affinity.

[0268]

[0342] The integrin β2 subfamily consists of four different integrin receptors, namely α M β2 (CD11b / CD18, Mac-1, CR3, Mo-1), α L β2 (CD11a / CD18, LFA-1), α X β2 (CD11c / CD18), and αD These leukocyte integrins consist of β2 (CD11d / CD18). They are involved in virtually all aspects of leukocyte function, including immune responses, adhesion to and transendothelium, phagocytosis of pathogens, and leukocyte activation.

[0269]

[0343] All β2 integrin α subunits contain an insertion region of approximately 200 amino acids, referred to as the I or A domain. The highly conserved I domain is found in several other integrin α subunits and other proteins, such as certain coagulation and complement proteins. The I domain mediates protein-protein interactions and is integrally involved in protein ligand binding in integrins. While the I domain governs the ligand-binding function of integrins, other regions of the α subunit influence ligand recognition. For example, α M In β2, mAb(OKM1) is outside the I domain but α M α integrins recognize epitopes in their subunits and inhibit ligand binding, and have an I domain in their α subunit. L In β2 and α2β1, the EF hand region contributes to ligand recognition. M The subunit, and possibly other α-subunits, contain lectin-like domains involved in the association of non-protein ligands, and their occupation may modulate the function of the I domain.

[0270]

[0344] Because integrins lack enzymatic activity, signal transduction is instead induced by the assembly of signaling complexes on the cytoplasmic surface of the plasma membrane. The formation of these complexes is achieved in two ways: 1. by receptor cluster formation, which increases the avidity of molecular interactions and thereby increases the binding rate of effector molecules; and 2. by induction of conformational changes in receptors that create or expose effector binding sites. Within the ECM, integrins have the ability to bind to fibronectin, laminin, collagen, tenascin, vitronectin, and thrombospondin. Integrin / ECM interaction clusters form focal adhesions, enriching intracellular cytoskeletal components and signaling molecules. The cytoplasmic tail of integrins serves as a binding site for α-actinin and talin, which later recruits vinculin, a protein involved in anchoring F-actin to the membrane. Talin is activated by kinases such as protein kinase C (PKCα).

[0271]

[0345] Integrins are activated by selectins. Leukocytes express L-selectin, activated platelets express P-selectin, and activated endothelial cells express E- and P-selectins. P-selectin-mediated adhesion allows for the activation of β2 integrins induced by chemokines or platelet activators, stabilizing adhesion. This also facilitates the release of chemokines from adherent leukocytes. The cytoplasmic domain of P-selectin glycoprotein ligand 1 formed a constitutive complex with Nef-related factor 1. After binding of P-selectin, Src kinase phosphorylates Nef-related factor 1, recruiting the phosphoinositide-3-OH kinase p85-p110σ heterodimer, resulting in the activation of leukocyte integrins. E-selectin ligand transmits signals that also affect β2 integrin function. Selectins induce the activation of Src family kinases. SFK activated by selectin association phosphorylates the immune receptor tyrosine-based activation motif (ITAM) in the cytoplasmic domains of DAP12 and FcRγ. In some respects, CD CD44 is sufficient to transmit signals from E-selectin. CD44 induces inside-out signaling of integrins. A common final step in integrin activation is the binding of taline to the cytoplasmic tail of the β-subunit. Kindrins, cytoplasmic adapters of another group, bind to different regions of the integrin β-tail. Kindrins enhance clustering of taline-activated integrins. Kindrins are responsive to selectin signaling, but most are found in hematopoietic cells such as neutrophils. Selectin signaling and signaling during integrin activation by chemokine components share components including SFK, Syk, and SLP-76.

[0272]

[0346] In some embodiments, the intracellular domain of the recombinant PSR fusion protein includes an integrin activation domain. The integrin activation domain includes the intracellular domain of a selectin, such as P-selectin, L-selectin, or E-selectin.

[0273]

[0347] In some embodiments, the intracellular domain of the recombinant PSR fusion protein includes the integrin activation domain of laminin.

[0348] In some embodiments, the intracellular domain of the recombinant PSR fusion protein includes an integrin activation domain related to talin activation.

[0274]

[0349] In some embodiments, the intracellular domain of the recombinant PSR fusion protein includes an integrin-activating domain fused to the cytoplasmic terminal of the phagocytic receptor ICD domain.

[0275] Chimeric receptors for enhancing antigen cross-presentation

[0350] In some embodiments, recombinant nucleic acids can enable cross-presentation of antigens. It encodes a domain. Generally, MHC class I molecules present self- or pathogen-derived antigens synthesized within the cell, while exogenous antigens obtained through endocytosis are loaded onto MHC class II molecules for presentation to CD4+ T cells. Peptides are MHC I-restricted presentation of endogenous antigens produced by the proteasome. However, in some cases, DCs can process exogenous antigens and introduce them into the MHC-I pathway for presentation to CD8+ T cells. This is called cross-presentation of antigens. Soluble or exogenous antigenic components can be degraded by lysosomal proteases in the vacuole and cross-presented by DCs instead of following the endocytosis pathway. In some cases, chaperones such as heat shock protein 90 (Hsp90) have been shown to assist in the cross-presentation of antigens by certain APCs. HSP-peptide complexes are known to be internally translocated by different groups of receptors compared to free polypeptides. These receptors are derived from the scavenger receptor family and include LOX-1, SREC-I / SCARF-I, and FEEL1 / Stabilin-1. Both SREC-I and LOX-1 mediate the cross-presentation of molecular chaperone-binding antigens, and CD8 + It has been shown to trigger the activation of T lymphocytes.

[0276]

[0351] SREC-1 (a scavenger receptor expressed by endothelial cells) does not have significant homology to other types of scavenger receptors, but it has a unique domain structure. SREC-1 contains 10 repeat sequences of an EGF-like cysteine-rich motif in its extracellular domain. Recently, the structure of SREC-1 has been shown to be similar to that of a transmembrane protein with 16 EGF-like repeat sequences encoded by the Caenorhabditis elegans gene ced-I, which functions as a cell surface phagocytic receptor that recognizes apoptotic cells.

[0277]

[0352] Cross-presentation of cancer antigens via the class-I MHC pathway results in an enhancement of the CD8+ T cell response, which is associated with cytotoxicity and therefore beneficial in tumor reduction. In some embodiments, the intracellular domain of CFP includes the SREC1 intracellular domain. In this state, the intracellular domain of CFP includes the SRECII intracellular domain.

[0278]

[0353] In some embodiments, the PSR subunit includes an intracellular domain containing a PSR intracellular signaling domain derived from SREC1 or SRECII.

[0354] In some embodiments, the PSR subunit includes (i) a transmembrane domain and (ii) an intracellular domain comprising a PSR intracellular signaling domain derived from SREC1 or SRECII.

[0279]

[0355] In some embodiments, the PSR subunit comprises (i) a transmembrane domain, (ii) an intracellular domain including a PSR intracellular signaling domain, and (iii) an extracellular domain derived from SREC1 or SRECII.

[0280] Transmembrane domain of CFP fusion protein

[0356] In some embodiments, the recombinant nucleic acid encoded by TM is a scavenger It includes a saturation (SR) domain. In some embodiments, TM may be a TM domain derived from or one or more of the following: lectin, dectin 1, mannose receptor (CD206), SRA1, MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, SRCRB4D, SSC5D, and CD169.

[0281]

[0357] In some embodiments, the TM domain is approximately 20-30 amino acids long. The TM domain of SR is approximately 20-30 amino acids long.

