Target DNA integration by a lentiviral vector and its use

Abstract

The present invention relates to a composition of lentiviral particles comprising a mutant integrase and an RNA-guided nuclease for target gene insertion.

Description

Technical Field

[0001] The present invention relates to the fields of gene editing and gene therapy, particularly targeted gene insertion.

Background Art

[0002] Pathological genetic defects can range from a few bases to large deletions. Targeted DNA integration of gene-sized fragments in the mammalian genome can enable the precise addition of therapeutic messages and new functions. Precise gene delivery methods based on non-homologous end joining (NHEJ), such as homology independent targeted integration (HITI), have been developed. This method has been demonstrated for insertions of several kilobases, but remains inefficient for very large edits. HITI can function to deliver exons, but may not be efficient enough to robustly deliver the cDNA of genes such as DMD (about 14 kb) or ABCA4 (about 6.8 kb). Recently, HITI has been extended to improve efficiency for DNA by fusion to a DNA binding domain. Precise gene delivery in bacteria has been demonstrated using CRISPR programmable transposons, but this technology is not yet available for mammalian cells.

[0003] The development of new methods for genome editing has enabled the expansion of human gene therapy (HGT) applications. Many HGT strategies are based on the addition of DNA payloads that can compensate for genetic defects and / or confer synthetic characteristics to recipient cells. Delivery of therapeutic payloads has conventionally been based on viral and non-viral vectors that integrate uncontrollably across the genome. While accurate delivery technologies have emerged, virus vector-mediated uncontrolled delivery has benefited a significant number of patients. After insertional mutagenesis events were observed and leukemia cases were associated with retrovirus-based SCID-X1 gene therapy, the first attempts at gene therapy trials were aborted. Vector-driven clonal expansion was recently observed again in leukemia cases associated with CAR-T therapy and the retrovirus-based drug Strimvelis. rAAV may cause insertional mutations and induce clonal expansion. Technologies that do not use viruses, such as PiggyBac transposase (PB), have been associated with carcinogenesis induction. Lentiviral vectors are therapeutically used for uncontrolled insertion of transgenes for both ex vivo and in vivo applications. The inventors have pointed out that by mutating the integrase protein and incorporating SpCas9 nuclease into lentiviral particles, the lentiviral vector can be reprogrammed to accurately integrate transgenic DNA.

[0004] The discovery and implementation of accurate delivery strategies using tools such as ZFN, TALEN, and CRISPR-Cas9 can potentially avoid unintended gene disruption or activation and can simultaneously perform gene inactivation and addition, which are important features for therapeutic genome modification. Such technologies have also raised concerns regarding safety. It has been reported that payload integration can be inaccurate, resulting in various unexpected integration outcomes such as donor integration at off-target cleavage sites and genomic rearrangements such as inversions or translocations.

[0005] Therefore, there is a need for an accurate and safe gene delivery strategy, especially for the delivery of very large arrays, which is not being met.

Summary of the Invention

[0006] The present invention relates to (i) a nucleic acid encoding a lentiviral vector having reduced integration activity, optionally comprising an aptamer-binding protein, (ii) a nucleic acid encoding a polypeptide or protein comprising an RNA-guided nuclease or nickase, (iii) a nucleic acid encoding a guide RNA (gRNA) fused to an aptamer, (iv) a nucleic acid encoding a transgene of interest, (v) optionally, · a first protein comprising or consisting of a GAG polyprotein, · a second protein comprising or consisting of an aptamer-binding protein, and · optionally, a third protein comprising or consisting of a POL polyprotein and a nucleic acid encoding a fusion protein comprising and a composition comprising

[0007] In some embodiments, the aptamer-binding protein is preferably an MS2 bacteriophage coat protein (MCP) sharing at least 75% identity with SEQ ID NO: 61, and the aptamer is preferably an MS2 RNA tetraloop-binding sequence sharing at least 75% identity with SEQ ID NO: 63.

[0008] In some embodiments, the RNA-guided nuclease or nickase is not fused to an integrase.

[0009] In some embodiments, the RNA-guided nuclease or nickase is a Cas protein.

[0010] In some embodiments, the RNA-guided nuclease or nickase is bound to a mutant hyperactive PiggyBac transposase.

[0011] In some embodiments, the integrase contained in the integrase of (i) and / or the POL protein of (v), if present, comprises at least one amino acid mutation at a position selected from the group consisting of positions 10, 11, 13, 64, 94, 116, 117, 119, 120, 122, 124, 128, 152, 164, 168, 170, 185, 186, 231, 264, 266 of the HIV-1 integrase of SEQ ID NO: 1 or the corresponding position in another integrase.

[0012] In some embodiments, the integrase of (i) and / or (v) comprises at least one amino acid mutation selected from the group consisting of D10K, E11K, E13K, D64A, D64E, G94D, G94E, G94R, G94K, D116A, D116E, N117D, N117E, N117R, N117K, S119A, S119P, S119T, S119G, S119D, S119E, S119R, S119K, N120D, N120E, N120R, N120K, T122K, T122I, T122V, T122A, T122R, A124D, A124E, A124R, A124K, A128T, E152A, E152D, D164N, Q168L, Q168A, E170G, F185K, K186E, R231G, R231K, R231D, R231E, R231S, K264R, K266R, and K273R of the HIV-1 integrase of SEQ ID NO: 1 or the amino acid mutation at the corresponding position in another integrase.

[0013] In some embodiments, the integration activity of the lentiviral vector of (i) is reduced or abolished by at least one mutation in the integrase sequence selected from the group consisting of D116N substitution, D164N substitution, insertion of a premature stop codon, and deletion of the integrase gene (ΔIN), numbered based on the HIV-1 integrase of SEQ ID NO: 1.

[0014] The present invention relates to (i) a step of transfecting lentivirus-producing cells cultured in a suitable culture medium with the composition according to the present invention; (ii) a step of recovering lentivirus particles in the culture medium of the lentivirus-producing cells and also relates to a method for producing lentivirus particles comprising the above steps.

[0015] The present invention also relates to a population of lentivirus particles obtained by the method for producing lentivirus particles according to the present invention.

[0016] The present invention relates to a. a lentivirus protein and / or gene or a part thereof having a reduced integration activity of the integrase of the lentivirus, b. an RNA-guided nuclease or nickase, preferably a Cas9 protein or a nucleic acid sequence encoding the same, c. an aptamer, preferably a guide RNA fused to at least one MS2 RNA tetraloop binding sequence, and d. optionally, (1) a first protein comprising or consisting of a GAG polyprotein, (2) a second protein comprising or consisting of an aptamer-binding protein, and optionally (3) a third protein comprising or consisting of a POL polyprotein, or a nucleic acid sequence encoding the same and also relates to a population of lentivirus particles comprising or consisting of the above lentivirus particles.

[0017] The present invention also relates to an in vitro method for site-specific integration of a target transgene into the genome of a cell, which comprises a step of infecting the cell with a population of lentivirus particles according to the present invention, wherein the population of lentivirus particles contains a target transgene or the target transgene is delivered to the cell before, simultaneously with or after the population of lentivirus particles.

[0018] The invention also relates to a population of lentiviral particles according to the invention for use as a medicament.

[0019] The invention also relates to a population of lentiviral particles according to the invention for use in the site-specific integration of a transgene of interest into the genome of a cell, wherein the population of lentiviral particles comprises the transgene of interest or the transgene of interest can be delivered to the cell before, simultaneously with or after the population of lentiviral particles.

[0020] The invention also relates to a population of lentiviral particles according to the invention for use in the treatment of a disease, preferably a genetic disease, in a subject in need thereof, wherein the population of lentiviral particles comprises the transgene of interest or the transgene of interest can be delivered to the cell before, simultaneously with or after the population of lentiviral particles.

[0021] Definitions In the present disclosure, any use of the singular forms “a”, “an” or “the” includes not only a single referent but also a plurality of referents unless the context clearly indicates otherwise. Thus, for example, reference to “a medicament” includes a single medicament and a plurality of such medicaments.

[0022] "Nucleic acid (sequence)" and "nucleotide sequence" can be used interchangeably to refer to any molecule consisting of or containing monomeric nucleotides. The nucleic acid may be an oligonucleotide or a polynucleotide, which may be DNA, RNA, or a mixture thereof. It may be chemically modified, i.e., artificial, for example, it includes peptide nucleic acid (PNA), morpholino, and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Each of these nucleic acids is distinguished from naturally occurring DNA or RNA by changes in the molecular backbone. Phosphorothioate nucleotides may also be used. Other deoxynucleotide analogs include, but are not limited to, methylphosphonate, phosphoramidate, phosphorodithioate, N3’P5’ phosphoramidate, and oligoribonucleotide phosphorothioate, as well as their 2’O-allyl analogs and 2’O-methyl ribonucleotide methylphosphonates that can be used in the nucleic acids of the present disclosure.

[0023] "Polypeptide", "peptide", "protein", and "amino acid sequence" are used interchangeably to refer to a polymer of amino acid residues. Unless specified otherwise, the polymer of amino acid residues may be of any length. The term also applies to amino acid polymers in which one or more of the amino acids are chemical analogs or modified derivatives of the corresponding naturally occurring amino acids.

[0024] "Nuclease" refers to an enzyme that catalyzes the hydrolysis of nucleic acids within a nucleic acid sequence. Nuclease activity can cleave single-stranded or double-stranded nucleic acid molecules, which may be DNA or RNA.

[0025] "Cas9" or "Cas9 nuclease" refers to an RNA-guided nuclease that includes the Cas9 protein or a fragment thereof (e.g., a protein containing the active or inactive DNA cleavage domain of Cas9 and / or the gRNA binding domain of Cas9). Cas9 nuclease is sometimes also referred to as casn1 nuclease or CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. CRISPR is an adaptive immune system that provides defense against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). The CRISPR cluster contains sequences complementary to spacers, i.e., precursor mobile elements, and targets invading nucleic acids. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). Correct processing of pre-crRNA in type II CRISPR systems requires a trans-encoded small RNA (tracrRNA), the endogenous ribonuclease 3 (rnc), and the Cas9 protein. TracrRNA functions as a guide for processing assisted by ribonuclease 3 of pre-crRNA. Subsequently, the Cas9 / crRNA / tracrRNA complex cleaves linear or circular dsDNA targets complementary to the spacer by endonuclease. The target strand not complementary to crRNA is first cleaved by endonuclease and then trimmed by exonuclease in the 3'-5' direction. Essentially, both a protein and both RNAs are typically required for DNA binding and cleavage. However, a single guide RNA ("sgRNA" or simply "gRNA") can be engineered to incorporate both sides of crRNA and tracrRNA into a single RNA species. Cas9 recognizes a short motif in the CRISPR repeat sequence (PAM or protospacer adjacent motif) to help distinguish self from non-self. The Cas9 nuclease sequence and structure are well known to those skilled in the art. Cas9 orthologs have been described in various species including, but not limited to, Streptococcus pyogenes and Thermophilus.Additional suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on the present disclosure, and such Cas9 nucleases and sequences include, for example, the Cas9 sequences from the organisms and loci disclosed in Chylinski et al., 2013. (RNA Biol. 10(5):726-37), the entire content of which is incorporated herein by reference. In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain. The nuclease-inactivated Cas9 protein can be interchangeably referred to as the "dCas9" (the "inactive form" of Cas9 with respect to the nuclease) protein. Methods for producing a Cas9 protein (or a fragment thereof) having an inactive DNA cleavage domain are known in the art (see Jinek et al., 2012. Science. 337(6096):816-821; Qi et al., 2013. Cell. 152(5):1173-83, the entire content of each of which is incorporated herein by reference).

[0026] "Exogenous" refers to any molecule that is not naturally present in a cell or organism of interest but can be introduced therein by one or more genetic, biochemical, or other means. The natural presence of a molecule in a cell or organism may also be determined with respect to a particular developmental stage and its environmental conditions. Thus, for example, a molecule that is present only during the development of a muscle embryo is an exogenous molecule with respect to adult muscle cells. Similarly, a molecule induced by heat shock is an exogenous molecule with respect to cells that have not been heat shocked. Exogenous molecules can include, for example, a functional version of a dysfunctional endogenous molecule or a dysfunctional version of a normally functioning endogenous molecule. In contrast, the term "endogenous" refers to any molecule that is normally present in a cell or organism at a particular developmental stage under specific environmental conditions.

[0027] "Fusion protein" refers to a single-chain hybrid polypeptide containing two or more amino acid sequences fused to each other (i.e., from two or more different proteins and / or peptides). The two or more amino acid sequences can be fused to each other directly via a peptide bond or indirectly via a peptide linker. The fusion protein may be encoded in particular entirely by a single nucleic acid sequence.

[0028] "Gene" typically refers to a DNA region encoding a protein (i.e., the coding region). The term may also include a DNA region that does not itself encode a protein (i.e., a non-coding region). Examples of the latter include regions transcribed into functional non-coding RNA molecules (such as transfer RNA, ribosomal RNA, regulatory RNA, etc.). Other non-coding regions function as regulatory elements controlling the transcription and translation of the coding region (i.e., regulatory elements), or as structural elements (such as scaffold / matrix attachment regions), as origins of DNA replication, as centromeres or telomeres, etc. Regulatory elements include, but are not limited to, promoter sequences, terminators, translation control sequences (such as ribosome binding sites [RBS] and internal ribosome entry sites [IRES]), enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

[0029] "Eukaryotic" refers to a cell that is not a bacterial cell (prokaryotic cell) or an organism consisting of such cells. Eukaryotic cells include, but are not limited to, fungal cells, plant cells, and animal cells (such as mammalian cells and human cells).

[0030] "Transfection" and all its inflected forms refer to the introduction of one or several nucleic acid molecules (DNA and / or RNA) into one or more cells by non-viral means, whether in vitro or in vivo. Methods for transfection are well known in the art and include, for example, lipofection and electroporation.

[0031] "Transfection" and all its inflected forms refer to the introduction of one or more nucleic acid molecules (DNA and / or RNA) into one or more cells using a viral vector carrier, such as, but not limited to, a retrovirus (including lentivirus), an adenovirus, a virus including an adeno-associated virus (AAV), a viral particle or a viral vector, and vectors derived therefrom.

[0032] "Cleavage" refers to the breaking of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand cleavage and double-strand cleavage are possible, and double-strand cleavage may result from two separate single-strand cleavage events. DNA cleavage may be due to the generation of either blunt ends or staggered ends.

[0033] "Specificity" refers to the ability to selectively bind to a sequence that shares a degree of sequence identity with a selected sequence.