[0358] The TM domain or ICD domain of the PSP does not originate from Megf10, Bai1, or MerTK. The ICD of the PSR does not contain the intracellular domain of CD3 zeta.

[0282]

[0359] In some embodiments, TM is derived from the same phagocytic receptor as ICD.

[0360] In some embodiments, the TM region is derived from a plasma membrane protein. TM can be selected from Fc receptors (FcRs). In some embodiments, a nucleic acid sequence encoding a domain derived from a specific FcR is used for cell-specific expression of the recombinant construct. The FCR alpha region containing the TM domain may be used for macrophage-specific expression of the construct. The FcRβ recombinant protein is expressed in mast cells.

[0283]

[0361] In some embodiments, the CFP includes a TM of FCR beta (FcRβ).

[0362] In some embodiments, the CFP includes both the FcRβTM domain and the ICD domain.

[0284]

[0363] In some embodiments, the TM domain is derived from CD8.

[0364] In some embodiments, TM is derived from CD2.

[0365] In some embodiments, TM is derived from FCR alpha.

[0285] Extracellular domain of CFP fusion protein

[0366] The extracellular domain is an antigen-binding domain that binds to one or more target antigens on target cells. The main domain is included. The target-binding domain is specific to the target. The extracellular domain may contain an antibody or antigen-binding domain selected from intrabody, peptide body, nanobody, single-domain antibody, SMIP, and multispecific antibody.

[0286]

[0367] In some embodiments, the extracellular domain includes a Fab-binding domain. In yet other such embodiments, the extracellular domain includes an scFv.

[0368] In some embodiments, the chimeric antigen receptor includes an extracellular antigen-binding domain derived from a group consisting of an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a nanobody, a VH domain, a VL domain, a single-domain antibody (sdAb), a VNAR domain, and a VHH domain, a bispecific antibody, a diabody, or any functional fragment thereof. In some embodiments, the antigen-binding fragment (Fab), the single-chain variable fragment (scFv), the nanobody, the VH domain, a VL domain, a single-domain antibody (sdAb), a VNAR domain, and a VHH domain, a bispecific antibody, a diabody, or any functional fragment thereof specifically binds to one or more antigens.

[0287]

[0369] In some embodiments, the antigen is a cancer antigen, and the target cell is a target cancer cell. In some embodiments, the antigens of the target cancer cells are CD3, CD4, CD5, CD7, CD19, CCR2, CCR4, CD30, CD37, TCRB1 / 2, TCRαβ, TCRγσ, CD22, HER2(ERBB2 / neu), mesothelin, PSCA, CD123, CD30, CD171, CD138, CS-1, CLECL1, CD33, CD79b, EGFRvIII, GD2, GD3, BCMA, PSMA, ROR1, FLT3, TAG72, CD38, CD44v6, CEA The group is selected from EPCAM, B7H3 (CD276), KIT (CD117), CD213A2, IL-1IRa, PRSS21, VEGFR2, CD24, MUC-16, PDGFR-beta, SSEA-4, CD20, MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, FAP, EphA2, GM3, TEM1 / CD248, TEM7R, CLDN6, TSHR, GPRC5D, CD97, CD179a, ALK, and IGLL1.

[0288]

[0370] Various cancer antigen targets can be selected from cancer antigens known to those skilled in the art. Depending on the cancer and cell type, the cancer antigen involved is a mutant native protein. The antigen-binding domain is screened for specificity to mutant / cancer antigens rather than native antigens.

[0289]

[0371] In some embodiments, for example, the cancer antigens of the target cancer cells are mutations / cancer antigens: MUC16, CCAT2, CTAG1A, CTAG1B, MAGEA1, MAGEA2, MAGEA3, MAGEA4, MAGEA6, PRAME, PCA3, MAGEC1, MAGEC2, MAGED2, AFP, MAGEA8, MAGE9, MAGEA11, MAGEA12, IL13RA2, PLAC1, SD It may be one or more of CCAG8, LSP1, CT45A1, CT45A2, CT45A3, CT45A5, CT45A6, CT45A8, CT45A10, CT47A1, CT47A2, CT47A3, CT47A4, CT47A5, CT47A6, CT47A8, CT47A9, CT47A10, CT47A11, CT47A12, CT47B1, SAGE1, and CT55.

[0290]

[0372] In some embodiments, for example, the cancer antigen of the target cancer cell may be one or more of the mutant / cancer antigens: CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD30, CD45, CD56, where the cancer is a T-cell lymphoma.

[0291]

[0373] In some embodiments, for example, the cancer antigen of the target cancer cell may be one or more of the mutations / cancer antigens: IDH1, ATRX, PRL3, or ETBR, where the cancer is glioblastoma.

[0292]

[0374] In some embodiments, for example, the cancer antigen of the target cancer cell may be one or more of the following: mutation / cancer antigen: CA125, beta-hCG, urinary gonadotropin fragment, AFP, CEA, SCC, inhibin, or extradiol, where the cancer is ovarian cancer.

[0293]

[0375] In some embodiments, the cancer antigen of the target cancer cell may be HER2.

[0376] In some embodiments, the cancer antigen of the target cancer cell may be EGFR variant III.

[0294]

[0377] In some embodiments, the cancer antigen of the target cancer cell may be CD19.

[0378] In some embodiments, the SR subunit region includes the extracellular domain (ECD) of the scavenger receptor. In some embodiments, the ECD of the scavenger receptor includes the ECD domain of the SR, which includes the ICD and TM domains. In some embodiments, the SR-ECD contributes to the binding of phagocytic cells to target cells and is subsequently activated to activate phagocytosis of the target cells.

[0295]

[0379] In some embodiments, the PSR domain optionally includes the ECD domain or a portion thereof of the respective scavenger receptor into which the ICD and TM domains are incorporated into the PSR. Thus, in some embodiments, the ECD encoded by the recombinant nucleic acid includes a domain selected from the group consisting of lectin, dectin 1, mannose receptor (CD206), scavenger receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), and CD169 receptor. The extracellular domains of most macrophage scavenger receptors contain scavenger receptors with broad binding specificity that can be used to distinguish self from non-self in nonspecific, antibody-independent recognition of foreign substances. Type I and II class A scavenger receptors (SR-AI1 and SR-AII) are trimer membrane glycoproteins with a small NH2-terminal intracellular domain and an extracellular component containing a short spacer domain, an α-helix-coiled domain, and a triple-helix collagen domain. In addition, type I receptors contain a cysteine-rich COOH-terminal (SRCR) domain. These receptors are present in macrophages in diverse tissues throughout the body and exhibit remarkably broad ligand-binding specificity. These receptors bind to a wide variety of polyanions, including chemically modified proteins such as modified LDL, and are associated with cholesterol deposition during atherogenesis. These receptors may also play a role in macrophage-associated host defense and cell adhesion processes in inflammatory states.

[0296]

[0380] In some embodiments, SR ECD is designed to bind to pro-apoptotic cells. In some embodiments, the scavenger receptor ECD includes a binding domain for cell surface molecules of cancer cells or infected cells.

[0297]

[0381] In some embodiments, the extracellular domain of the PR subunit is linked by a linker to a target cell-binding domain, such as an antibody or a portion thereof, that is specific to the cancer antigen.

[0382] In some embodiments, the extracellular antigen-binding domain includes one antigen-binding domain. In some embodiments, the extracellular antigen-binding domain includes two or more binding domains. In some embodiments, the binding domain is an scFv. In some embodiments, the binding domain is a single-domain antibody (sdAb). In some embodiments, the binding domain fuses with recombinant PR in the extracellular domain. In some embodiments, the binding domain (e.g., scFv) and the extracellular domain of PR are linked via a linker.

[0298]

[0383] In some embodiments, the ECD antigen-binding domain can bind to an intracellular antigen. In some embodiments, the intracellular antigen is a cancer antigen.

[0384] In some embodiments, the extracellular antigen-binding domain binds to the target ligand with an affinity of less than 1000 nM. In some embodiments, the extracellular antigen-binding domain binds to the target ligand with an affinity of less than 500 nM. In some embodiments, the extracellular antigen-binding domain The extracellular antigen-binding domain binds to the target ligand with an affinity of less than 450 nM. In some embodiments, the extracellular antigen-binding domain binds to the target ligand with an affinity of less than 400 nM. In some embodiments, the extracellular antigen-binding domain binds to the target ligand with an affinity of less than 350 nM. In some embodiments, the extracellular antigen-binding domain binds to the target ligand with an affinity of less than 250 nM. In some embodiments, the extracellular antigen-binding domain binds to the target ligand with an affinity of less than 200 nM. In some embodiments, the extracellular antigen-binding domain binds to the target ligand with an affinity of less than 100 nM. In some embodiments, the extracellular antigen-binding domain binds to the target ligand with an affinity in the range of greater than 200 nM to 1000 nM. In some embodiments, the extracellular antigen-binding domain binds to the target ligand with an affinity in the range of greater than 300 nM to 1.5 mM. In some embodiments, the antigen-binding domain binds to the target ligand with an affinity of greater than 200 nM, greater than 300 nM, or greater than 500 nM.

[0299] Peptide linker

[0385] In some embodiments, the extracellular antigen-binding domain scfv is linked by a linker. The SCFV is linked to an M domain or other extracellular domain. In some embodiments where two or more SCFVs have extracellular antigen-binding domains, the two or more SCFVs are linked to each other by a linker.

[0300]

[0386] In some embodiments, the linker is mobile. In some embodiments, the linker includes a hinge region. The linker is typically a short peptide sequence. In some embodiments, the linker is a segment of glycine and one or more serine residues. Other amino acids preferred for short peptide linkers, but not limited to these, include threonine (Thr), serine (Ser), proline (Pro), glycine (Gly), aspartic acid (Asp), lysine (Lys), glutamine (Gln), asparagine (Asn), and alanine (Ala), arginine (Arg), phenylalanine (Phe), and glutamic acid (Glu). Of these, Pro, Thr, and Gln are frequently used amino acids for natural linkers. Pro is a unique amino acid with a cyclic side chain that results in a very restricted conformation. Pro-rich sequences are used as interdomain linkers, including the linker (GA2PA3PAKQEA3PAPA2KAEAPA3PA2KA) between the lipoyl domain and the E3-binding domain in pyruvate dehydrogenase. For the purposes of this disclosure, empirical linkers may be mobile linkers, rigid linkers, and cleavage linkers. Sequences such as (G4S)x (where x is a part of multiple copies represented as 1, 2, 3, 4, etc.) include mobile linker sequences. Other mobile sequences used herein include several repeat sequences of glycine, e.g., (Gly)6 or (Gly)8. On the other hand, rigid linkers may also be used; for example, the linker (EAAAK)x (where x is an integer such as 1, 2, 3, 4, etc.) produces a rigid linker.

[0301]

[0387] In some embodiments, the linker contains at least two or at least three amino acids. In some embodiments, the linker contains four amino acids. In some embodiments, the linker contains five amino acids. In some embodiments, the linker contains six amino acids. In some embodiments, the linker contains seven amino acids. In some embodiments, the linker contains eight amino acids. In some embodiments, the linker contains nine amino acids. In some embodiments, the linker contains eight amino acids. In some embodiments, the linker contains ten amino acids. In some embodiments, the linker contains eleven amino acids. In some embodiments, the linker contains twelve amino acids. In some embodiments, the linker contains thirteen amino acids. In some embodiments, the linker contains fourteen amino acids. In some embodiments, the linker contains fifteen amino acids. In some embodiments, the linker contains sixteen amino acids. In some embodiments, the linker contains seventeen amino acids. In some embodiments, the linker contains eighteen amino acids. In some embodiments, the linker contains nineteen amino acids. In some embodiments, the linker contains twenty amino acids.

[0302]

[0388] As intended herein, any suitable ECD, TM, or ICD domain may be cloned interchangeably with any one suitable portion of the CARP receptor described herein to obtain a protein having enhanced phagocytosis compared to the endogenous receptor.

[0303] Characteristics of the fusion protein:

[0389] CFP can be structurally incorporated into the cell membrane of cells that express CFP. Specific leader sequences in nucleic acid constructs such as signal peptides can be used to guide the plasma membrane expression of coding proteins. The transmembrane domain encoded by the construct can integrate the expressed protein into the cell's plasma membrane.

[0304]

[0390] In some embodiments, the transmembrane domain includes the TM domain of the FcR alpha receptor, which dimerizes with the endogenous FcR gamma receptor in macrophages, ensuring macrophage-specific expression.

[0305]

[0391] CFP can make cells expressing CFP highly phagocytic. When recombinant nucleic acid encoding CFP is expressed in cells, the cells can exhibit improved phagocytosis of target cells having the target cell antigen compared to cells that do not express the recombinant nucleic acid. When recombinant nucleic acid is expressed in cells, the cells can exhibit improved phagocytosis of target cells having the target cell antigen compared to cells that do not express the recombinant nucleic acid. In some embodiments, when recombinant nucleic acid is expressed in cells, the cells exhibit at least 2-fold improved phagocytosis of target cells having the target cell antigen compared to cells that do not express the recombinant nucleic acid. In some embodiments, when recombinant nucleic acid is expressed in cells, the cells exhibit at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, or at least 5-fold improved phagocytosis of target cells having the target cell antigen compared to cells that do not express the recombinant nucleic acid.

[0306]

[0392] In some embodiments, SIRP-ΔICD expression enhances phagocytosis of cells expressing SIRP-ΔICD by 1.1 times, 1.2 times, 1.3 times, 4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 8 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, and 100 times or more compared to cells that do not express SIRP-ΔICD.

[0307]

[0393] In some embodiments, cells co-expressing SIRP-ΔICD and a CFP encoding the phagocytic receptor described herein exhibit increased phagocytosis compared to cells that do not express either protein. In some embodiments, co-expression of SIRP-ΔICD and a CFP encoding the phagocytic receptor described herein results in an improvement in phagocytic ability (measured by a multiplier change in the phagocytic index) of more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 times compared to cells that do not express either SIRP-ΔICD or the CFP encoding the phagocytic receptor.

[0308]

[0394] In some embodiments, the expression of either the extracellular domain of SIRPα's CD47 blocking domain or the intracellular domain of the phagocytic receptor CFP results in a phagocytic activity of cells expressing it being at least 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 8 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, or 70 times greater compared to cells not expressing CFP or cells expressing SIRP-ΔICD. It increases by more than 0 times, more than 90 times, or more than 100 times.

[0309]

[0395] In some embodiments, the enhancement of target cell phagocytosis by cells expressing SIRP-ΔICD is significantly improved compared to phagocytic cells that do not express SIRP-ΔICD.

[0396] In some embodiments, the enhancement of target cell phagocytosis by cells expressing CFP, which includes the extracellular domain of SIRPα's CD47 blocking domain and the intracellular domain of the phagocytic receptor, is significantly improved compared to control phagocytic cells that do not express the fusion protein or control phagocytic cells that express SIRP-ΔICD.