[0034] "Insertion" and "integration" refer to the addition of a nucleic acid sequence to a second nucleic acid sequence or to a genome or a part thereof. An insertion may be a "specific", "site-specific", "targeted" or "on-target" insertion, and these adjectives define the insertion of a nucleic acid into a specific site of a second nucleic acid or of a genome or a part thereof (i.e., a site intentionally selected for insertion). Conversely, the adjectives "random", "non-targeted" or "off-target" refer to non-specific and / or unintended insertion of a nucleic acid into an undesired site. The term "total" or "overall" refers to the total number of insertions.

[0035] "Mutation" refers to the substitution of a residue by another residue within a sequence, e.g., within a nucleic acid or amino acid sequence, and / or the deletion or insertion of one or more residues within a nucleic acid or amino acid sequence. Mutations are typically described herein by identifying the original residue, then the position of the residue within the sequence, and then the identity of the newly substituted residue. Various methods for making amino acid substitutions (mutations) provided herein are well known in the art and are provided, for example, by Green & Sambrook, 2012 ("Molecular cloning: a laboratory manual" (4th edition). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0036] "Integrase" refers to an enzyme produced by the genome of a retrovirus (e.g., HIV) that catalyzes the integration of a nucleic acid sequence into the genome of a host cell. In contrast to a transiently expressed nucleic acid sequence such as a transfected plasmid, an integrated nucleic acid sequence is stably expressed by the cell independent of mitotic events and is replicated along with the rest of the genome.

[0037] "Transposase" refers to an enzyme that binds to the ends of a transposon and catalyzes its movement to another part of the genome by a cut-and-paste mechanism or a replicative transposition mechanism.

[0038] "Mutant" means, with respect to a sequence (e.g., an amino acid sequence or a nucleic acid sequence), that the sequence is different from a reference sequence such as a wild-type sequence. Typically, a mutant sequence contains at least one of a substitution, addition, or deletion of one or several residues as compared to a reference sequence such as the corresponding wild-type sequence.

[0039] "Linker" refers to a chemical group or molecule that connects two adjacent molecules or moieties.

[0040] "Identity" or "identical," when used in the context of the relationship between two or more amino acid sequences or two or more nucleic acid sequences, refers to the degree of sequence relatedness between amino acid sequences or nucleic acid sequences determined by the number of matches between the sequences of two or more amino acid residues or nucleic acid residues. "Identity" is a measure of the percentage of identity between the shorter sequences of two or more sequences with gap alignment (if any) handled by a specific mathematical model or computer program (i.e., "algorithm"). The identity of related amino acid sequences or nucleic acid sequences can be easily calculated by known methods.Such methods include, but are not limited to, those described in Lesk A.M. (1988). "Computational molecular biology: Sources and methods for sequence analysis". New York, NY: Oxford University Press; Smith D.W. (1993). "Biocomputing: Informatics and genome projects". San Diego, CA: Academic Press; Griffin A.M. & Griffin H.G. (1994). "Computer analysis of sequence data, Part 1". Totowa, NJ: Humana Press; von Heijne G. (1987). "Sequence analysis in molecular biology: treasure trove or trivial pursuit". San Diego, CA: Academic press; Gribskov M.R. & Devereux J. (1991). "Sequence analysis primer". New York, NY: Stockton Press; Carillo et al., 1988. SIAM J Appl Math. 48(5):107 - 382. Preferred methods for determining identity are designed to give the maximum match between the sequences being tested. Methods for determining identity are described in publicly available computer programs.Preferred computer program-based methods for determining identity between two arrays include the GCG program package, including GAP (Genetics Computer Group, University of Wisconsin, Madison, Wisconsin; Devereux et al., 1984. Nucleic Acids Res. 12(1 Pt 1):387-95), BLASTP, BLASTN, and FASTA (Altschul et al., 1990. J Mol Biol. 215(3):403-10). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al., NCB / NLM / NIH, Bethesda, Maryland 20894). Identity may also be determined using the well-known Smith Waterman algorithm.

[0041] "Subject" refers to a mammal, preferably a human. The subject may be a "patient" who is waiting to receive or is currently receiving medical care, or who has been or is currently or will be the subject of a medical treatment, or who is being monitored for the occurrence of a disease, i.e., a warm-blooded animal, more preferably a human. The term "mammal" as used herein refers to any mammal, including, for example, humans, domestic animals, zoo animals, sports animals, or pet animals such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Preferably the mammal is a primate, more preferably a human.

[0042] "Treatment", "alleviation", "cure" and all their inflected forms refer to therapeutic treatments excluding preventive measures, the purpose of which is to (partially or completely) delay, alleviate, arrest or even reverse the targeted pathological condition or disease. Those in need of treatment include those already suffering from the disease and those suspected of having the disease. The subject shows a successful "treatment" for the targeted pathological condition or disease when there is an observable and / or measurable decrease or absence of one or more symptoms associated with the pathological condition or disease after treatment, i.e., to some extent, alleviation, reduction in morbidity and mortality and / or improvement in quality of life problems. The above parameters for evaluating the success of treatment and the improvement of the disease are easily measurable by the usual procedures with which physicians are familiar.

[0043] "Prevention" and all its inflected forms refer to preventive measures, the purpose of which is to reduce the probability that a subject will develop a given pathological condition or disease over a given period. Such a reduction may be reflected, for example, in a delay in the onset of at least one symptom of the pathological condition or disease in the subject.

DETAILED DESCRIPTION OF THE INVENTION

[0044] The present invention relates to (i) a nucleic acid encoding a lentiviral vector having reduced integration activity, optionally comprising an aptamer-binding protein, (ii) a nucleic acid encoding a polypeptide or protein comprising an RNA-guided nuclease or nickase, (iii) a nucleic acid encoding a guide RNA (gRNA) fused to an aptamer, (iv) a nucleic acid encoding a transgene of interest, (v) optionally, · a first protein comprising or consisting of a GAG polyprotein, · a second protein comprising or consisting of an aptamer-binding protein, and · optionally, a third protein comprising or consisting of a POL polyprotein A nucleic acid encoding a fusion protein comprising and a composition comprising the same.

[0045] In some embodiments, the nucleic acid of the composition is deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a combination thereof. In some embodiments, the nucleic acid of the composition is DNA. In some embodiments, the nucleic acid of the composition is RNA.

[0046] In some embodiments, the nucleic acid of the composition comprises or consists of natural nucleotides, unnatural nucleotides, or a combination thereof. In some embodiments, the nucleic acid of the composition comprises or consists of natural nucleotides. In some embodiments, the natural nucleotides are selected from the group consisting of or comprising adenine, guanine, cytosine, thymine, and uracil. In some embodiments, the natural nucleotides are selected from the group consisting of or comprising adenine, guanine, cytosine, and thymine. In some embodiments, the natural nucleotides are selected from the group consisting of or comprising adenine, guanine, cytosine, and uracil. In some embodiments, the nucleic acid of the composition comprises or consists of unnatural nucleotides (e.g., chemically modified ones).

[0047] In some embodiments, the nucleic acid of the composition is single-stranded, double-stranded, or a combination thereof. In some embodiments, the nucleic acid of the composition is single-stranded. In some embodiments, the nucleic acid of the composition is double-stranded.

[0048] In some embodiments, the nucleic acid of the composition is selected from the group consisting of or comprising a plasmid, messenger RNA (mRNA), naked DNA, cosmid, fosmid, prokaryotic chromosome (e.g., bacterial artificial chromosome), eukaryotic chromosome (e.g., yeast artificial chromosome or human artificial chromosome), or combinations thereof. In some embodiments, the nucleic acid of the composition is a plasmid, mRNA, or a combination thereof. In some embodiments, the nucleic acid of the composition is a plasmid. In some embodiments, the nucleic acid of the composition is mRNA.

[0049] In some embodiments, each nucleic acid of the composition is contained within its own vector (e.g., plasmid, cosmid, fosmid, chromosome, etc.). Alternatively, two nucleic acids of the composition may be contained within a single vector. For example, the nucleic acids of (i) and (ii), or (i) and (iii), or (i) and (iv), or (i) and (v), or (ii) and (iii), or (ii) and (iv), or (ii) and (v), or (iii) and (iv), or (iii) and (v), or (iv) and (v) may be contained within a single vector. Alternatively, three nucleic acids of the composition may be contained within a single vector. For example, the nucleic acids of (i) and (ii) and (iii), or (i) and (ii) and (iv), or (i) and (ii) and (v), or (i) and (iii) and (iv), or (i) and (iii) and (v), or (i) and (iv) and (v), or (ii) and (iii) and (iv), or (ii) and (iii) and (v), or (ii) and (iv) and (v), or (iii) and (iv) and (v) may be contained within a single vector. Alternatively, four nucleic acids of the composition may be contained within a single vector. For example, the nucleic acids of (i) and (ii) and (iii) and (iv), or (i) and (ii) and (iii) and (v), or (i) and (ii) and (iv) and (v), or (i) and (iii) and (iv) and (v), or (ii) and (iii) and (iv) and (v) may be contained within a single vector. Alternatively, five nucleic acids of the composition may be contained within a single vector.

[0050] In some embodiments, the nucleic acid of the composition comprises at least one coding sequence and optionally at least one non-coding sequence, such as a control sequence or an origin of replication sequence.

[0051] In some embodiments, the nucleic acid of the composition encodes a protein (including polyproteins and pre-polypeptides that are cleaved and / or modified after translation), a nucleic acid, or a combination thereof. In other words, the nucleic acid of the composition comprises at least one expression cassette.

[0052] The proteins or nucleic acids encoded by the nucleic acids of the composition are further described herein.

[0053] According to the present invention, the composition comprises a lentiviral vector having reduced integration activity.

[0054] As used herein, the expression "reduced integration activity" means that the integrase of the lentiviral vector has reduced or lost integration activity compared to a wild-type lentiviral vector. Preferably, the lentiviral vector has lost integration activity compared to a wild-type lentiviral vector. To achieve reduced or lost integration activity, the integrase of the lentiviral vector has been genetically modified, for example, by one or several amino acid substitutions that change its integration activity, or by partial or complete deletion of the integrase coding sequence.

[0055] In some embodiments, the integrase of the lentiviral vector comprises at least one amino acid mutation compared to wild-type (WT) integrase (e.g., when compared to wild-type HIV-1 integrase having SEQ ID NO: 1). As used herein, the term "at least one" means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. As used herein, the term "amino acid mutation" encompasses substitutions, additions, deletions, etc.

[0056] Unless otherwise specified, the numbering of amino acid residues herein is based on the sequence of HIV-1 integrase having SEQ ID NO:1. One of ordinary skill in the art can readily determine the corresponding positions in the sequences of other integrases, for example, by sequence alignment. SEQ ID NO:1: HIV-1 integrase FLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASCDKCQLKGEAMHGQVDCSPGIWQLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTVHTDNGSNFTSTTVKAACWWAGIKQEFGIPYNPQSQGVIESMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIVDIIATDIQTKELQKQITKIQNFRVYYRDSRDPVWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDED

[0057] In some embodiments, the integrase of the lentiviral vector comprises at least one amino acid mutation at a position selected from the group consisting of or comprising 10, 11, 13, 64, 94, 116, 117, 119, 120, 122, 124, 128, 152, 164, 168, 170, 185, 186, 231, 264, 266 and 273.

[0058] In some embodiments, the integrase of the lentiviral vector comprises at least one amino acid mutation selected from the group consisting of or consisting of E10K, E11K, E13K, D64V, D64A, D64E, G94D, G94E, G94R, G94K, D116N, D116A, D116E, N117D, N117E, N117R, N117K, S119A, S119P, S119T, S119G, S119D, S119E, S119R, S119K, N120D, N120E, N120R, N120K, T122K, T122I, T122V, T122A, T122R, A124D, A124E, A124R, A124K, A128T, E152V, E152A, E152D, D164N, Q168L, Q168A, E170G, F185K, K186Q, K186E, R231G, R231K, R231D, R231E, R231S, K264R, K266R and K273R.

[0059] In some embodiments, the integrase of the lentiviral vector comprises at least one amino acid mutation at a position selected from the group consisting of or consisting of 10, 13, 64, 116, 119, 152, 164, 186 and 231. In some embodiments, the integrase of the lentiviral vector comprises at least one amino acid mutation selected from the group consisting of or consisting of E10K, E13K, D64V, D64A, D64E, D116N, D116A, D116E, S119A, S119P, S119T, S119G, S119D, S119E, S119R, S119K, E152V, E152A, E152D, D164N, K186Q, K186E, R231G, R231K, R231D, R231E and R231S.

[0060] In some embodiments, the integrase of the lentiviral vector comprises at least one amino acid mutation selected from the group consisting of or consisting of E10K, E13K, D64V, D116N, S119G, E152V, D164N, K186Q and R231G.

[0061] In some embodiments, the integrase of the lentiviral vector comprises at least one mutation that interferes with its interaction with p75. In some embodiments, the integrase of the lentiviral vector comprises at least one amino acid mutation selected from the group consisting of or comprising the double mutations E10K / E13K and K186Q. In some embodiments, the integrase of the lentiviral vector comprises the amino acid mutations E10K and E13K, and typically the integrase of the lentiviral vector has the amino acid sequence of SEQ ID NO: 2. In some embodiments, the integrase of the lentiviral vector comprises the amino acid mutation K186Q, and typically the integrase of the lentiviral vector has the amino acid sequence of SEQ ID NO: 8.

[0062] In some embodiments, the integrase of the lentiviral vector comprises at least one mutation in its catalytic site. In some embodiments, the integrase of the lentiviral vector comprises at least one amino acid mutation selected from the group consisting of or comprising D64V, D116N, and E152V. In some embodiments, the integrase of the lentiviral vector comprises the amino acid mutation D64V, and typically the integrase of the lentiviral vector has the amino acid sequence of SEQ ID NO: 3. In some embodiments, the integrase of the lentiviral vector comprises the amino acid mutation D116N, and typically the integrase of the lentiviral vector has the amino acid sequence of SEQ ID NO: 4. In some embodiments, the integrase of the lentiviral vector comprises the amino acid mutation E152V, and typically the integrase of the lentiviral vector has the amino acid sequence of SEQ ID NO: 6.

[0063] In some embodiments, the integrase of the lentiviral vector comprises at least one mutation that retargets viral integration away from gene-dense regions. In some embodiments, the integrase of the lentiviral vector comprises at least one amino acid mutation selected from the group consisting of or comprising S119G and R231G. In some embodiments, the integrase of the lentiviral vector comprises the amino acid mutation S119G, and typically the integrase of the lentiviral vector has the amino acid sequence of SEQ ID NO: 5. In some embodiments, the integrase of the lentiviral vector comprises the amino acid mutation R231G, and typically the integrase of the lentiviral vector has the amino acid sequence of SEQ ID NO: 9.

[0064] In some embodiments, the integrase of the lentiviral vector comprises at least one amino acid mutation at a position selected from the group consisting of or comprising 94, 117, 119, 120, 122, 124, and 231. In some embodiments, the integrase of the lentiviral vector comprises at least one amino acid mutation selected from the group consisting of or comprising G94D, G94E, G94R, G94K, N117D, N117E, N117R, N117K, S119A, S119P, S119T, S119G, S119D, S119E, S119R, S119K, N120D, N120E, N120R, N120K, T122K, T122I, T122V, T122A, T122R, A124D, A124E, A124R, A124K, R231G, R231K, R231D, R231E, and R231S.

[0065] In some embodiments, the integrase of the lentiviral vector comprises at least one amino acid mutation at a position selected from the group consisting of or comprising 264, 266, and 273. In some embodiments, the integrase of the lentiviral vector comprises at least one amino acid mutation selected from the group consisting of or comprising K264R, K266R, and K273R.

[0066] In some embodiments, the integrase of the lentiviral vector comprises at least one amino acid mutation at a position selected from the group consisting of 10, 13, 64, 116, 128, 152, 168, and 170. In some embodiments, the integrase of the lentiviral vector comprises at least one amino acid mutation selected from the group consisting of E10K, E13K, D64A, D64E, D116A, D116E, A128T, E152A, E152D, Q168L, Q168A, and E170G.

[0067] In some embodiments, the integrase of the lentiviral vector comprises at least one amino acid mutation at position 168. In some embodiments, the integrase of the lentiviral vector comprises at least one amino acid mutation Q168L or Q168A.

[0068] In some embodiments, the integrase of the lentiviral vector comprises at least one mutation selected from the group consisting of D116N substitution, D164N substitution, insertion of a premature stop codon into its coding sequence, and deletion of its coding sequence (ΔIN).

[0069] In some embodiments, the integrase of the lentiviral vector has the amino acid sequence of SEQ ID NO: 4. In some embodiments, the integrase of the lentiviral vector has the amino acid sequence of SEQ ID NO: 68.

[0070] In some embodiments, the nucleic acid sequence encoding the integrase of the lentiviral vector comprises a premature stop codon (i.e., UAA, UAG, or UGA). In some embodiments, the resulting integrase is truncated and non-functional. In some embodiments, the resulting integrase is degraded, for example, by the cell's quality control system.

[0071] In some embodiments, the nucleic acid sequence encoding integrase is deleted from the pol gene of the lentiviral vector, which is also referred to as "ΔIN".

[0072] In some embodiments, the lentiviral vector encodes at least one lentiviral polyprotein selected from the group consisting of POL, GAG, and ENV. In some embodiments, the lentiviral vector encodes GAG and POL.

[0073] In some embodiments, the lentiviral vector comprises a lentiviral vector genome comprising at least one gene selected from the group consisting of gag and pol. In some embodiments, the lentiviral vector comprises a lentiviral vector genome comprising at least one gene selected from the group consisting of gag, pol, and vpr. In some embodiments, the lentiviral vector comprises a lentiviral vector genome comprising at least one gene selected from the group consisting of gag, pol, env, and vpr. In some embodiments, the lentiviral vector comprises a lentiviral vector genome comprising at least one gene selected from the group consisting of gag, pol, env, vpr, vpu, vif, nef, tat, and rev.

[0074] In some embodiments, the lentiviral vector is derived from human immunodeficiency virus type 1 (HIV-1) or human immunodeficiency virus type 2 (HIV-2). In some embodiments, the lentiviral vector is derived from HIV-1.

[0075] In some embodiments, the lentiviral vector further comprises at least one non-lentiviral protein or a nucleic acid encoding the same, particularly at least one non-lentiviral surface protein. In some embodiments, the non-lentiviral protein can bind to the surface proteins of eukaryotic cells, preferably mammalian cells, more preferably human cells.

[0076] According to the present invention, a lentiviral vector having reduced integration activity optionally contains an aptamer-binding protein.

[0077] In some embodiments, the aptamer-binding protein is fused to the GAG polyprotein of the lentiviral vector. In some embodiments, the C-terminus of the aptamer-binding protein is optionally fused to the N-terminus of the GAG polyprotein via a linker. In some embodiments, the N-terminus of the aptamer-binding protein is optionally fused to the C-terminus of the GAG polyprotein via a linker.

[0078] In some embodiments, the aptamer-binding protein is fused to the POL polyprotein of the lentiviral vector. In some embodiments, the C-terminus of the aptamer-binding protein is optionally fused to the N-terminus of the POL polyprotein via a linker. In some embodiments, the N-terminus of the aptamer-binding protein is optionally fused to the C-terminus of the POL polyprotein via a linker.

[0079] In some embodiments, the aptamer-binding protein is selected from the group consisting of or comprising the MS2 bacteriophage coat protein (MCP), the PP7 coat protein (PCP), the λN22 peptide, and COM.

[0080] In some embodiments, the aptamer-binding protein is the MS2 bacteriophage coat protein (MCP).

[0081] In some embodiments, the MCP has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 61 (e.g., that encoded by the nucleic acid sequence having SEQ ID NO: 60). SEQ ID NO: 60: MS2 bacteriophage coat protein - DNA coding sequence atggcttcaaactttactcagttcgtgctcgtggacaatggtgggacaggggatgtgacagtggctccttctaatttcgctaatggggtggcagagtggatcagctccaactcacggagccaggcctacaaggtgacatgcagcgtcaggcagtctagtgcccagaagagaaagtataccatcaaggtggaggtccccaaagtggctacccagacagtgggcggagtcgaactgcctgtcgccgcttggaggtcctacctgaacatggagctcactatcccaattttcgctaccaattctgactgtgaactcatcgtgaaggcaatgcaggggctcctcaaagacggtaatcctatcccttccgccatcgccgctaactcaggtatctac SEQ ID NO: 61: MS2 bacteriophage coat protein MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY

[0082] In some embodiments, the aptamer-binding protein is preferably an MS2 bacteriophage coat protein (MCP) that shares at least 75% identity with SEQ ID NO: 61, and the aptamer is preferably an MS2 RNA tetraloop-binding sequence that shares at least 75% identity with SEQ ID NO: 63.

[0083] According to the present invention, the composition comprises a nucleic acid encoding a polypeptide or protein comprising an RNA-guided nuclease or nickase.

[0084] In some embodiments, the RNA-guided nuclease or nickase is a nuclease protein comprising an active DNA cleavage domain and a guide RNA binding domain.

[0085] In some embodiments, the RNA-guided nuclease or nickase is a Cas protein, including but not limited to Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Campylobacter jejuni Cas9 (CjCas9), or variants thereof, nickase Cas9 (nCas9), inactive Cas9 (dCas9), Cas12a protein, Cas12b protein, Cas12f protein, Cpf1 protein, or CasX protein (including variants and functional fragments thereof).

[0086] In some embodiments, the RNA-guided nuclease or nickase is a Cas9 protein (including variants and functional fragments thereof).

[0087] In some embodiments, the RNA-guided nuclease or nickase is a Cas9 protein.

[0088] In some embodiments, the Cas9 protein has at least 80%, 90%, 95%, 99%, or at least 100% amino acid sequence identity with the sequence of a protein selected from the group consisting of Streptococcus pyogenes Cas9 (SpCas9) of SEQ ID NO: 34, Staphylococcus aureus Cas9 (SaCas9) of SEQ ID NO: 35, Cpf1 of SEQ ID NO: 36, Campylobacter jejuni Cas9 (CjCas9) of SEQ ID NO: 37, Streptococcus pyogenes Cas9 nickase (nCas9) of SEQ ID NO: 38, and Staphylococcus aureus Cas9 nickase of SEQ ID NO: 39.

[0089] In some embodiments, the Cas9 protein has at least 80%, 90%, 95%, 99%, or at least 100% amino acid sequence identity with the sequence of a protein selected from the group consisting of Staphylococcus aureus Cas9 (SaCas9) of SEQ ID NO: 34 and Streptococcus pyogenes Cas9 (SpCas9) of SEQ ID NO: 35.

[0090] In some embodiments, the Cas9 protein is Staphylococcus aureus Cas9 (SaCas9) of SEQ ID NO: 34. In some embodiments, the Cas9 protein is Streptococcus pyogenes Cas9 (SpCas9) of SEQ ID NO: 35.

[0091] In some embodiments, the Cas9 variant comprises the amino acid sequence of a Cas9 protein having one or several amino acid substitutions.

[0092] In some embodiments, the RNA-guided nuclease or nickase is a variant or a functional fragment of the Cas9 protein.

[0093] In some embodiments, the Cas9 variant is a humanized Cas9 (hCas9) or a functional fragment thereof. As used herein, the term "humanized Cas9" or "hCas9" refers to a sequence-optimized Cas9 protein for human cells. In some embodiments, the hCas9 protein has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with SEQ ID NO: 40.

[0094] In some embodiments, the RNA-guided nuclease or nickase is a CasX protein. In some embodiments, CasX has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with SEQ ID NO: 41.

[0095] In some embodiments, the RNA-guided nuclease or nickase is a deadCas9 protein. In some embodiments, deadCas9 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with SEQ ID NO: 42.

[0096] In some embodiments, the RNA-guided nuclease or nickase is a TnpB (transposase B from transposon PsiTn554) protein. In some embodiments, TnpB has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with SEQ ID NO: 43.

[0097] In some embodiments, the RNA-guided nuclease or nickase is a Cas12f protein. In some embodiments, the Cas12f protein is derived from the bacterium Acidibacillus sulfuroxidans (AsCas12f). In some embodiments, Cas12f has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with SEQ ID NO: 78.

[0098] In some embodiments, the RNA-guided nuclease or nickase is bound to a transposase.

[0099] In some embodiments, the RNA-guided nuclease or nickase is bound to a mutant hyperactive PiggyBac transposase (hyPB). In some embodiments, the RNA-guided nuclease or nickase is bound to a hyperactive PiggyBac transposase having at least 75% amino acid sequence identity with SEQ ID NO: 10. SEQ ID NO: 10: Hyperactive PiggyBac transposase MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKPTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMGLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKASASCKKCKKVICREHNIDMCQSCF

[0100] As used herein, the term "mutant hyPB" refers to a transposase that contains one or more amino acid substitutions, typically 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid substitutions, when compared to hyPB having the amino acid sequence of SEQ ID NO: 10. More specifically, mutant hyPB contains (i) one or more amino acid substitutions for increasing excision activity when compared to hyPB having the amino acid sequence of SEQ ID NO: 10, and / or (ii) one or more amino acid substitutions for decreasing DNA binding activity when compared to hyPB having the amino acid sequence of SEQ ID NO: 10. In some embodiments, mutant hyPB contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 10.

[0101] In some embodiments, mutant hyperactive PiggyBac contains one or more amino acid mutations for increasing excision activity.

[0102] In some embodiments, mutant hyperactive PiggyBac contains one or more amino acid mutations for increasing excision activity selected from among amino acid mutations within a region defined by amino acid position numbers [194-200], [214-222], [434-442], or [446-456], such as amino acid substitutions at positions D198, D201, R202, M212, and / or S213, wherein the position numbers correspond to the amino acid numbers of wild-type hyperactive PiggyBac of SEQ ID NO: 10.

[0103] In some embodiments, mutant hyperactive PiggyBac contains one or more amino acid mutations for increasing excision activity selected from among amino acid mutations at positions 450, 560, 564, 573, 589, 592, and / or 594, wherein the position numbers correspond to the amino acid numbers of wild-type hyperactive PiggyBac of SEQ ID NO: 10.

[0104] In some embodiments, the mutant hyperactive PiggyBac contains one or more amino acid mutations selected from amino acid mutations at positions M194 and / or D450 to increase the excision activity, wherein the position numbers correspond to the amino acid numbers of the wild-type hyperactive PiggyBac of SEQ ID NO: 10, preferably corresponding to amino acid substitutions selected from M194V and / or D450N.

[0105] In some embodiments, the mutant hyperactive PiggyBac contains one or more amino acid mutations to reduce the DNA binding activity.

[0106] In some embodiments, the mutant hyperactive PiggyBac contains one or more amino acid mutations selected from amino acid mutations at positions 254, 275, 277, 347, 372, 375 and / or 465 to reduce the DNA binding activity, wherein the position numbers correspond to the amino acid numbers of the wild-type hyperactive PiggyBac of SEQ ID NO: 10.

[0107] In some embodiments, the mutant hyperactive PiggyBac contains one or more amino acid mutations selected from R275, N347, R372, K375, R376, E377 and E380 to reduce the DNA binding activity, wherein the position numbers correspond to the amino acid numbers of the wild-type hyperactive PiggyBac of SEQ ID NO: 10.

[0108] In some embodiments, the mutant hyperactive PiggyBac contains one or more amino acid mutations selected from R372, K375, R376, E377 and E380 to reduce the DNA binding activity, wherein the position numbers correspond to the amino acid numbers of the wild-type hyperactive PiggyBac of SEQ ID NO: 10, preferably selected from amino acid substitutions of R372A, K375A, R376A, E377A and / or E380A.

[0109] In some embodiments, the mutant hyperactive PiggyBac contains one or more amino acid mutations for reducing DNA binding activity selected from among N347, R372, and K375, where the position numbers correspond to the amino acid numbers of the non-mutant hyperactive PiggyBac of SEQ ID NO: 10, preferably selected from among the amino acid substitutions N347S, N347A, R372A, K375A, and more preferably selected from among the amino acid substitutions N347S, N347A.

[0110] In some embodiments, the mutant hyperactive PiggyBac contains one or more amino acid mutations for increasing the excision activity as defined above and one or more amino acid mutations for reducing the DNA binding activity as defined above.

[0111] In some embodiments, the mutant hyperactive PiggyBac contains at least one amino acid substitution at position D450 for increasing the excision activity and at least two amino acid substitutions at positions N347, R372, and K375 for reducing the DNA binding activity. Preferably, the mutant transposase of the hyperactive PiggyBac contains the double mutation N347S and D450N or the triple mutation D450N, R372A, and K375A, where the position numbers correspond to the amino acid numbers of the non-mutant hyperactive PiggyBac of SEQ ID NO: 10. In a more preferred embodiment, the mutant transposase of the hyperactive PiggyBac contains the double mutation N347S and D450N, where the position numbers correspond to the amino acid numbers of the non-mutant hyperactive PiggyBac of SEQ ID NO: 10.