[0310]

[0397] In some embodiments, when recombinant nucleic acids described herein are expressed in cells, the cells exhibit enhanced cytokine production. The cytokines may include any one of IL-1, IL-6, IL-12, IL-23, TNF, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, and interferon.

[0311]

[0398] In some embodiments, when the recombinant nucleic acids described herein are expressed in cells, the cells exhibit enhanced cell migration.

[0399] In some embodiments, when the recombinant nucleic acids described herein are expressed in cells, the cells exhibit enhanced immune activity. In some embodiments, when the recombinant nucleic acids are expressed in cells, the cells exhibit increased expression of MHC II. In some embodiments, when the recombinant nucleic acids are expressed in cells, the cells exhibit increased expression of CD80. In some embodiments, when the recombinant nucleic acids are expressed in cells, the cells exhibit increased expression of CD86. In some embodiments, when the recombinant nucleic acids are expressed in cells, the cells exhibit enhanced iNOS production.

[0312]

[0400] In some embodiments, when recombinant nucleic acids are expressed in cells, the cells exhibit reduced trogocytosis of target cells expressing the target cell antigen compared to cells that do not express recombinant nucleic acids.

[0313]

[0401] In several embodiments, the chimeric receptor may be glycosylated, PEGylated, and / or otherwise post-translationally modified. In further embodiments, glycosylation, PEGylated, and / or other post-translational modifications may be carried out in vivo or in vitro, and / or using chemical techniques. In additional embodiments, any glycosylation, PEGylated, and / or other post-translational modifications may be N-linked or O-linked. In several embodiments, any one of the chimeric receptors may be enzymatically or functionally active, and as a result, when the extracellular domain is bound by a ligand, a signal is transmitted to polarize macrophages.

[0314]

[0402] In some embodiments, the chimeric fusion protein (CFP) includes an extracellular domain (ECD) (CD5-binding domain) targeted to bind to CD5, which includes a heavy chain variable region (VH) having, for example, the amino acid sequence described in SEQ ID NO: 1. In some embodiments, the chimeric CFP includes a CD5-binding heavy chain variable domain having an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity with SEQ ID NO: 1. In some embodiments, the extracellular domain (ECD) (CD5-binding domain) targeted to bind to CD5 includes a light chain variable domain (VH) having the amino acid sequence described in SEQ ID NO: 2. L ) includes. In some embodiments, the chimeric CFP includes a CD5-binding light chain variable domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity with SEQ ID NO: 2.

[0315]

[0403] In some embodiments, CFP is a heavy-chain variable domaine, for example, as described in Sequence ID No. 8. The CFP includes an extracellular domain (HER2-binding domain) targeted to bind to HER2, having an amino acid sequence and a light chain variable domain amino acid sequence described in Sequence ID No. 9. In some embodiments, the CFP includes a HER2-binding heavy chain variable domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity with Sequence ID No. 8. In some embodiments, the CFP includes a HER2-binding light chain variable domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity with Sequence ID No. 9.

[0316]

[0404] In some embodiments, the CFP includes a hinge that connects the ECD transmembrane (TM). In some embodiments, the hinge includes the amino acid sequence of the hinge region of the CD8 receptor. In some embodiments, the CFP may include a hinge having the amino acid sequence described in SEQ ID NO: 7 (CD8α chain hinge domain). In some embodiments, the PFP hinge region includes an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity with SEQ ID NO: 7.

[0317]

[0405] In some embodiments, the CFP includes a CD8 transmembrane region having, for example, the amino acid sequence described in SEQ ID NO: 6. In some embodiments, the CFP™ region includes an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity with SEQ ID NO: 6.

[0318]

[0406] In some embodiments, CFP includes an intracellular domain having an FcR domain. In some embodiments, CFP includes an intracellular domain with an FcR domain that includes the amino acid sequence described in SEQ ID NO: 3, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 3.

[0319]

[0407] In some embodiments, CFP includes an intracellular domain having a PI3K recruiting domain. In some embodiments, the PI3K recruiting domain includes the amino sequence described in SEQ ID NO: 4. In some embodiments, the PI3K recruiting domain includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity with SEQ ID NO: 4.

[0320]

[0408] In some embodiments, the CFP includes an intracellular domain having a CD40 intracellular domain. In some embodiments, the CD40 ICD includes the amino sequence described in SEQ ID NO: 5. In some embodiments, the CD40 ICD includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity with SEQ ID NO: 5.

[0321]

[0409] In some embodiments, the CD5 binding domain is (i) a variable heavy chain (V) having at least 90% sequence identity with SEQ ID NO: 1. H (ii) a sequence, and (ii) a variable light chain (V) having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or to sequence number 2. L ) Includes an scFv containing the sequence. In some embodiments, the CD5 binding domain includes SEQ ID NO: 33 or includes an scFv having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 33. In some embodiments, the HER2 binding domain includes (i) a variable heavy chain (V) having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 8. H ) sequences, and (ii) of sequence number 9, or sequence number 9 and at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , or a variable light chain (V) with 99% sequence identity L ) comprises an scFv containing the sequence. In some embodiments, the CD5-binding domain comprises SEQ ID NO: 32 or an scFv having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 32. In some embodiments, the CFP further comprises an intracellular domain, where the intracellular domain comprises one or more intracellular signaling domains, and the wild-type protein containing the intracellular domain does not contain an extracellular domain.

[0322]

[0410] In some embodiments, the extracellular domain further comprises a hinge domain derived from CD8, which operatively ligates with the transmembrane domain and the anti-CD5 binding domain. In some embodiments, the extracellular hinge domain comprises a sequence of SEQ ID NO: 7, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 7.

[0323]

[0411] In some embodiments, the CFP includes an extracellular domain fused with a transmembrane domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 30. In some embodiments, the CFP includes an extracellular domain fused with a transmembrane domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 31.

[0324]

[0412] In some embodiments, the transmembrane domain includes a CD8 transmembrane domain. In some embodiments, the transmembrane domain includes a sequence of sequence number 6 or 29, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with sequence number 6 or 29. In some embodiments, the transmembrane domain includes a sequence of sequence number 18, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with sequence number 18. In some embodiments, the transmembrane domain includes a sequence having sequence identity with SEQ ID NO: 34, or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO: 34. In some embodiments, the transmembrane domain includes a sequence having sequence identity with SEQ ID NO: 19, or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO: 19.

[0325]

[0413] In some embodiments, CFP includes one or more intracellular signaling domains, including a phagocytic signaling domain. In some embodiments, the phagocytic signaling domain includes intracellular signaling domains derived from receptors other than Megf10, MerTk, FcRα, and Bai1. In some embodiments, the phagocytic signaling domain includes intracellular signaling domains derived from receptors other than Megf10, MerTk, FcR, and Bai1. In some embodiments, the phagocytic signaling domain includes intracellular signaling domains derived from receptors other than CD3ζ. In some embodiments, the phagocytic signaling domain includes intracellular signaling domains derived from FcRγ, FcRα, or FcRε. In some embodiments, the phagocytic signaling domain includes an intracellular signaling domain derived from CD3ζ. In some embodiments, the CFP is sequence-identical to any one of sequence numbers 3, 20, 27, and 28, or to any one of sequence numbers 3, 20, 27, and 28 by at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. It includes an intracellular signaling domain having a sex. In some embodiments, one or more intracellular signaling domains further include a pro-inflammatory signaling domain. In some embodiments, the pro-inflammatory signaling domain includes a PI3 kinase (PI3K) mobilization domain. In some embodiments, the pro-inflammatory signaling domain includes a sequence of SEQ ID NO: 4, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 4. In some embodiments, the pro-inflammatory signaling domain is derived from an intracellular signaling domain of CD40. In some embodiments, the pro-inflammatory signaling domain includes a sequence of SEQ ID NO: 5, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 5. In some embodiments, the CFP includes an intracellular signaling domain having sequence identity with or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of sequence identity with SEQ ID NO: 21. In some embodiments, the CFP includes an intracellular signaling domain having sequence identity with or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of sequence identity with SEQ ID NO: 23.