[0112] In some embodiments, the mutant hyperactive PiggyBac as disclosed in the previous embodiments further contains at least one mutation in the region defined by amino acid position numbers [158-169], such as A166S, and / or at least one mutation at positions Y527, R518, K525, N463.

[0113] Typically, the mutant hyperactive PiggyBac comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity with the mutant hyperactive PiggyBac of SEQ ID NO: 1.

[0114] In some embodiments, the mutant hyperactive PiggyBac is a variant of the hyperactive PiggyBac of SEQ ID NO: 10 having one or more amino acid substitutions, typically 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or fewer amino acid substitutions when compared to SEQ ID NO: 10.

[0115] In some embodiments, the mutant hyperactive PiggyBac further comprises one or more of the amino acid mutations at positions 34, 43, 117, 202, 230, 245, 268, 275, 277, 287, 290, 315, 325, 341, 346, 347, 350, 351, 356, 357, 388, 409, 411, 412, 432, 447, 460, 461, 465, 517, 560, 564, 571, 573, 576, 586, 587, 589, 592 and / or 594, where this position number corresponds to the amino acid number of the hyperactive PiggyBac sequence (SEQ ID NO: 10).

[0116] In some embodiments, the mutant hyperactive PiggyBac has the following mutations or combinations of mutations: V34M, T43I, Y177H, R202K, S230N, R245A, D268N, K287A, K290A, K287A / K290A, R315A, G325A, R341A, D346N, N347A, N347S, T350A, S351E, S351P, S351A, K356E, N357A, R388A, K409A, A411T, K412A, K432A, D447A, D447N, D450N, R460A, K461A, W465A, S517A, T560A, S564P, S571N, S573A, K576A, H586A, I587A, M589V, S592G or F594L, D450N / R372A / K375A, R275A / R277A, K409A / K412A, R460A / K461A, R275A / R277A / N347S / K375A / T560A / S573A / M589V / S592G and R245A / R275A / R277A / R372A / W465A.

[0117] In some embodiments, the mutant hyperactive PiggyBac has the following amino acid substitutions or combinations of amino acid substitutions: · R372A / K375A / D450N, · R372A / K375A / R376A / D450N, · K375A / R376A / E377A / E380A / D450N, · R372A / K375A / R376A / E377A / E380A / D450N, · M194V, · M194V / R372A / K375A, · S351A / R372A / K375A / R388A / D450N / W465A / S573A / M589V / S592G / F594L, · R245A / R275A / R277A / R372A / W465A / M589V, · R275A / 325A / R372A / T560A, · N347A / D450N, ·N347S / D450N / T560A / S573A / F594L, ·R202K / R275A / N347S / R372A / D450N / T560A / F594L, R275A / N347S / K375A / D450N / S592G, ·R275A / N347S / R372A / D450N / T560A / F594L, ·R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L, ·R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G, ·R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L, ·V34M / R275A / G325A / N347S / S351A / R372A / K375A / D450N / T560A / S564P, ·G325A / N347S / K375A / D450N / S573A / M589V / S592G, ·S230N / R277A / N347S / K375A / D450N, ·T43I / R372A / K375A / A411T / D450N, ·G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, and ·Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G are included, and these position numbers correspond to the amino acid numbers of the highly active PiggyBac sequence (SEQ ID NO: 10).

[0118] Preferred mutant highly active PiggyBac transposases for use according to the present disclosure include the following combinations of amino acid substitutions: ·R372A / K375A / D450N, ·S351A / R372A / K375A / R388A / D450N / W465A / S573A / M589V / S592G / F594L, · R245A / R275A / R277A / R372A / W465A / M589V, · N347A / D450N, · N347S / D450N / T560A / S573A / F594L, · R202K / R275A / N347S / R372A / D450N / T560A / F594L, · R275A / N347S / K375A / D450N / S592G, · R275A / N347S / R372A / D450N / T560A / F594L, · R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L, · R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G, · R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L, · V34M / R275A / G325A / N347S / S351A / R372A / K375A / D450N / T560A / S564P, · G325A / N347S / K375A / D450N / S573A / M589V / S592G, · S230N / R277A / N347S / K375A / D450N, · T43I / R372A / K375A / A411T / D450N, · G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, · Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G, and · R275A / 325A / R372A / T560A include mutant highly active PiggyBac, and these position numbers correspond to the amino acid numbers of the highly active PiggyBac sequence (SEQ ID NO: 10).

[0119] In some embodiments, the mutant highly active PiggyBac has the following amino acid substitutions or combinations of amino acid substitutions: ·R245A / R275A / R277A / R372A / W465A / M589V, ·R275A / 325A / R372A / T560A, ·N347A / D450N, ·N347S / D450N / T560A / S573A / F594L, ·R202K / R275A / N347S / R372A / D450N / T560A / F594L, ·R275A / N347S / K375A / D450N / S592G, ·R275A / N347S / R372A / D450N / T560A / F594L, ·R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L, ·R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G, ·R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L, ·G325A / N347S / K375A / D450N / S573A / M589V / S592G, ·S230N / R277A / N347S / K375A / D450N, ·G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, and ·Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G wherein these position numbers correspond to the amino acid numbers of the highly active PiggyBac sequence (SEQ ID NO: 10).

[0120] In a more preferred embodiment, the mutant highly active PiggyBac has the following combination of amino acid substitutions: ·N347A / D450N, · N347S / D450N / T560A / S573A / F594L, · R202K / R275A / N347S / R372A / D450N / T560A / F594L, · R275A / N347S / K375A / D450N / S592G, · R275A / N347S / R372A / D450N / T560A / F594L, · R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L, · R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G, · R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L, · G325A / N347S / K375A / D450N / S573A / M589V / S592G, · S230N / R277A / N347S / K375A / D450N, · G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, · Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G including, and these position numbers correspond to the amino acid numbers of the highly active PiggyBac sequence (SEQ ID NO: 10).

[0121] In some embodiments, the mutant transposase includes the following combination of amino acid substitutions: R372A / K375A / D450N, and the position numbers correspond to the amino acid numbers of the non-mutant highly active PiggyBac of SEQ ID NO: 10. In some embodiments, the mutant transposase has the amino acid sequence of SEQ ID NO: 11.

[0122] In some embodiments, the mutant transposase has an amino acid sequence selected from any of SEQ ID NOs: 12 - 33.

[0123] In some embodiments, the mutant transposase has an amino acid sequence selected from any of SEQ ID NO: 12 to SEQ ID NO: 20.

[0124] In some embodiments, the mutant transposase has an amino acid sequence selected from any of SEQ ID NO: 21 to SEQ ID NO: 33.

[0125] In some embodiments, the mutant transposase can include one or more mutations to hyPB involved in the conserved catalytic triad at, for example, amino acids 268 and / or 346 (such as D268N and / or D346N) corresponding to the amino acid numbering of SEQ ID NO: 10.

[0126] In some embodiments, the mutant transposase can include one or more mutations to hyPB essential for removal at, for example, amino acids 287, 287 / 290 and / or 460 / 461 (such as K287A, K287A / K290A and / or R460A / K461A) corresponding to the amino acid numbering of SEQ ID NO: 10.

[0127] In some embodiments, the mutant transposase can include one or more mutations to hyPB involved in target binding at, for example, amino acids 351, 356 and / or 379 (such as S351E, S351P, S351A and / or K356E) corresponding to the amino acid numbering of SEQ ID NO: 10.

[0128] In some embodiments, the mutant transposase can include one or more mutations to hyPB essential for integration at, for example, amino acids 560, 564, 571, 573, 589, 592 and / or 594 (such as T560A, S564P, S571N, S573A, M589V, S592G and / or F594L) corresponding to the amino acid numbering of SEQ ID NO: 10.

[0129] In some embodiments, the mutant transposase can include one or more mutations to hyPB involved in alignment at amino acids 325, 347, 350, 357 and / or 465 corresponding to the amino acid numbering of SEQ ID NO: 10 (e.g., G325A, N347A, N347S, T350A and / or W465A).

[0130] In some embodiments, the mutant transposase can include one or more mutations to well-conserved hyPB at amino acids 576 and / or 587 corresponding to the amino acid numbering of SEQ ID NO: 10 (e.g., K576A and / or I587A).

[0131] In some embodiments, the mutant transposase can include one or more mutations to hyPB involved in Zn 2+ binding at 586 corresponding to the amino acid numbering of SEQ ID NO: 10 (e.g., H586A).

[0132] In some embodiments, the programmable transposase can include one or more mutations to hyPB involved in integration at 315, 341, 372 and / or 375 corresponding to the amino acid numbering of SEQ ID NO: 10 (e.g., R315A, R341A, R372A and / or K375A).

[0133] In some embodiments, the mutant hyperactive PiggyBac comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence set forth in SEQ ID NO: 10. In some embodiments, the mutant hyperactive PiggyBac is selected for its high specificity of DNA integration into the genome as compared to wild-type PiggyBac. In some embodiments, the mutant hyperactive PiggyBac comprises an amino acid sequence having one or more of the modifications disclosed herein relative to SEQ ID NO: 10 and retains at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the sequences set forth in SEQ ID NOs: 12-33.

[0134] In some embodiments, the mutant hyperactive PiggyBac transposase can comprise one or more mutations selected from amino acids 245, 275, 277, 325, 347, 351, 372, 375, 388, 450, 465, 560, 564, 573, 589, 592, 594 corresponding to the amino acid numbering of SEQ ID NO: 10.

[0135] In some embodiments, the mutant hyperactive PiggyBac transposase mutations can comprise one or more amino acid modifications selected from R245A, R275A, R277A, R275A / R277A, G325A, N347A, N347S, S351E, S351P, S351A, R372A, K375A, R388A, D450N, W465A, T560A, S564P, S573A, M589V, S592G or F594L corresponding to the amino acid numbering of SEQ ID NO: 10.

[0136] In one embodiment, the mutant hyperactive PiggyBac transposase comprises the amino acid modification D450N corresponding to the amino acid numbering of SEQ ID NO: 10.

[0137] In one embodiment, the mutant hyperactive PiggyBac transposase comprises the amino acid modifications R245A and D450 corresponding to the amino acid numbering of SEQ ID NO: 10.

[0138] In one embodiment, the mutant hyperactive PiggyBac transposase comprises the amino acid modifications R245A, G325A, and S573P corresponding to the amino acid numbering of SEQ ID NO: 10.

[0139] In one embodiment, the mutant hyperactive PiggyBac transposase comprises the amino acid modifications R245A, G325A, D450, and S573P corresponding to the amino acid numbering of SEQ ID NO: 10.

[0140] In one embodiment, the mutant hyperactive PiggyBac transposase comprises the amino acid modification N347S or N347A corresponding to the amino acid numbering of SEQ ID NO: 10.

[0141] In one embodiment, the mutant hyperactive PiggyBac transposase comprises the amino acid modifications N347S and D450N corresponding to the amino acid numbering of SEQ ID NO: 10.

[0142] In another embodiment, the mutant hyperactive PiggyBac transposase comprises the amino acid modifications N347A and D450N corresponding to the amino acid numbering of SEQ ID NO: 10. In some embodiments, this mutant hyperactive PiggyBac transposase comprises the amino acid sequence of SEQ ID NO: 21.

[0143] In some embodiments, the mutant hyperactive PiggyBac transposase comprises SEQ ID NO: 10 of the amino acid sequence, wherein · the amino acid residue at position 34 is V or M, · the amino acid residue at position 43 is T or I, · the amino acid residue at position 177 is Y or H, · the amino acid residue at position 202 is R or K, · The amino acid residue at position 230 is S or N, · The amino acid residue at position 245 is A, · The amino acid residue at position 268 is D or N, · The amino acid residue at position 277 is R or A, · The amino acid residue at position 275 is R or A, · The amino acid residue at position 277 is R or A, · The amino acid residue at position 325 is A or G, · The amino acid residue at position 347 is S or A, · The amino acid residue at position 351 is E, P or A, · The amino acid residue at position 372 is R or A, · The amino acid residue at position 375 is K or A, · The amino acid residue at position 388 is R or A, · The amino acid residue at position 409 is K or A, · The amino acid residue at position 411 is A or T, · The amino acid residue at position 412 is K or A, · The amino acid residue at position 450 is D or N, · The amino acid residue at position 460 is R or A, · The amino acid residue at position 465 is W or A, · The amino acid residue at position 517 is S or A, · The amino acid residue at position 560 is T or A, · The amino acid residue at position 564 is P or S, · The amino acid residue at position 571 is S or N, · The amino acid residue at position 573 is S or A, · The amino acid residue at position 576 is K or A, · The amino acid residue at position 586 is H or A, · The amino acid residue at position 587 is I or A, The amino acid residue at position 589 is M or V, the amino acid residue at position 592 is G or S, and / or the amino acid residue at position 594 is L or F.

[0144] In some embodiments, the mutant hyperactive PiggyBac transposase comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% sequence identity to an amino acid sequence selected from the group consisting of or comprising SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33.

[0145] In some embodiments, the mutant transposase is not a Himar1C9 mutant.

[0146] In some embodiments, the RNA-guided nuclease or nickase and hyPB are linked to each other in the form of a fusion protein. In some embodiments, the RNA-guided nuclease or nickase and hyPB are included in a fusion protein. In some embodiments, the fusion protein further comprises a linker.

[0147] In some embodiments, the RNA-guided nuclease / nickase-hyPB fusion protein is (i) a first protein comprising or consisting of the RNA-guided nuclease or nickase described above in this specification, (ii) optionally a linker, and (iii) A second protein comprising or consisting of a transposase that is a mutant hyPB containing one or more amino acid mutations when compared to hyPB of SEQ ID NO: 10 described above in this specification Comprising or consisting of them.

[0148] In some embodiments, the RNA-guided nuclease / nickase-hyPB fusion protein contains an RNA-guided nuclease or nickase that is the Cas9 protein described above in this specification.

[0149] In some embodiments, the RNA-guided nuclease / nickase-hyPB fusion protein (i) A first protein comprising or consisting of the Cas9 protein or a variant thereof described above in this specification, (ii) Optionally a linker, and (iii) A second protein comprising or consisting of a transposase that is a mutant hyPB containing one or more amino acid mutations when compared to hyPB of SEQ ID NO: 10 described above in this specification Comprising or consisting of them.

[0150] In some embodiments, the first protein and the second protein can be oriented in the fusion protein in either order.

[0151] In some embodiments, the N-terminus of the RNA-guided nuclease or nickase is fused directly or indirectly via a linker to the C-terminus of hyPB. In another embodiment, the N-terminus of hyPB is fused directly or indirectly via a linker to the C-terminus of the RNA-guided nuclease or nickase.

[0152] In some embodiments, the RNA-guided nuclease or nickase (e.g., Cas9 protein) is fused to a lentiviral protein, preferably a structural lentiviral protein (e.g., capsid or matrix protein), to facilitate its packaging into lentiviral particles.

[0153] In some embodiments, the RNA-guided nuclease or nickase is bound to the viral protein R (VPR). In some embodiments, VPR has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% sequence identity with SEQ ID NO: 67. SEQ ID NO: 67 MEQAPEDQGPQREPYNEWTLELLEELKREAVRHFPRPWLHGLGQHIYETYGDTWTGVEAIIRILQRLLFVHFRIGCQHSRIGILRQRRARNGASRS

[0154] In some embodiments, the VPR-RNA-guided nuclease / nickase fusion protein comprises (i) a first protein comprising or consisting of VPR or a variant thereof, (ii) optionally a linker, and (iii) a second protein comprising or consisting of an RNA-guided nuclease or nickase as described hereinabove and consists of or is composed of them.

[0155] In some embodiments, the RNA-guided nuclease or nickase and VPR are bound to each other in the form of a fusion protein. In some embodiments, the RNA-guided nuclease or nickase comprised in the fusion protein is the Cas9 protein as described hereinabove.

[0156] In some embodiments, the VPR-Cas9 fusion protein (i) A first protein comprising or consisting of VPR or a variant thereof, (ii) Optionally a linker, and (iii) A second protein comprising or consisting of the Cas9 protein or a variant thereof described herein comprises or consists of these.

[0157] In some embodiments, the first protein and the second protein can be oriented in a fusion protein in either order.

[0158] In some embodiments, the N-terminus of Cas9 is fused directly or indirectly via a linker to the C-terminus of VPR. In some embodiments, the VPR-Cas9 fusion protein has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% sequence identity with SEQ ID NO: 66.

[0159] In some embodiments, the C-terminus of Cas9 is fused directly or indirectly via a linker to the N-terminus of VPR.

[0160] In some embodiments, the VPR-Cas9 fusion protein is further fused to an RNA-guided nuclease or nickase described herein.

[0161] In some embodiments, the RNA-guided nuclease or nickase is alternatively bound to the lens epithelium-derived growth factor (LEDGF), also known as P75.

[0162] In some embodiments, the P75-RNA-guided nuclease / nickase fusion protein (i) A first protein comprising or consisting of P75 or a variant thereof, (ii) Optionally a linker, and (iii) A second protein comprising or consisting of an RNA-guided nuclease or nickase described above in this specification comprising or consisting of them.

[0163] In some embodiments, the RNA-guided nuclease or nickase and P75 are linked to each other in the form of a fusion protein. In some embodiments, the RNA-guided nuclease or nickase comprised in the fusion protein is the Cas9 protein described above in this specification.

[0164] In some embodiments, the P75-Cas9 fusion protein (i) A first protein comprising or consisting of P75 or a variant thereof, (ii) Optionally a linker, and (iii) A second protein comprising or consisting of the Cas9 protein or a variant thereof described above in this specification comprising or consisting of them.

[0165] In some embodiments, the first protein and the second protein can be oriented in the fusion protein in either order.

[0166] In some embodiments, the N-terminus of Cas9 is fused to the C-terminus of P75 either directly or indirectly via a linker. In another embodiment, the C-terminus of Cas9 is fused to the N-terminus of P75 either directly or indirectly via a linker.

[0167] In some embodiments, the P75-Cas9 fusion protein has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to any one of SEQ ID NOs: 69-73. In some embodiments, the P75-Cas9 fusion protein has an amino acid sequence selected from the group consisting of or comprising SEQ ID NOs: 69-73. In some embodiments, the P75-Cas9 fusion protein has the amino acid sequence of SEQ ID NO: 69. In some embodiments, the P75-Cas9 fusion protein has the amino acid sequence of SEQ ID NO: 70. In some embodiments, the P75-Cas9 fusion protein has the amino acid sequence of SEQ ID NO: 71. In some embodiments, the P75-Cas9 fusion protein has the amino acid sequence of SEQ ID NO: 72. In some embodiments, the P75-Cas9 fusion protein has the amino acid sequence of SEQ ID NO: 73.

[0168] In some embodiments, the P75-Cas9 fusion protein is further fused to an RNA-guided nuclease or nickase described hereinabove.

[0169] In some embodiments, the RNA-guided nuclease or nickase is not fused to an integrase. In some embodiments, the RNA-guided nuclease or nickase is not fused to the integrase of the lentiviral vector of (i). In some embodiments, the RNA-guided nuclease or nickase, if present, is not fused to the integrase of the fusion protein encoded by the nucleic acid of (v).

[0170] In some embodiments, the RNA-guided nuclease or nickase is bound to an integrase or a mutant thereof as defined herein.

[0171] In some embodiments, the integrase-RNA-guided nuclease / nickase fusion protein is (i) A first protein comprising or consisting of an integrase or a mutant thereof as defined herein, (ii) Optionally a linker, and (iii) A second protein comprising or consisting of an RNA-guided nuclease or nickase as described herein comprises or consists of them.

[0172] In some embodiments, the RNA-guided nuclease or nickase and integrase or mutant thereof as defined herein are linked to each other in the form of a fusion protein. In some embodiments, the RNA-guided nuclease or nickase comprised in the fusion protein is the Cas9 protein as described herein.

[0173] In some embodiments, the integrase-Cas9 fusion protein (i) A first protein or variant thereof comprising or consisting of an integrase or a mutant thereof as defined herein, (ii) Optionally a linker, and (iii) A second protein comprising or consisting of the Cas9 protein or a variant thereof as described herein comprises or consists of them.

[0174] In some embodiments, the first protein and the second protein can be oriented in the fusion protein in either order.

[0175] In some embodiments, the N-terminus of Cas9 is fused to the C-terminus of an integrase or a mutant thereof as defined herein, either directly or indirectly via a linker. In another embodiment, the C-terminus of Cas9 is fused to the N-terminus of an integrase or a mutant thereof as defined herein, either directly or indirectly via a linker.

[0176] In some embodiments, the integrase-Cas9 fusion protein is further fused to an RNA-guided nuclease or nickase described hereinabove.

[0177] In some embodiments, the RNA-guided nuclease or nickase is bound to GAG and / or POL.

[0178] In some embodiments, the GAG and / or POL-RNA-guided nuclease / nickase fusion protein (i) comprises or consists of a first protein comprising GAG and / or POL or a variant thereof, (ii) optionally a linker, and (iii) a second protein comprising or consisting of an RNA-guided nuclease or nickase described hereinabove. comprises or consists of these.

[0179] In some embodiments, the RNA-guided nuclease or nickase and GAG and / or POL are bound to each other in the form of a fusion protein. In some embodiments, the RNA-guided nuclease or nickase comprised in the fusion protein is the Cas9 protein described hereinabove.

[0180] In some embodiments, the GAG and / or POL-Cas9 fusion protein (i) comprises or consists of a first protein comprising GAG and / or POL or a variant thereof, (ii) optionally a linker, and (iii) a second protein comprising or consisting of the Cas9 protein or a variant thereof described hereinabove. comprises or consists of these.

[0181] In some embodiments, the first protein and the second protein can be oriented in the fusion protein in either order.

[0182] In some embodiments, the N-terminus of Cas9 is fused directly or indirectly via a linker to the C-terminus of GAG and / or POL. In another embodiment, the C-terminus of Cas9 is fused directly or indirectly via a linker to the N-terminus of GAG and / or POL.

[0183] In some embodiments, the GAG and / or POL-Cas9 fusion protein is further fused to an RNA-guided nuclease or nickase as described hereinabove.

[0184] According to the present invention, the composition comprises a nucleic acid encoding a guide RNA (gRNA) fused to at least one aptamer.

[0185] As used herein, the term "at least one" means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0186] In some embodiments, the aptamer is an RNA sequence comprising a tetraloop. The term "tetraloop" is used interchangeably with the terms "stem loop" and "hairpin loop".

[0187] In some embodiments, the aptamer-binding protein described herein can bind to at least one aptamer.

[0188] In some embodiments, at least one tetraloop is an MS2 RNA tetraloop-binding sequence. In some embodiments, at least one MS2 RNA tetraloop-binding sequence has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 63 (e.g., that encoded by the DNA sequence of SEQ ID NO: 62). SEQ ID NO: 62: MS2 RNA tetraloop-binding sequence - DNA coding sequence ggggccactagggacaggatgttttagagctaggccaacatgaggatcacccatgtctgcagggcctagcaagttaaaataaggctagtccgttatcaacttggccaacatgaggatcacccatgtctgcagggccaagtggcaccgagtcggtgctttttt Sequence number 63: MS2 RNA Tetraloop binding sequence ggggccacuagggacaggauguuuuagagcuaggccaacaugaggaucacccaugucugcagggccuagcaaguuaaaauaaggcuaguccguuaucaacuuggccaacaugaggaucacccaugucugcagggccaaguggcaccgagucggugcuuuuuu

[0189] In some embodiments, the gRNA can form a complex with an RNA-guided nuclease or nickase described herein, for example, it can form a fusion protein comprising Cas9 protein or Cas9.

[0190] In some embodiments, the gRNA can target an RNA-guided nuclease or nickase described herein to a specific sequence or region of the genome of a cell, preferably a mammalian cell, more preferably a human cell. In some embodiments, the specific sequence targeted by the gRNA is adjacent to a protospacer adjacent motif (PAM) specific for the Cas9 protein.

[0191] In some embodiments, the specific sequence targeted by the gRNA may be within a safe harbor locus of the cell's genome. A "safe harbor locus" refers to a region of the cell's genome that can appropriately express the integrated material without disturbing the endogenous gene structure or function. Examples of safe harbor loci include, but are not limited to, AAVS1 (intron 1 of PPP1R12C), HPRT, HI1, hRosa26, albumin, and the F-A region. The safe harbor locus may be an exon or intron of a gene that is ubiquitously expressed and / or has tissue-specific expression (e.g., muscle). The safe harbor locus can be selected from the group consisting of exon 1, intron 1, or exon 2 of PPP1R12C, exon 1, intron 1, or exon 2 of HPRT, and exon 1, intron 1, or exon 2 of hRosa26, and intron 1 of the albumin gene. The safe harbor locus may include a region of the genome that does not contain an endogenous gene that enables the expression of the transgene inserted without inhibiting the genomic structure or function and has open chromatin.

[0192] In some embodiments, the specific sequence targeted by the gRNA may be within or very close to a gene in the cell's genome. For example, the specific sequence targeted by the gRNA is considered to be within or very close to a gene that requires gene repair or gene modification (by integration of an exogenous copy of the gene brought about by the nucleic acid encoding the transgene for the purpose of (iv)).

[0193] According to the present invention, the composition contains a nucleic acid encoding a transgene of interest.

[0194] As used herein, the term "introduced gene of interest" refers to an exogenous nucleic acid sequence that is inserted into the genome of a cell, preferably a eukaryotic cell, more preferably a mammalian cell, containing at least one open reading frame. Typically, the introduced gene of interest is a coding sequence, i.e., it encodes at least one protein or polypeptide. However, the introduced gene of interest may also be a non-coding sequence.

[0195] In some embodiments, the introduced gene of interest comprises at least one gene and at least one non-coding sequence, such as a regulatory element or a promoter.

[0196] In some embodiments, the introduced gene of interest comprises at least one gene. In some embodiments, the at least one gene is not present in the genome of the cell. In some embodiments, the at least one gene is an allele of a gene in the genome of the cell, and this allele is not endogenous to the genome of the cell.

[0197] In some embodiments, the introduced gene of interest comprises at least one gene from the same species as the cell. In another embodiment, the introduced gene of interest comprises at least one gene from a species different from the cell.

[0198] In some embodiments, the introduced gene of interest has a length of at least 500 base pairs (bp). As used herein, at least 500 means 500, 600, 700, 800, 900, 1,000, 10,000, 20,000 bp or more.

[0199] In some embodiments, the introduced gene of interest is a DNA molecule, an RNA molecule, or a combination thereof. In some embodiments, the introduced gene of interest is a DNA molecule. In some embodiments, the introduced gene of interest is an RNA molecule.

[0200] In some embodiments, the target transgene may be flanked by inverted terminal repeat (ITR) sequences. This is particularly desirable when an RNA-guided nuclease or nickase is conjugated to a transposase as described above.

[0201] According to the present invention, the composition optionally comprises a nucleic acid encoding a fusion protein comprising (1) a first protein comprising or consisting of a GAG polyprotein, (2) a second protein comprising or consisting of an aptamer-binding protein, and optionally (3) a third protein comprising or consisting of a POL polyprotein.

[0202] As used herein, the term "GAG" refers to a retroviral polyprotein. GAG polyproteins are typically processed by a viral protease (PR) after translation into MA (matrix), CA (capsid), and NC (nucleocapsid) moieties and sometimes more. For example, the GAG polyprotein of the HIV-1 virus is processed after translation into the matrix protein p17, the capsid protein p24, the nucleocapsid proteins p7 and p6-gag. In some embodiments, the fusion protein comprises a protein comprising or consisting of an HIV-1 GAG polyprotein.

[0203] As used herein, the term "POL" refers to a retroviral polyprotein. POL polyproteins are typically processed by a viral protease (PR) after translation into PR (protease), RT (reverse transcriptase), p15 (ribonuclease H), and INT (integrase) moieties. This protease itself is released by autocatalytic cleavage. In some embodiments, the fusion protein comprises a protein comprising or consisting of an HIV-1 POL polyprotein.

[0204] In some embodiments, the fusion protein comprises the POL protein, and thus the composition may include nucleic acid molecules encoding two distinct integrases, namely (i) one integrase having reduced integration activity in the lentiviral vector and (v) one integrase in the fusion protein.

[0205] In some embodiments, the aptamer-binding protein is fused to the GAG protein. In some embodiments, the C-terminus of the aptamer-binding protein is optionally fused to the N-terminus of the GAG protein via a linker. In some embodiments, the N-terminus of the aptamer-binding protein is optionally fused to the C-terminus of the GAG protein via a linker.