[0326]

[0414] In some embodiments, the CFP includes a sequence having sequence identity with sequence number 14, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with sequence number 14. In some embodiments, the CFP includes a sequence having sequence identity with sequence number 15, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with sequence number 15. In some embodiments, the CFP includes a sequence having sequence identity with sequence number 16, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with sequence number 16. In some embodiments, the CFP includes a sequence having sequence identity with sequence number 24, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with sequence number 24. In some embodiments, the CFP includes a sequence having sequence identity with sequence number 25, or a sequence having at least 70%, 75%, 80%, 85%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with sequence number 25.

[0327]

[0415] In some embodiments, the CFP comprises an extracellular domain comprising (a)(i) scFv that specifically binds to CD5, (ii) an extracellular domain comprising a hinge domain derived from CD8; a hinge domain derived from CD28; or at least a portion of an extracellular domain derived from CD68, (b) a CD8 transmembrane domain, a CD28 transmembrane domain, a CD2 transmembrane domain; or a CD68 transmembrane domain, and (c) an intracellular domain comprising at least two intracellular signaling domains, wherein the at least two intracellular signaling domains comprise (i) a first intracellular signaling domain derived from FcRα, FcRγ, or FcRε, and (ii) a second intracellular signaling domain comprising (A) a PI3K mobilization domain or (B) a second intracellular signaling domain derived from CD40. In some embodiments, the CFP includes an intracellular domain comprising at least two intracellular signaling domains, wherein the at least two intracellular signaling domains comprise (i) a first intracellular signaling domain derived from a phagocytic receptor intracellular domain, and (ii) a second intracellular signaling domain comprising (A) a PI3K mobilization domain or (B) a scavenger receptor phagocytic receptor intracellular domain derived from CD40. Exemplary scavenger receptors from which the intracellular signaling domains may originate. This can be found in Table 2. In some embodiments, the CFP includes an intracellular signaling domain derived from the intracellular signaling domain of the innate immune receptor.

[0328]

[0416] In some embodiments, the recombinant polynucleic acid is mRNA. In some embodiments, the recombinant polynucleic acid is circRNA. In some embodiments, the recombinant polynucleic acid is a viral vector. In some embodiments, the recombinant polynucleic acid is delivered via a viral vector.

[0329]

[0417] In some embodiments, the myeloid cells are CD14+ cells, CD14+ / CD16- cells, CD14+ / CD16+ cells, CD14- / CD16+ cells, CD14- / CD16- cells, dendritic cells, M0 macrophages, M2 macrophages, M1 macrophages, or mosaic myeloid cells / macrophages / dendritic cells.

[0330]

[0418] In one embodiment, a method for treating cancer in a human subject requiring treatment for cancer is provided herein, comprising the step of administering a pharmaceutical composition to the human subject, wherein the pharmaceutical composition comprises (a) myeloid cells comprising a recombinant polynucleic acid sequence, the polynucleic acid sequence comprising a sequence encoding a chimeric fusion protein (CFP), the CFP comprising (i) an extracellular domain comprising an anti-CD5 binding domain, and (ii) a transmembrane domain operatively linked to the extracellular domain, and (b) a pharmaceutically acceptable carrier, wherein the myeloid cells express the CFP.

[0331]

[0419] In some embodiments, the binding of CFP to CD5 expressed by target cancer cells increases the killing or phagocytic activity of myeloid cells by more than 20% compared to myeloid cells that do not express CFP. In some embodiments, tumor growth is inhibited in human subjects.

[0332]

[0420] In some embodiments, the cancer is CD5+ cancer. In some embodiments, the cancer is leukemia, T-cell lymphoma, or B-cell lymphoma. In some embodiments, the CFP comprises one or more sequences shown in Table A and / or Table B below. [Table A-1] [Table A-2] [Table A-3] [Table A-4] [Table A-5] [Table A-6] [Table B]

[0333] IV. Non-coding exogenous sequences for delivery and integration into the cell genome

[0421] Non-coding sequences can be delivered to cells and incorporated into the cell's genome. It can be designed in such a way. Non-coding sequences, as used herein, are sequences that do not produce translated protein products but may have regulatory elements, such as transcripts, such as interfering RNA. In some embodiments, such sequences may be miRNA sequences. In some embodiments, sequences may be sequences for siRNA generation. In some embodiments, sequences may include intron sequences or binding sites that can be created, to which one or more DNA-binding proteins can bind and influence the properties and behavior of adjacent regions. In some embodiments, sequences may be transcription factor binding sites. In some embodiments, sequences may include enhancer binding sites. In some embodiments, sequences may include binding sites for topoisomerase, gyrase, reverse transcriptase, polymerase, poly(A)-binding protein, guanylyl cyclase, ligase, restriction enzyme, DNA methylase, HDAC enzyme, etc. In some embodiments, non-coding sequences may be intended to manipulate heterochromatin. Non-coding insertion sequences, as sometimes referred to herein, may be a few nucleotides to 5 kB in length.

[0334] V. Plasmid design including insert sequences and recombinant nucleic acid design

[0422] 1 x Nucleic acid constructs containing sequences are incorporated into plasmids for transcription and mRNA generation. mRNA can be transcribed in a vitro system using a cell extract synthesis system. Alternatively, mRNA can be generated and collected in cells. These cells may be prokaryotic cells, such as bacterial cells. In some embodiments, the cells are eukaryotic cells. In some embodiments, transcription is performed in a synthesis system. Exemplary plasmid designs are provided herein.

[0335]

[0423] In some embodiments of various aspects of this disclosure, a plasmid is designed for the expression of an mRNA molecule containing a heterologous sequence of interest encoding a protein or polypeptide. The plasmid includes, among other things, a sequence relating to a genomic integration element for the integration of the heterologous sequence of interest encoding a protein or polypeptide; a sequence containing a transgene or a fragment thereof that operably ligates to separate promoters and regulatory elements required for expression in the host (e.g., the subject to which the mRNA is administered) after integration into the host genome; one or more regulatory elements for mRNA transcription and generation, including a promoter and a 3' stabilizing element for mRNA expression in, for example, bacterial cells or cell extracts; and a sequence relating to one or more detection markers and / or selection markers.

[0336]

[0424] As is well known to those skilled in the art, the plasmid backbone can be an available vector, such as an in-house or commercially developed vector, which can be improved in various ways for the best expression of the transcription sequence, for example (but not limited to) by introducing one or more desired restriction digestion sites into a multi-cloning site (MCS), introducing a desired promoter for transcription of the entire mRNA, such as a T7 promoter, replacing a sequence present in the plasmid vector with one or more desired sequences, or introducing one or more desired segments, such as a selection marker sequence.