[0206] In some embodiments, the C-terminus of the aptamer-binding protein is optionally fused to the N-terminus of the polyprotein GAG via a linker, and typically the fusion protein has the amino acid sequence of SEQ ID NO: 64. SEQ ID NO: 64 MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYSQNYPIVQSGLRSRAQASNSAVDGTAGPGSMDSGRDFLTLHGLQDDEDLQALLKGSQLLKVKSSSWRRERFYKLQEDCKTIWQESRKVMRTPESQLFSIEDIQEVRMGHRTEGLEKFARDVPEDRCFSIVFKDQRNTLDLIAPSPADAQHWVLGLHKIIHHSGSMDQRQKLQHWIHSCLRKADKNKDNKMSFKELQNFLKELNIQPRAEFTLAARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALDKIEEEQNKSKKKAQQAAADTGHSSQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKARVLAEAMSQVTNTATIMMQRGNFRNQRKMVKCFNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSYKGRPGNFLQSRPEPTAPPFLQSRPEPTAPPEESFRSGVETTTPPQKQEPIDKELYPLTSLRSLFGNDPSSQ

[0207] In some embodiments, the aptamer-binding protein, if present, is fused to the POL polyprotein. In some embodiments, the C-terminus of the aptamer-binding protein is optionally fused, via a linker, to the N-terminus of the POL polyprotein, preferably the GAG-POL polyprotein. In some embodiments, the N-terminus of the aptamer-binding protein is optionally fused, via a linker, to the C-terminus of the POL polyprotein, preferably the GAG-POL polyprotein.

[0208] In some embodiments, the C-terminus of the aptamer-binding protein is optionally fused, via a linker, to the N-terminus of the polyprotein GAG-POL, and typically the fusion protein has the amino acid sequence of SEQ ID NO: 65. SEQ ID NO: 65

[0209] Although the aptamer-binding protein has been described above, this description applies here with the necessary modifications.

[0210] In some embodiments, if the fusion protein comprises a third protein that comprises or consists of a POL polyprotein, the integrase of the POL polyprotein may have reduced integration activity.

[0211] Although mutations including substitutions and deletions for reducing the integration activity of integrase have been described, this description applies here with the necessary modifications.

[0212] In some embodiments, the integrase of the POL polyprotein may comprise the same or different mutations as the integrase of the lentiviral vector of (i).

[0213] In some embodiments, the fusion protein comprises one or several linkers.

[0214] In some embodiments, the linker is a peptide linker. In one embodiment, the peptide linker is (GGS) n , (GGGGS) n , (G) n , (EAAAK) n , an XTEN linker and (XP) n (wherein n is independently an integer from 1 to 50) motifs and combinations thereof, and is selected from the group consisting of or consisting of any of these.

[0215] In some embodiments, the linker is 1 to 24 amino acids in length or is encoded by a nucleic acid sequence 3 to 72 nucleotides in length. In one embodiment, the linker is 1 to 12 amino acids in length or is encoded by a nucleic acid sequence 3 to 36 nucleotides in length. In one embodiment, the linker is 1 to 6 amino acids in length or is encoded by a nucleic acid sequence 3 to 18 nucleotides in length.

[0216] In some embodiments, the linker is an XTEN linker or (GGS) n linker.

[0217] In some embodiments, the linker is selected from among the linkers shown in Table 1.

Table 1

[0218] In some embodiments, the linker comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, each of which is encoded by an exemplary nucleic acid sequence of SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, or any combination thereof.

[0219] In some embodiments, the linker comprises or consists of the amino acid sequence of SEQ ID NO: 45 encoded by the exemplary nucleic acid sequence of SEQ ID NO: 44.

[0220] In some embodiments, the aptamer-binding protein is fused to the mutant integrase in any order. In some embodiments, the aptamer-binding protein is fused to the mutant integrase via the N-terminus. In some embodiments, the aptamer-binding protein is fused to the integrase via the C-terminus. In some embodiments, the N-terminus of the aptamer-binding protein is optionally fused to the C-terminus of the mutant integrase via a linker. In some embodiments, the C-terminus of the aptamer-binding protein is optionally fused to the N-terminus of the mutant integrase via a linker.

[0221] In some embodiments, the fusion protein further comprises viral protein R (VPR). In some embodiments, the aptamer-binding protein is fused to the integrase of the POL polyprotein, and the integrase of the POL polyprotein is further fused to VPR. In some embodiments, VPR has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% sequence identity with SEQ ID NO: 67. In some embodiments, the aptamer-binding protein is fused to the GAG polyprotein, and the GAG polyprotein is further fused to VPR. In some embodiments, the aptamer-binding protein is fused to the GAG-POL polyprotein, and the GAG-POL polyprotein is further fused to VPR.

[0222] In some embodiments, the N-terminus of the integrase of the POL polyprotein is optionally fused to the C-terminus of VPR via a linker, and the N-terminus of the aptamer-binding protein is optionally fused to the C-terminus of the integrase of the POL polyprotein via a linker. In some embodiments, the N-terminus of the integrase of the POL polyprotein is optionally fused to the C-terminus of the aptamer-binding protein via a linker, and the N-terminus of VPR is optionally fused to the C-terminus of the integrase of the POL polyprotein via a linker.

[0223] In some embodiments, the N-terminus of the GAG polyprotein is optionally fused to the C-terminus of VPR via a linker, and the N-terminus of the aptamer-binding protein is optionally fused to the C-terminus of the GAG polyprotein via a linker. In some embodiments, the N-terminus of the GAG polyprotein is optionally fused to the C-terminus of the aptamer-binding protein via a linker, and the N-terminus of VPR is optionally fused to the C-terminus of the GAG polyprotein via a linker.

[0224] In some embodiments, the N-terminus of the GAG-POL protein is optionally fused to the C-terminus of VPR via a linker, and the N-terminus of the aptamer-binding protein is optionally fused to the C-terminus of the GAG-POL protein via a linker. In some embodiments, the N-terminus of the GAG-POL protein is optionally fused to the C-terminus of the aptamer-binding protein via a linker, and the N-terminus of VPR is optionally fused to the C-terminus of the GAG-POL protein via a linker.

[0225] The present invention further relates to a population of lentiviral particles obtained by a method for producing the lentiviral particles described above.

[0226] In some embodiments, the population of lentiviral particles (i) the lentiviral proteins and / or genes or portions thereof described herein having reduced integrase-mediated integration activity of the lentivirus, (ii) optionally an RNA-guided nuclease or nickase, preferably a Cas9 protein or a nucleic acid sequence encoding the same, fused to a mutant hyPB and / or VPR and / or P75 and / or GAG and / or POL and / or integrase or mutants thereof described herein, (iii) a guide RNA (gRNA) fused to an aptamer described herein, preferably at least one MS2 RNA tetraloop binding sequence, and (iv) optionally a fusion protein comprising or consisting of (1) a first protein comprising or consisting of a GAG protein, (2) a second protein comprising or consisting of an aptamer-binding protein, and optionally (3) a third protein comprising or consisting of a POL protein described herein, or a nucleic acid sequence encoding the same comprising or consisting of lentiviral particles.

[0227] In some embodiments, the lentiviral particles in a population of lentiviral particles further comprise the transgene for the purpose described above.

[0228] In some embodiments, the lentiviral particles are suspended and stored in a suitable solvent, preferably a biocompatible solvent (such as biological buffers and physiological sera, etc.).

[0229] In some embodiments, the lentiviral particles are stored at a temperature suitable for preventing nucleic acid degradation. In some embodiments, the lentiviral particles are stored at a temperature included in 4°C to -200°C. As used herein, the expression "4°C to -200°C" includes 4, 3, 2, 1, 0, -1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, -18, -19, -20, -30, -40, -50, -60, -70, -80, -90, -100, -120, -140, -160, -180, -200°C. In some embodiments, the lentiviral particles are stored at a temperature included in 4°C to -80°C. In some embodiments, the lentiviral particles are stored at a temperature included in 4°C to -20°C. In some embodiments, the lentiviral particles are stored at a temperature included in 4°C to 0°C. In some embodiments, the lentiviral particles are stored at a temperature included in 0°C to -200°C. In some embodiments, the lentiviral particles are stored at a temperature included in -20°C to -200°C. In some embodiments, the lentiviral particles are stored at a temperature included in -80°C to -200°C. In some embodiments, the lentiviral particles are stored at a temperature of -196°C.

[0230] In some embodiments, the lentiviral particles are substantially pure.

[0231] In some embodiments, the lentiviral particles further comprise at least one therapeutic agent.

[0232] In some embodiments, when the lentiviral particle contains a fusion protein comprising a GAG polyprotein, an aptamer-binding protein, and optionally a POL polyprotein, this fusion protein (or the nucleic acid encoding it) and an RNA-guided nuclease or nickase (or the nucleic acid encoding it) may be contained in separate lentiviral particles.

[0233] In some embodiments, when the lentiviral particle contains a fusion protein comprising a GAG polyprotein, an aptamer-binding protein, and optionally a POL polyprotein, this fusion protein (or the nucleic acid encoding it) and an RNA-guided nuclease or nickase (or the nucleic acid encoding it) are contained in two separate lentiviral particles.

[0234] In some embodiments, a population of lentiviral particles (i) a lentiviral protein and / or gene or a part thereof as described hereinabove, having reduced integration activity of the lentiviral integrase, (ii) optionally, an RNA-guided nuclease or nickase, preferably a Cas9 protein or a nucleic acid sequence encoding the same, fused to a mutant hyPB and / or VPR and / or P75 and / or GAG and / or POL and / or integrase or a mutant thereof as described hereinabove, and (iii) a guide RNA (gRNA) fused to an aptamer as described hereinabove, preferably at least one MS2 RNA tetraloop-binding sequence comprising or consisting of a first lentiviral particle, and (i) a lentiviral protein and / or gene or a part thereof as described hereinabove, having reduced integration activity of the lentiviral integrase, and (ii) A fusion protein or a nucleic acid sequence encoding the same, comprising: (1) a first protein comprising or consisting of a GAG polyprotein, (2) a second protein comprising or consisting of an aptamer-binding protein, and optionally (3) a third protein comprising or consisting of a POL polyprotein, as described hereinabove and a second lentiviral particle comprising or consisting of them comprises.

[0235] In some embodiments, the first lentiviral particle, the second lentiviral particle, or both contain the transgene of interest described hereinabove.

[0236] In some embodiments, the population of lentiviral particles (i) a lentiviral protein and / or gene or a part thereof as described hereinabove, having reduced integration activity of the lentiviral integrase, and (ii) optionally an RNA-guided nuclease or nickase fused to a mutant hyPB and / or VPR and / or P75 and / or GAG and / or POL and / or integrase or a mutant thereof as described hereinabove, preferably a Cas9 protein or a nucleic acid sequence encoding the same and a first lentiviral particle comprising or consisting of them (i) a lentiviral protein and / or gene or a part thereof as described hereinabove, having reduced integration activity of the lentiviral integrase, (ii) a fusion protein or a nucleic acid sequence encoding the same, comprising: (1) a first protein comprising or consisting of a GAG polyprotein, (2) a second protein comprising or consisting of an aptamer-binding protein, and optionally (3) a third protein comprising or consisting of a POL polyprotein, as described hereinabove, and (iii) An aptamer described above in this specification, preferably a guide RNA (gRNA) fused to at least one MS2 RNA tetraloop binding sequence comprising or consisting of a second lentiviral particle and comprises.

[0237] In some embodiments, the first lentiviral particle, the second lentiviral particle, or both contain the transgene of interest described above in this specification.

[0238] In some embodiments, when the lentiviral particle contains a fusion protein comprising a GAG polyprotein, an aptamer-binding protein, and optionally a POL polyprotein, this fusion protein (or the nucleic acid encoding it) and an RNA-guided nuclease or nickase (or the nucleic acid encoding it) are contained in three separate lentiviral particles.

[0239] In some embodiments, the population of lentiviral particles (i) A lentiviral protein and / or gene or a part thereof described above in this specification having reduced integration activity of the lentiviral integrase, and (ii) Optionally, an RNA-guided nuclease or nickase, preferably a Cas9 protein or a nucleic acid sequence encoding it, fused to a mutant hyPB and / or VPR and / or P75 and / or GAG and / or POL and / or integrase or a mutant thereof described above in this specification comprising or consisting of a first lentiviral particle and (i) A lentiviral protein and / or gene or a part thereof described above in this specification having reduced integration activity of the lentiviral integrase, and (ii) A fusion protein or a nucleic acid sequence encoding the same, which comprises or consists of: (1) a first protein comprising or consisting of a GAG polyprotein, (2) a second protein comprising or consisting of an aptamer-binding protein, and optionally (3) a third protein comprising or consisting of a POL polyprotein, as described above in the present specification A second lentiviral particle comprising or consisting of the same, and (i) A lentiviral protein and / or gene or a part thereof as described above in the present specification, having reduced integrase-mediated integration activity of the lentivirus, and (ii) A guide RNA (gRNA) as described above in the present specification, preferably fused to an aptamer, preferably at least one MS2 RNA tetraloop-binding sequence A third lentiviral particle comprising or consisting of the same and Comprising the same.

[0240] In some embodiments, the first lentiviral particle, the second lentiviral particle, the third lentiviral particle, or two or all three of them comprise the transgene of interest as described above in the present specification.

[0241] In some embodiments, when the lentiviral particle comprises a fusion protein comprising a GAG polyprotein, an aptamer-binding protein, and optionally a POL polyprotein, this fusion protein (or the nucleic acid encoding the same) and an RNA-guided nuclease or nickase (or the nucleic acid encoding the same) are contained in four separate lentiviral particles.

[0242] In some embodiments, the population of lentiviral particles (i) A lentiviral protein and / or gene or a part thereof as described above in the present specification, having reduced integrase-mediated integration activity of the lentivirus, and (ii) An RNA-guided nuclease or nickase optionally fused to a mutant hyPB and / or VPR and / or P75 and / or GAG and / or POL and / or integrase or a mutant thereof described hereinabove, preferably a Cas9 protein or a nucleic acid sequence encoding the same A first lentiviral particle comprising or consisting of the same, and (i) A lentiviral protein and / or gene or a part thereof described hereinabove having reduced integrase-mediated integration activity, and (ii) A fusion protein comprising or consisting of (1) a first protein comprising or consisting of a GAG polyprotein, (2) a second protein comprising or consisting of an aptamer-binding protein, and optionally (3) a third protein comprising or consisting of a POL polyprotein described hereinabove, or a nucleic acid sequence encoding the same A second lentiviral particle comprising or consisting of the same, and (i) A lentiviral protein and / or gene or a part thereof described hereinabove having reduced integrase-mediated integration activity, and (ii) A guide RNA (gRNA) described hereinabove fused to an aptamer, preferably at least one MS2 RNA tetraloop-binding sequence A third lentiviral particle comprising or consisting of the same, and (i) A lentiviral protein and / or gene or a part thereof described hereinabove having reduced integrase-mediated integration activity, and (ii) A transgene of interest described hereinabove A fourth lentiviral particle comprising or consisting of the same and is included.

[0243] The present invention further provides (i) A step of transfecting lentivirus-producing cells cultured in a suitable culture medium with the composition according to the present invention, and (ii) the step of recovering lentiviral particles in the medium of the lentivirus-producing cells, and relates to a method for producing lentiviral particles, which comprises the above.

[0244] In some embodiments, the lentivirus-producing cells are transfected with 1, 2, 3, 4 or 5 nucleic acids of the present composition described above. For example, in order to produce different lentiviral particles, it is conceivable to transfect different batches of lentivirus-producing cells with different nucleic acids of the present composition described above.

[0245] The cells may be transfected by methods known in the art, and non-limiting examples include lipofection, electroporation, sonoporation, gene gun, microinjection or chemical substance-based ones such as calcium phosphate.

[0246] In some embodiments, the cells are transfected simultaneously and / or separately with the nucleic acid molecules of the present composition. In some embodiments, the cells are transfected simultaneously with the nucleic acid molecules of the present composition. In some embodiments, the cells are transfected separately with the nucleic acid molecules of the present composition.

[0247] In some embodiments, the lentiviral particles are obtained by a split system, such as a trans-complementation system (vector / packaging system), by transfecting permissive cells (e.g., HEK293T cells) in vitro with a plasmid containing components of a lentiviral vector genome comprising gag, pol, and env sequences encoding polyproteins GAG, POL, and envelope polypeptides or a portion of these polypeptides sufficient to allow the formation of retroviral particles.

[0248] Suitable cells for the production of lentiviral particles include, but are not limited to, eukaryotic and prokaryotic cells and / or cell lines. Non-limiting examples of such cells or cell lines produced from such cells include, for example, COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0, SP2 / 0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T) and perC6 cells and insect cells such as Spodoptera fugiperda (Sf) or fungal cells such as Saccharomyces, Pichia and Schizosaccharomyces.

[0249] In some embodiments, lentiviral particles are secreted by lentiviral-producing cells into their medium. In some embodiments, lentiviral particles are recovered in the culture medium of lentiviral-producing cells.

[0250] In some embodiments, lentiviral particles are recovered 12 hours to 1 week after transfection, preferably 24 to 72 hours after transfection.

[0251] In some embodiments, lentiviral particles are purified from the culture medium of lentiviral-producing cells. Purification of lentiviral particles to increase concentration can be achieved by any suitable method such as density gradient purification (e.g., cesium chloride (CsCl)), chromatography techniques (e.g., column or batch chromatography) or ultracentrifugation. For example, the lentiviral particles of the present invention can be subjected to two or three CsCl density gradient purification steps. Lentiviral particles are preferably purified from infected cells using a method that includes lysing the cells, applying the lysate to a chromatography resin, eluting the lentiviral particles from the chromatography resin, and recovering the fraction containing the lentiviral particles.

[0252] The present invention further relates to producer cells comprising (i.e., transfected therewith) the composition according to the present invention.

[0253] In some embodiments, the producer cells comprising the composition according to the present invention produce lentiviral particles. Typically, the lentiviral particles bud from the plasma membrane of the cells.

[0254] In some embodiments, the producer cells are derived from an immortalized cell line. In some embodiments, the producer cells are derived from a primary cell culture.

[0255] In some embodiments, the producer cells are eukaryotic cells. In some embodiments, the producer cells are animal or plant cells. In some embodiments, the producer cells are animal cells. In some embodiments, the producer cells are vertebrate or insect cells. In some embodiments, the producer cells are vertebrate cells. In some embodiments, the producer cells are mammalian cells.

[0256] In some embodiments, the producer cells are non-human mammalian cells (e.g., non-human primates, mice, rats). In some embodiments, the producer cells are human cells. Cultured cell lines are well known in the art (e.g., HeLa cells, HEK293 cells, and HAP-1 cells, etc.). In some embodiments, the producer cells are derived from the HEK293 cell line. In some embodiments, the producer cells are derived from the HEK293T cell line.

[0257] In some embodiments, the producer cells contain one or more mutations compared to the cell line from which they are derived. In some embodiments, the producer cells are unmodified compared to the cell line from which they are derived.

[0258] Methods for maintaining cells in culture are well known in the art. In some embodiments, the producer cells are maintained in a suitable culture medium, such as Dulbecco's Modified Eagle Medium (DMEM). In some embodiments, the producer cells are typically maintained in 95% air and 5% CO2 Culture at 37°C.

[0259] The present invention further relates to a method for site-specific integration of a target transgene into the genome of a target cell, which comprises infecting said cell with a population of the lentiviral particles described above.

[0260] In some embodiments, the method is an in vitro method.

[0261] In some embodiments, the lentiviral particles contain the transgene of interest. In some embodiments, some of the lentiviral particles in the population of lentiviral particles contain the transgene of interest.

[0262] In some embodiments, the transgene of interest is contained in one or some or all of the lentiviral particles of the population of lentiviral particles. In some embodiments, the lentiviral particles do not contain the transgene of interest to be delivered to the target cells by a suitable method such as transfection (e.g., lipofection and electroporation, etc.). In some embodiments, the transgene of interest is delivered to the target cells before the lentiviral particles. In some embodiments, the transgene of interest is delivered to the target cells simultaneously with the lentiviral particles. In some embodiments, the transgene of interest is delivered to the target cells after the lentiviral particles.

[0263] In some embodiments, the method is particularly aimed at editing the genome of the target cell in order to perform targeted insertion of the transgene of interest.

[0264] In some embodiments, target cells can be infected with the lentiviral particles of the present invention. In some embodiments, the target cells express on their extracellular surface at least one protein or a portion thereof or a protein complex that can be recognized by the lentiviral particles, particularly by the surface proteins of the lentiviral particles. In some embodiments, this at least one protein or protein complex enables the binding (or immobilization) of the lentiviral particles to the target cells and / or their entry into the interior.

[0265] In some embodiments, the target cells express CD4, CCR5, and / or CXCR4 surface proteins. Typically, CD4, CCR5, and / or CXCR4 interact with the HIV-1 surface proteins Gp120 and / or Gp41.

[0266] In some embodiments, the target cells are primary cells or cells from a cell line suitable for lentiviral infection, such as HEK293T cells, CHO cells, HSC cells, iPS cells, primary immune cells, primary T cells, primary NK cells, primary macrophages, primary hepatocytes, K-562 cells, and Jurkat-T cells.

[0267] In some embodiments, the target cells artificially express at least one surface protein that enables the binding and / or entry of the lentiviral particles. "Artificially express" means that the cells are modified or manipulated to transiently or stably express the surface protein although they do not endogenously express the surface protein.

[0268] In some embodiments, the target cells are cells isolated from an organism, preferably a mammalian organism, more preferably a human organism. In some embodiments, the target cells are myeloid cells. In some embodiments, the target cells are lymphocytes.

[0269] In some embodiments, the method is used to perform targeted gene insertion into a population of target cells extracted from an organism, maintain them in vitro, and reintroduce them into the organism. According to this embodiment, the method is said to be ex vivo.

[0270] When the contents of the lentiviral particles are delivered into the target cells, an RNA-guided nuclease or nickase (e.g., Cas9) that forms a complex with a gRNA containing at least one aptamer (e.g., an MS2 RNA tetraloop binding sequence) interacts with an aptamer-binding protein (e.g., MCP) fused to an integrase (from a fusion protein containing a lentiviral vector with reduced integration activity and / or a third protein containing or consisting of the POL polyprotein if present as described above). It should be understood that this interaction in particular occurs through an aptamer / aptamer-binding protein interaction (e.g., MS2 / MCP).

[0271] In some embodiments, the site specificity of the gRNA directs the RNA-guided nuclease or nickase / integrase complex activity towards a specific region or site in the genome of the target cell (e.g., a safe harbor locus or within or in close proximity to a gene within the genome of the target cell).

[0272] In some embodiments, the transgene of interest is inserted at a site specifically recognized by the gRNA. In some embodiments, the transgene of interest is inserted 1 to 20 (where "1 to 20" includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20) nucleotides away from the sequence specifically recognized by the gRNA.

[0273] In some embodiments, the mutant integrase activity results in targeted gene integration, also referred to as site-specific integration of the transgene of interest.

[0274] In some embodiments, the introduced gene of interest is not inserted into the coding and / or control sequences of the genome of the target cell. In some embodiments, the target gene insertion resulting from the use of the methods of the invention does not interfere with, change, or reduce the expression of one or more genes.

[0275] In some embodiments, the introduced gene of interest is inserted into the coding and / or control sequences of the genome of the target cell, resulting in partial or complete inactivation of the coding sequence.

[0276] In some embodiments, the introduced gene of interest is inserted by non-homologous end joining (NHEJ). In some embodiments, the introduced gene of interest is inserted by homologous recombination (HR).

[0277] In some embodiments, the inserted introduced gene of interest is expressed by the cell. In some embodiments, the inserted introduced gene of interest is stably expressed by the cell. In some embodiments, the expression of the inserted introduced gene of interest is under the control of a promoter or regulatory element.

[0278] In some embodiments, site-specific integration of the introduced gene of interest is controlled by sequencing the region of the genome surrounding the target site, i.e., the site recognized by the gRNA.

[0279] In some embodiments, the ratio of the number of lentiviral particles to the number of cells (multiplicity of infection, MOI) is from 0.5 MOI to 40 MOI.

[0280] The invention further relates to a population of lentiviral particles as described above for use as a medicament (+ optionally, the gene of interest if the gene of interest is not included within the lentiviral particles of the population of lentiviral particles).

[0281] The present invention further relates to a population of lentiviral particles as described above for site-specific integration of a desired transgene into the genome of a target cell (+ optionally, the desired transgene if the desired transgene is not included within the lentiviral particles of the population of lentiviral particles).

[0282] The present invention further relates to a population of lentiviral particles as described above for use in the treatment of a disease in a subject in need thereof (+ optionally, the desired transgene if the desired transgene is not included within the lentiviral particles of the population of lentiviral particles). The present invention also relates to a method for treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a population of lentiviral particles as described above (+ optionally, the desired transgene if the desired transgene is not included within the lentiviral particles of the population of lentiviral particles).

[0283] In some embodiments, the treatment of the disease is effected by site-specific integration of a desired transgene into the genome of the cells of a subject in need thereof.

[0284] In some embodiments, the disease is a genetic disease. In some embodiments, the genetic disease is characterized in that at least one gene is partially or completely repressed or inactivated or is dysfunctional. In some embodiments, the genetic disease is characterized in that at least one gene in the cells of a subject in need thereof encodes at least one non-functional protein. In some embodiments, the genetic disease is characterized in that at least one gene in the cells of a subject in need thereof encodes at least one protein or polypeptide that has an adverse effect on the cell.

[0285] In some embodiments, the disease is cancer. In some embodiments, the cancer is a hematological cancer (e.g., leukemia).

[0286] Typically, target gene insertion restores the normal expression and function of at least one gene involved in the disease.

[0287] In some embodiments, the population of lentiviral particles can be administered before, at the same time as, or after the second therapeutic agent.

Brief Description of the Drawings

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Example

[0289] The present invention is further illustrated by the following examples.

[0290] Example 1: Target DNA integration by lentiviral vector Materials and methods Plasmid constructs The following plasmids: · hCas9 (plasmid #41815), a pcDNA™ 3.3-TOPO™ plasmid expressing human codon-optimized Cas9 nuclease under the control of the CMV promoter, · psPAX2 (plasmid #12260), an empty second-generation lentiviral packaging plasmid containing the gag, pol, tat, and rev genes, · pCMV-VSV-G (plasmid #8454), a plasmid expressing the G glycoprotein of vesicular stomatitis virus under the control of the CMV promoter, · pMDLg / pRRE (plasmid #12251), a third-generation lentiviral packaging plasmid expressing HIV-1 Gag (encoding the major structural protein of the viral particle), HIV-1 Pol (encoding HIV-1 protease, HIV-1 reverse transcriptase, and HIV-1 integrase), and the HIV-1 Rev response element (RRE, the binding site for the Rev protein that facilitates the export of RNA from the nucleus), · pRSV-Rev (plasmid #12253), a third-generation lentiviral packaging plasmid expressing the Rev protein were obtained from Addgene.

[0291] Mutations were obtained in integrase using the QuikChange Lightning Multi Site Directed Mutagenesis Kit (Agilent #210513). The sequences of the integrase mutants are disclosed in SEQ ID NO: 2 to SEQ ID NO: 9.

[0292] All remaining vectors were constructed by Golden Gate Assembly using Esp3I and T4 ligase. Plasmid pSICO of SEQ ID NO: 74 (Addgene reference plasmid #41815; a common vector containing the gene of interest), pRRL dual of SEQ ID NO: 75 (a common vector containing the gene of interest), psMCP-GAG of SEQ ID NO: 76, and psMCP-GAGPOL of SEQ ID NO: 77 were used.

[0293] Cell culture hEK293T cells (ATCC CRL-3216) were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with high glucose (Gibco, Thermo Fisher), 10% fetal bovine serum, 2 mM glutamine, 100 U penicillin, and 0.1 mg / mL streptomycin.

[0294] K-562 cells (ATCC CRL-3343) and Jurkat-T cells (Clone E6-1, ATCC IB-152) were grown in RPMI 1640 medium (Gibco) supplemented with 10% FBS, 1% penicillin-streptomycin (Gibco), and 1% GlutaMAX (100×) (Gibco).

[0295] All cell lines were grown at 37 °C in 95% air and 5% CO 2 2.