[0337]

[0425] A plasmid contains transcriptional regulatory elements such as a promoter and a 3' stabilizing element in the 5' region. In some embodiments, the promoter is selected to enhance mRNA transcription in desired cells, such as E. coli bacterial cells. In some embodiments, the promoter for plasmid transcription is the T7 promoter, Sp6 promoter, pL (lambda) promoter, T3 promoter, trp promoter, or araBad promoter. The promoter is selected from T7, lac, or Ptac promoters. In some embodiments, the promoter is the T7 promoter. The T7 or Sp6 promoters are constitutive promoters and are useful for high-level transcription or in vitro transcription. In some embodiment...

Claims

1. A method for incorporating a nucleic acid sequence into the genome of a cell, comprising the step of introducing recombinant mRNA or an mRNA-encoding vector into a cell, wherein the mRNA is (a) An insertion sequence, (i) Foreign arrangement, (ii) Sequences that are the reverse complementary strands of foreign sequences Includes, an inserted sequence, (b) A 5'UTR sequence and a 3'UTR sequence downstream of the 5'UTR sequence, wherein the 5'UTR sequence or the 3'UTR sequence contains a binding site for a human ORF protein. Includes, A method by which an insertion sequence is incorporated into the cell's genome.

2. The method according to claim 1, wherein the 5'UTR sequence or the 3'UTR sequence contains a binding site for human ORF2p.

3. A method for incorporating a nucleic acid sequence into the genome of immune cells, comprising the step of introducing recombinant mRNA or an mRNA-encoding vector, wherein the mRNA is (a) an insertion sequence comprising (i) a foreign sequence or (ii) a reverse complementary sequence of a foreign sequence, (b) A 5'UTR sequence and a 3'UTR sequence located downstream of the 5'UTR sequence, wherein the 5'UTR sequence or the 3'UTR sequence includes an endonuclease binding site and / or a reverse transcriptase binding site. Includes, A method for integrating a transgene sequence into the genome of immune cells.

4. A method for incorporating a nucleic acid sequence into the genome of a cell, comprising the step of introducing recombinant mRNA or an mRNA-encoding vector, wherein the mRNA (a) an insertion sequence comprising (i) a foreign sequence or (ii) a reverse complementary sequence of a foreign sequence, (b) A 5'UTR sequence, a sequence of a human retrotransposon downstream of the 5'UTR sequence, and a 3'UTR sequence downstream of the human retrotransposon sequence, wherein the 5'UTR sequence or the 3'UTR sequence includes an endonuclease binding site and / or a reverse transcriptase binding site. Includes, The sequence of the human retrotransposon encodes two proteins translated from a single RNA containing two ORFs. A method by which an insertion sequence is incorporated into the cell's genome.

5. The method according to claim 3 or 4, wherein the 5'UTR sequence or 3'UTR sequence includes an ORF2p binding site.

6. The method according to claim 2 or 5, wherein the ORF2p binding site is a poly(A) sequence in the 3'UTR sequence.

7. The method according to any one of claims 1 to 3, wherein the mRNA comprises a sequence of a human retrotransposon.

8. The method according to claim 7, wherein the sequence of the human retrotransposon is downstream of the 5'UTR sequence.

9. The method according to claim 7 or 8, wherein the sequence of the human retrotransposon is upstream of the 3'UTR sequence.

10. The method according to any one of claims 7 to 9, wherein the sequence of a human retrotransposon codes for two proteins translated from a single RNA containing two ORFs.

11. The method according to claim 4 or 10, wherein the two ORFs are non-overlapping ORFs.

12. The method according to claim 4, 10, or 11, wherein the two ORFs are ORF1 and ORF2.

13. The method according to claim 12, wherein ORF1 codes ORF1p and ORF2 codes ORF2p.

14. The method according to any one of claims 4 to 13, wherein the sequence of the human retrotransposon includes the sequence of a non-LTR retrotransposon.

15. The method according to any one of claims 4 to 13, wherein the sequence of the human retrotransposon includes the LINE-1 retrotransposon.

16. The method according to claim 15, wherein the LINE-1 retrotransposon is a human LINE-1 retrotransposon.

17. The method according to any one of claims 4 to 16, wherein the sequence of the human retrotransposon comprises a sequence encoding an endonuclease and / or reverse transcriptase.

18. The method according to claim 17, wherein the endonuclease and / or reverse transcriptase is ORF2p.

19. The method according to claim 17, wherein the reverse transcriptase is a group II intron reverse transcriptase domain.

20. The method according to claim 17, wherein the endonuclease and / or reverse transcriptase is minke whale endonuclease and / or reverse transcriptase.

21. The method according to any one of claims 4 to 16 or 20, wherein the sequence of the human retrotransposon includes a sequence encoding ORF2p.

22. The method according to claim 21, wherein the insertion sequence is integrated into the genome at a poly-T site using the specificity of the endonuclease domain of ORF2p.

23. The method according to claim 22, wherein the polyT portion includes the sequence TTTTTA.

24. The method according to any one of claims 4 to 23, wherein (i) the sequence of the human retrotransposon includes a sequence encoding ORF1p, (ii) the mRNA does not include a sequence encoding ORF1p, or (iii) the mRNA includes a substitution sequence of the ORF1p-encoding sequence having a 5' UTR sequence derived from a complementary gene.

25. The method according to any one of claims 1 to 24, wherein the mRNA comprises a first mRNA molecule encoding ORF1p and a second mRNA molecule encoding an endonuclease and / or reverse transcriptase.

26. The method according to any one of claims 1 to 24, wherein the mRNA is an mRNA molecule comprising a first sequence encoding ORF1p and a second sequence encoding an endonuclease and / or reverse transcriptase.

27. The method according to claim 26, wherein a first sequence encoding ORF1p and a second sequence encoding an endonuclease and / or reverse transcriptase are separated by a linker sequence.

28. The method according to claim 27, wherein the linker sequence includes an internal ribosome entry sequence (IRES).

29. The method according to claim 28, wherein IRES is IRES derived from CVB3 or EV71.

30. The method according to claim 27, wherein the linker sequence encodes a self-cleaving peptide sequence.

31. The method according to claim 27, wherein the linker sequence codes for a T2A, E2A, or P2A sequence.

32. The method according to any one of claims 1 to 31, wherein the sequence of the human retrotransposon comprises a sequence encoding ORF1p fused with an additional protein sequence and / or a sequence encoding ORF2p fused with an additional protein sequence.

33. The method according to claim 32, wherein ORF1p and / or ORF2p fuse with the nuclear retained sequence.

34. The method according to claim 33, wherein the nuclear retained sequence is an Alu sequence.

35. The method according to claim 32, wherein ORF1p and / or ORF2p are fused with the MS2 coat protein.

36. The method according to any one of claims 1 to 35, comprising at least one, two, three, or more MS2 hairpin sequences, each containing a 5'UTR sequence or a 3'UTR sequence.

37. The method according to any one of claims 17 to 36, wherein the 5'UTR sequence or 3'UTR sequence comprises a sequence that promotes or enhances the interaction between the polyA tail of mRNA and an endonuclease and / or reverse transcriptase.

38. The method according to any one of claims 17 to 37, wherein the 5'UTR sequence or 3'UTR sequence comprises a sequence that promotes or enhances the interaction between poly(A)-binding protein (PABP) and endonuclease and / or reverse transcriptase.

39. The method according to any one of claims 17 to 38, wherein the 5'UTR sequence or 3'UTR sequence includes a sequence that enhances the specificity of the endonuclease and / or reverse transcriptase to mRNA compared to another mRNA expressed by the cell.

40. Claims 1 to 3, wherein the 5'UTR sequence or 3'UTR sequence includes an Alu element sequence. The method described in any one of item 2.

41. The method according to any one of claims 26 to 40, wherein a first sequence encoding ORF1p and a second sequence encoding an endonuclease and / or reverse transcriptase have the same promoter.

42. The method according to any one of claims 24 to 41, wherein the inserted sequence has a promoter different from the promoter of the first sequence encoding ORF1p.

43. The method according to any one of claims 17 to 42, wherein the inserted sequence has a promoter different from the promoter of a second sequence encoding an endonuclease and / or reverse transcriptase.

44. The method according to any one of claims 26 to 43, wherein the first sequence encoding ORF1p and / or the second sequence encoding an endonuclease and / or reverse transcriptase has a promoter or transcription start site selected from the group consisting of an inducible promoter, a CMV promoter or transcription start site, a T7 promoter or transcription start site, an EF1a promoter or transcription start site, and combinations thereof.

45. The method according to any one of claims 1 to 44, wherein the inserted sequence has a promoter or transcription start site selected from the group consisting of an inducible promoter, a CMV promoter or transcription start site, a T7 promoter or transcription start site, an EF1a promoter or transcription start site, and combinations thereof.

46. The method according to any one of claims 26 to 45, wherein a first sequence encoding ORF1p and a second sequence encoding an endonuclease and / or reverse transcriptase are codon-optimized for expression in human cells.

47. The method according to any one of claims 1 to 46, wherein the mRNA comprises a WPRE element.

48. The method according to any one of claims 1 to 47, wherein the mRNA comprises a selection marker.

49. The method according to any one of claims 1 to 48, wherein the mRNA includes a sequence encoding an affinity tag.

50. The method according to claim 49, wherein the affinity tag is linked to a sequence encoding an endonuclease and / or reverse transcriptase.

51. The method according to any one of claims 1 to 50, wherein the 3'UTR contains a polyA sequence, or the polyA sequence is added to the mRNA in vitro.

52. The method according to claim 51, wherein the polyA sequence is downstream of a sequence encoding an endonuclease and / or reverse transcriptase.

53. The method according to claim 51 or 52, wherein the insertion sequence is upstream of the poly-A sequence.

54. The method according to any one of claims 1 to 53, wherein the 3'UTR sequence includes an insertion sequence.

55. Claims that the inserted sequence includes a sequence which is the reverse complementary strand of the sequence encoding the foreign polypeptide. The method described in any one of items 1 to 54.

56. The method according to any one of claims 1 to 55, wherein the inserted sequence includes a polyadenylation site.

57. The method according to any one of claims 1 to 56, wherein the inserted sequence includes an SV40 polyadenylation site.

58. The method according to any one of claims 1 to 57, wherein the inserted sequence includes a polyadenylation site upstream of a sequence that is the reverse complementary chain of a sequence encoding an exogenous polypeptide.

59. The method according to any one of claims 1 to 58, wherein the insertion sequence is incorporated into the genome at a locus other than a ribosome locus.

60. The method according to any one of claims 1 to 58, wherein the insertion sequence is incorporated into a gene or a regulatory region of a gene, thereby disrupting the gene or downregulating the expression of the gene.

61. The method according to any one of claims 1 to 58, wherein the insertion sequence is incorporated into a gene or a regulatory region of a gene, thereby upregulating gene expression.

62. The method according to any one of claims 1 to 58, wherein the insertion sequence is incorporated into the genome and replaces a gene.

63. The method according to any one of claims 1 to 62, wherein the insertion sequence is stably incorporated into the genome.

64. The method according to any one of claims 1 to 63, wherein the inserted sequence retrotransfers into the genome.

65. The method according to any one of claims 1 to 64, wherein the insertion sequence is incorporated into the genome by cleavage of the DNA strand at a target site by an mRNA-encoded endonuclease.

66. The method according to any one of claims 1 to 65, wherein the insertion sequence is incorporated into the genome via reverse transcription (TPRT) using a target as a primer.

67. The method according to any one of claims 1 to 65, wherein the insertion sequence is incorporated into the genome via reverse splicing of mRNA to a DNA target site in the genome.

68. The method according to any one of claims 1 or 4 to 67, wherein the cells are immune cells.

69. The method according to claim 3 or 68, wherein the immune cells are T cells or B cells.

70. The method according to claim 3 or 68, wherein the immune cells are bone marrow cells.

71. The method according to claim 3 or 68, wherein the immune cells are selected from the group consisting of monocytes, macrophages, dendritic cells, dendritic progenitor cells, and macrophage progenitor cells.

72. The method according to any one of claims 1 to 71, wherein the mRNA is self-integrated mRNA.

73. The method according to any one of claims 1 to 72, comprising the step of introducing mRNA into cells.

74. The method according to any one of claims 1 to 72, comprising the step of introducing a vector encoding mRNA into a cell.

75. The method according to any one of claims 1 to 74, comprising the step of introducing mRNA or an mRNA-encoding vector into a cell in ex vivo.

76. The method according to claim 75, further comprising the step of administering the cells to a human subject.

77. The method according to any one of claims 1 to 74, comprising the step of administering mRNA or an mRNA-encoding vector to a human subject.

78. The method according to claim 76 or 77, wherein no immune response is induced in human subjects.

79. The method according to claim 76 or 77, wherein the mRNA or vector is substantially non-immunogenic.

80. The method according to any one of claims 1 to 79, wherein the vector is a plasmid or a viral vector.

81. The method according to any one of claims 1 to 79, wherein the vector comprises a non-LTR retrotransposon.

82. The method according to any one of claims 1 to 79, wherein the vector comprises a human L1 element.

83. The method according to any one of claims 1 to 79, wherein the vector comprises the L1 retrotransposon ORF1 gene.

84. The method according to any one of claims 1 to 79, wherein the vector comprises the L1 retrotransposon ORF2 gene.

85. The method according to any one of claims 1 to 79, wherein the vector comprises an L1 retrotransposon.

86. The method according to any one of claims 1 to 85, wherein the mRNA is at least about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 kilobases.

87. The method according to any one of claims 1 to 86, wherein the mRNA is at most about 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5 kilobases.

88. The method according to any one of claims 1 to 87, wherein the mRNA comprises a sequence that inhibits or prevents the degradation of the mRNA.

89. The method according to claim 88, wherein the sequence that inhibits or prevents mRNA degradation inhibits or prevents mRNA degradation by an exonuclease or RNAse.

90. The method according to claim 88, wherein the sequence that inhibits or prevents mRNA degradation is a G4 structure, a pseudoknot, or a triple-stranded sequence.

91. The method according to claim 88, wherein the sequence that inhibits or prevents mRNA degradation is an exoribonuclease-resistant RNA structure derived from flavivirus RNA or an ENE element derived from KSV.

92. The method according to claim 88, wherein a sequence that inhibits or prevents mRNA degradation inhibits or prevents mRNA degradation by deadenylase.

93. The method according to claim 88, wherein the sequence that inhibits or prevents the degradation of mRNA includes a non-adenosine nucleotide inside or at the end of the poly-A tail of the mRNA.

94. The method according to claim 88, wherein a sequence that inhibits or prevents the degradation of mRNA improves the stability of mRNA.

95. The method according to any one of claims 1 to 94, wherein the foreign sequence includes a sequence encoding a foreign polypeptide.

96. The method according to claim 95, wherein the sequence encoding the foreign polypeptide is not in frame with the sequence encoding the endonuclease and / or reverse transcriptase.

97. The method according to claim 95 or 96, wherein the sequence encoding the foreign polypeptide is not in frame with the sequence encoding the endonuclease and / or reverse transcriptase.

98. The method according to any one of claims 95 to 97, wherein the foreign sequence does not contain an intron.

99. The method according to any one of claims 95 to 98, wherein the foreign sequence comprises a sequence encoding a foreign polypeptide selected from the group consisting of enzymes, receptors, transport proteins, structural proteins, hormones, antibodies, contractile proteins, and storage proteins.

100. The method according to any one of claims 95 to 98, wherein the foreign sequence comprises a sequence encoding a foreign polypeptide selected from the group consisting of chimeric antigen receptors (CARs), ligands, antibodies, receptors, and enzymes.

101. The method according to any one of claims 1 to 94, wherein the foreign sequence includes a regulatory sequence.

102. The method according to claim 101, wherein the regulatory sequence includes a cis-acting regulatory sequence.

103. The method according to claim 101, wherein the regulatory sequence includes a cis-acting regulatory sequence selected from the group consisting of enhancers, silencers, promoters, or response elements.

104. The method according to claim 101, wherein the regulatory sequence includes a trans-acting regulatory sequence.

105. The method according to claim 101, wherein the regulatory sequence includes a trans-acting regulatory sequence encoding a transcription factor.

106. The method according to any one of claims 1 to 105, wherein the incorporation of the insertion sequence does not adversely affect the health of the cells.

107. The method according to any one of claims 1 to 106, wherein the endonuclease, reverse transcriptase, or both are capable of site-specific insertion of the insertion sequence.

108. The method according to any one of claims 1 to 107, wherein the mRNA comprises a sequence encoding an additional nuclease domain or a nuclease domain not derived from ORF2.

109. The method according to any one of claims 1 to 107, wherein the mRNA comprises a sequence encoding a megaTAL nuclease domain, a TALEN domain, a Cas9 domain, a zinc finger binding domain derived from the R2 retroelement, or a DNA binding domain that binds to a repetitive sequence such as Rep78 derived from AAV.

110. The method according to any one of claims 17 to 109, wherein the endonuclease comprises a mutation that reduces the activity of the endonuclease compared to an endonuclease without the mutation.

111. The method according to claim 110, wherein the endonuclease is ORF2p endonuclease and the mutation is S228P.

112. The method according to any one of claims 17 to 111, wherein the mRNA comprises a sequence encoding a domain that improves the accuracy and / or processing ability of the reverse transcriptase.

113. The method according to any one of claims 17 to 111, wherein the reverse transcriptase is a reverse transcriptase having higher accuracy and / or processing ability compared to a reverse transcriptase derived from a retroelement other than ORF2, or a reverse transcriptase of ORF2p.

114. The method according to claim 113, wherein the reverse transcriptase is a group II intronic reverse transcriptase.

115. The method according to claim 114, wherein the group II intron reverse transcriptase is a group IIA intron reverse transcriptase, a group IIB intron reverse transcriptase, or a group IIC intron reverse transcriptase.

116. The method according to claim 114, wherein the group II intron reverse transcriptase is TGIRT-II or TGIRT-III.

117. The method according to any one of claims 1 to 116, wherein the mRNA comprises a sequence including an Alu element and / or a ribosome-binding aptamer.

118. The method according to any one of claims 1 to 117, wherein the mRNA comprises a sequence encoding a polypeptide including a DNA-binding domain.

119. The method according to any one of claims 1 to 118, wherein the 3'UTR sequence is derived from a viral 3'UTR or a beta-globin 3'UTR.

120. A composition comprising recombinant mRNA or a vector encoding mRNA, wherein the mRNA is (i) Human LINE-1 transposon 5'UTR sequence, (ii) The sequence encoding ORF1p located downstream of the human LINE-1 transposon 5'UTR sequence, (iii) ORF linker sequence downstream of the sequence encoding ORF1p, (iv) A sequence encoding ORF2p downstream of the ORF interlinker sequence, and (v) A 3'UTR sequence derived from a human LINE-1 transposon downstream of the sequence encoding ORF2p. It contains a human LINE-1 transposon sequence, A composition in which the 3'UTR sequence includes an insertion sequence, the insertion sequence being the reverse complementary chain of a sequence encoding a foreign polypeptide or the reverse complementary chain of a sequence encoding a foreign regulatory element.

121. The composition according to claim 120, wherein the insertion sequence is incorporated into the cell's genome when introduced into a cell.

122. The composition according to claim 121, wherein the insertion sequence is incorporated into a gene associated with a condition or disease, thereby disrupting the gene or downregulating its expression.

123. The composition according to claim 121, wherein the insertion sequence is incorporated into a gene, thereby upregulating gene expression.

124. The composition according to claim 121, wherein the mRNA comprises a sequence having at least 80% sequence identity with a sequence selected from the group consisting of sequence numbers 35 to 50.

125. The composition according to claim 121, wherein recombinant mRNA or an mRNA-encoding vector is isolated or purified.

126. A composition comprising (a) a long-chain scattered repeat sequence (LINE) polypeptide comprising human ORF1p and human ORF2p, and (b) a nucleic acid comprising an insertion sequence which is the reverse complementary chain of a sequence encoding a foreign polypeptide or the reverse complementary chain of a sequence encoding a foreign regulatory element, and which is substantially non-immunogenic.

127. The composition according to claim 126, comprising human ORF1p and human ORF2p proteins.

128. The composition according to claim 126 or 127, comprising ribonucleoprotein (RNP) containing human ORF1p and human ORF2p that form a complex with the nucleic acid.

129. The composition according to claim 127 or 128, wherein the nucleic acid is mRNA.

130. A composition comprising cells comprising the composition according to any one of claims 120 to 129.

131. The composition according to claim 130, wherein the cells are immune cells.

132. The composition according to claim 131, wherein the immune cells are T cells or B cells.

133. The composition according to claim 131, wherein the immune cells are bone marrow cells.

134. The composition according to claim 131, wherein the immune cells are selected from the group consisting of monocytes, macrophages, dendritic cells, dendritic progenitor cells, and macrophage progenitor cells.

135. The composition according to any one of claims 120 to 134, wherein the inserted sequence is the reverse complementary chain of a sequence encoding an exogenous polypeptide, and the exogenous polypeptide is a chimeric antigen receptor (CAR).

136. A pharmaceutical composition comprising the composition according to any one of claims 120 to 135 and a pharmaceutically acceptable excipient.

137. A pharmaceutical composition according to claim 136 for use in gene therapy.

138. A pharmaceutical composition according to claim 136 for use in the manufacture of a pharmaceutical for treating a disease or condition.

139. A pharmaceutical composition according to claim 136 for use in treating a disease or condition.

140. A method for treating a disease in a subject, comprising the step of administering the pharmaceutical composition according to claim 136 to a subject having the disease or condition.

141. The method according to claim 140, for increasing the amount or activity of a protein or functional RNA in a target.

142. The method according to claim 140 or 141, wherein the subject has an insufficient amount or activity of protein or functional RNA.

143. The method according to claim 142, wherein an insufficient amount or activity of a protein or functional RNA is associated with or causes a disease or condition.

144. The method according to any one of claims 140 to 143, further comprising the step of administering a drug that inhibits the human silencing hub (HUSH) complex, a drug that inhibits FAM208A, or a drug that inhibits TRIM28.

145. The method according to claim 144, wherein the agent inhibiting the human silencing hub (HUSH) complex is an agent inhibiting perifilin, TASOR, and / or MPP8.

146. The method according to claim 144, wherein a drug that inhibits the human silencing hub (HUSH) complex inhibits the assembly of the HUSH complex.

147. The method according to any one of claims 140 to 146, further comprising the step of administering a drug that inhibits the Fanconi anemia complex.

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