[0296] Lentivirus production Lentiviral vectors were produced according to the protocol available on the Addgene website at https: / / www.addgene.org / protocols / lentivirus-production / and replicated as follows. Introduction Using this protocol, lentivirus can be produced from lentiviral vectors transfected into Lenti-X 293T cells using a polyethyleneimine (PEI) transfection protocol. This procedure can be modified for other packaging cell lines or transfection reagents. Once the lentivirus is produced, it can be used for various downstream applications such as the production of stable cell lines. Work Schedule Day 0: Seed 293T packaging cells. Day 1 (pm): Transfect packaging cells. Day 2 (am): 18 hours after transfection. Remove the medium and replace it with fresh medium. Days 3 - 4 (am): Harvest the virus. Reagent Preparation 1) DMEM Complete: 10% v / v FBS and 4 mM L-alanyl-L-glutamine Add 55 mL heat-inactivated FBS and 11 mL L-alanyl-L-glutamine (200 mM) to a 500 mL bottle of high-glucose DMEM. Store at 4°C. 2) Chloroquine diphosphate (25 mM) Dissolve 0.129 g of chloroquine diphosphate in 10 mL of sterile water. Filter-sterilize through a 0.22 μm filter. Aliquot into 50 - 100 μL aliquots and store at -20°C. An aliquot can be thawed before use and stored at 4°C. Thawed aliquots must be discarded after 1 - 2 months. 3) 1 mg / mL PEI, linear MW 25,000 Da Dissolve 100 mg of the powder in 100 mL of deionized water. While stirring, slowly add hydrochloric acid until the solution becomes clear. Check the pH of the solution. Adjust the pH to 7.0 using hydrochloric acid or sodium hydroxide. Typically, this solution will be basic and will initially require adjustment with hydrochloric acid. Mix this solution for 10 minutes and then recheck the pH to ensure it has not fluctuated. Filter this solution through a 0.22 μm membrane. Aliquot into 500 - 1000 μL aliquots into sterile tubes. Store this tube at -80 °C. After thawing, this solution can be stored at 4 °C for up to 2 months. After 2 months, discard this tube and thaw a new working stock. The optimal DNA mass:PEI mass ratio needs to be determined based on experience for each new batch of 1 mg / mL PEI and for each cell line. Procedure Seed 293T packaging cells at 3.8×10 6 cells per plate in DMEM Complete in a 10 cm tissue culture plate. Incubate the cells at 37 °C in 5% CO 2 for approximately 20 hours. Gently aspirate the medium, add 10 mL of fresh DMEM Complete containing 25 μM chloroquine diphosphate, and incubate for approximately 5 hours. For 10 mL of DMEM Complete, add 10 μL of chloroquine diphosphate (25 mM). Prepare a mixture of the three transfection plasmids. · psPAX2: 1.3 pmol · pMD2.G: 0.72 pmol · Transfer plasmid: 1.64 pmol · OptiPro SFM until a total volume of 500 μL is reached Dilute the above 500 μL of the mixture with sufficient PEI in 500 μL of PEI - OptiPro SFM so that the ratio of DNA (μg):PEI (μg) is 1:3 (total 1000 μL per 10 cm dish). When using the transfer plasmid pHAGE TRE dCas9 - KRAB (27.8 μg total μg of plasmid DNA), this results in 83.4 μL (1 mg / mL) of PEI in 416.6 μL of OptiPro SFM per 10 cm dish. Gently add the diluted PEI to the diluted DNA. While gently flicking the diluted DNA tube with your finger, add the diluted PEI dropwise. Incubate the mixture at room temperature for 15 - 20 minutes. Carefully transfer the transfection mixture to the Lenti - X 293T packaging cells. Add the transfection mixture dropwise, taking care not to push the cells away. Incubate the cells for 18 hours or until the next morning. The next morning, carefully aspirate the medium. Replace the medium with 15 mL of DMEM Complete. Incubate the cells. The virus can be harvested 48, 72, and 96 hours after transfection, either as individual harvests or as a single harvest in which all individual harvests are pooled. When pooling harvests, transfer the harvested medium to a polypropylene storage tube and store at 4 °C during collection. Centrifuge the virus supernatant at approximately 500 g for 5 minutes to pellet any packaging cells collected during harvest. Filter the supernatant through a 0.45 μm PES filter. The virus supernatant can be stored at 4 °C for several hours, but to avoid loss of titer, it must be aliquoted into aliquots as soon as possible, snap - frozen in liquid nitrogen, and stored at - 80 °C.

[0297] One day before transfection, 0.72 pmol of pCMV-VSV-G envelope plasmid, 1.64 pmol of pSICO or pRRL dual payload (transfer plasmid) and 1.30 pmol of psPAX2 plasmid (wild type or containing pol mutation) were used to produce cells in a 10 cm dish seeded with 4.9×10 6 HEK293T cells.

[0298] For nuclease packaging, 0.65 pmol of psPAX2 and 0.65 pmol of psMCP-GAG or psMCP-GAGPOL plasmid were used together with 0.65 pmol of nuclease plasmid.

[0299] For transfection, the plasmids were mixed in 500 μL of Optimem and 100 mg of polyethyleneimine (PEI). Two days after plasmid transfection, the supernatant was collected, filtered, and centrifuged overnight at 4000 g and 4 °C. The supernatant was discarded and the lentivirus particles were resuspended to achieve a 100× vector concentration.

[0300] Determination of nuclease activity For VPR / IN / LEDGF nuclease fusion activity, a Traffic Light Reporter (TLR) (Certo et al., 2011. Nat Methods. 8(8):671-676) HEK293T cell line containing an out-of-frame mCherry ORF that becomes reconstituted by NHEJ activity upon Cas9 targeting was used. Fluorescence was measured by flow cytometry using a BD LSR Fortessa instrument (561 nm yellow-green laser with a 610 / 20 filter).

[0301] Cells were transfected with gRNA in p12 cells using 240,000 cells / well, and gRNA-mediated SaCas9, AsCas12f, and TnpB editing as well as MS2 scaffolds were evaluated in HEK293T. Three days after nuclease addition, cells were pelleted and DNA was extracted by KAPPA rapid extraction. PCR amplification of each target site was performed, and sequencing was carried out by Illumina sequencing on the synthetic MiSeq™ platform using the MiSeq Reagent Kit v2 (300 cycles, 2×150 configuration).

[0302] Determination of On-Target Integration A reporter cell line (Hershey) was constructed to evaluate target integration, where on-target integration resulted in a fluorescent signal (GFP) measurable by the reconstitution of the fluorescent protein ORF.

[0303] A viral payload plasmid containing 1 / 2 Emerald GFP (emGFP) and 1 / 2 intron was packaged into a lentiviral vector using the corresponding packaging system and / or integrase mutant. A reporter cell line was constructed by randomly inserting the C-terminal (C-t) half of emGFP without a promoter preceding the splicing acceptor into the genome of HEK293T cells. A "target site" was added upstream of C-t emGFP.

[0304] For experiments, 20,000 HEK293T Hershey reporter cells were seeded at 12 wells / plate and infected with lentiviral particles the next day, simultaneously with transfection that caused emGFP reconstitution upon addition of the payload plasmid.

[0305] Lentiviruses were produced using standard protocols. Four days after infection, emGFP fluorescence signals were measured by FACS (BD LSR Fortessa instrument; blue 488 nm laser equipped with a 530 / 30 filter, yellow-green 561 nm laser equipped with a 610 / 20 filter).

[0306] Sequencing of integrated junctions The INSERT-seq method combines target amplification of integrated DNA, UMI-based correction of PCR bias, and Oxford Nanopore long-read sequencing for robust analysis of DNA integration in the genome. INSERT-seq can detect events that occur at frequencies as low as 0.1%. INSERT-seq provides complete handling of all insertions independent of repeat size.

[0307] Library preparation and sequencing DNA was extracted using the Nanobind kit (Circulomics, catalog number NB-900-001-01) and sheared into fragments of approximately 2 kb using a g-TUBE (trademark) (Covaris, catalog number 520079). The WGP primer mixture from the Nanopore PCR Barcoding Kit (SQK-PBK004) was further added to the second PCR. Sequencing was performed on a Flongle R9.4.1 flow cell to obtain a total output of approximately 300,000 reads. For calculation of the limit of detection (LOD), monoclonal samples from HEK293T cells with one true lentiviral insertion were diluted at ratios of 1 / 100, 1 / 1000, and 1 / 10,000 with WT genomic material. The dilutions were sequenced on a Flongle R9.4.1 flow cell to obtain sequencing outputs of 500,000, 500,000, and 150,000 reads, respectively. Analyses were performed according to the INSERT-seq protocol.

[0308] Analysis of integration sites Base calling was performed using Guppy 4.0.11 (available from their website https: / / community.nanoporetech.com by ONT) with nanopore raw reads.

[0309] Read quality was evaluated using NanoStats, NanoQC, and NanoPlot from NanoPack (De Coster et al., 2018. Bioinformatics. 34(15):2666 - 2669). Reads were filtered by quality (>10) and length (>200) using NanoFilt from NanoPack. Reads were clustered by UMI using a combination and adaptation of two previously published pipelines (the pipeline - umi - unitig - amplicon distributed by ONT at https: / / github.com / nanoporetech / pipeline - umi - amplicon). Clustering was performed by extracting the UMI sequences using Python scripting, and the sequences were clustered using VSearch (Rognes et al., 2016. PeerJ. 4:e2584). The consensus sequence of the clusters was obtained by performing two rounds of polishing with Racon (Vaser et al., 2017. Genome Res. 27(5):737 - 746) and two rounds of medaka (https: / / github.com / nanoporetech / medaka).

[0310] For the analysis of insertions, cutadapt (Martin, 2011. EMBnet j. 17(1):10-12) was used to filter reads for the presence of the adapters used and trim them to remove the adapters and the insert sequences from the reads. Reads were mapped to the reference genome using minimap2 (Li, 2018. Bioinformatics. 34(18):3094-3100) with the "map-ont" default parameters, filtered using Python scripting, and reads that mapped uniquely with a mapping quality of over 30 were selected. bedtools (Quinlan & Hall, 2010. Bioinformatics. 26(6):841-842) was used to return the first output in bed format containing all the mapped reads. Subsequently, a peak calling step was performed using Python scripting to filter peaks by shape. Peaks were considered to have passed the shape filter if the residual sum of squares (RSS) fitting the peak coverage to a beta distribution was less than 1.

[0311] Implementation of the optimized INSERT-seq protocol Genomic DNA was extracted, fragmented, end-repaired, A-tailed, and then ligated with an adapter containing a UMI for read clustering and a barcode for sample demultiplexing in the computational pipeline. Reads were clustered by UMI, the integrated sequences and adapters were filtered and trimmed, the reads were mapped to the reference genome, and significant peaks were reported and annotated.

[0312] Results Nuclease packaging strategy Considering that the DNA of the lentivirus is integrated in a double-strand break (DSB) state, it was inferred that targeted integration would occur by packaging functional Cas9-gRNA into lentiviral particles.

[0313] The following six different packaging system configurations: (i) Co-expression of SpCas9 during vector production, (ii) Co-expression of a fusion of SpCas9 with the previously described viral particle target protein Vpr p6-GAG interaction domain (Indikova & Indik, 2020. Nucleic Acids Res. 48(14):8178-8187), (iii) Fusion of SpCas9 with the integrase protein, (iv) Previously described fusion to VPR integrase (Montagna et al., 2018. Mol Ther Nucleic Acids. 12:453-462), (v) Fusion to the integrase-binding domain of the previously described LEDGF-p75 chromatin docking factor (Hare et al., 2009. PLoS Pathog. 5(1):e1000259), and (vi) Direct fusion to the GAGPOL polyprotein in the packaging plasmid were tested to achieve targeted integration by packaging of the SpCas9 protein into lentivirus.

[0314] The SpCas9 protein was detected in all packaging systems, but no non-homologous end joining (NHEJ) editing was detected upon administration of lentivirus to cultured cells. One hypothesis was that the lack of editing might be due to a deficiency in SpCas9 gRNA, as it was found that U6-driven expression localizes the gRNA to the nucleus and cytoplasmic gRNA expression rescues gRNA packaging in lentivirus-derived vesicles.

[0315] The MS2 stem loop containing the MCP-GAGPOL packaging fusion and SpCas9 gRNA was used to recruit the gRNA into lentiviral particles. Using this construct, the editing activity upon transduction with lentiviral particles was rescued (Figure 1A).

[0316] The efficiency of target integration was compared between lentiviral particles containing the aptamer-binding protein MS2 coat protein (MCP) fused to either the lentiviral polyprotein GAG-POL or the polyprotein GAG alone (Figure 1B). Similar target integration rates were obtained under both conditions.

[0317] Target integration was observed in Jurkat-T cells during co-lentiviral infection and Cas9 RNP delivery. Programmable lentiviruses (PILVs) were produced using four different packaging strategies. Cas9 was co-packaged with NLS (NLS-Cas9), integrase fused to viral protein R (VPRIN-Cas9), LEDGF (p75-LEDGF-Cas9), or Gag-Pol (GAGPOL-Cas9). NLS-Cas9 and VPRIN-Cas9 resulted in the highest insertion rates (Figure 1C).

[0318] Several integrase (IN) mutants were tested, and all showed better target integration performance than wild-type integrase (Figure 1D).

[0319] Finally, using the MS2 system, the proportion of NHEJ-activated cells was compared when delivering DNA payloads using an editing tool that was either Cas9 nuclease or a fusion protein containing Cas9 and a programmable transposase (PT condition) in lentiviral particles. The editing tool was also provided in either DNA, RNA, or protein (RNP) form.

[0320] Compatibility with other nucleases Considering the vast repertoire of programmable nucleases described since SpCas9, the programmable lentiviral packaging system (PILV) was tested using different programmable nucleases (AsCas12 and TnpB). Target integration was detected in all of these systems (Figure 2), indicating that this system is compatible with any nuclease enzyme.

[0321] Co-delivery with programmable transposase Using the MCP-MS2 system, either Cas9 or a fusion protein containing Cas9 and highly active PiggyBac transposase (with FiCAT, R372A / K375A / D450N substitutions) was loaded into lentiviral particles. Gene editing activity was measured in traffic light reporter HEK293T cells transduced with lentiviral particles loaded with a gene editor in either mRNA or protein (RNP) form (Figure 3).

[0322] The results showed gene editing activity for both conditions, but better results were obtained when the gene editor was delivered in protein form.

[0323] Measurement of on-target integration As shown in Figures 4B and 4C, on-target integration was measured using the INSERT-seq technique.

[0324] As shown in Figure 4A, overall and on-target integration activities were compared between wild-type lentiviral particles (WT-LV) and programmable lentiviral (PILV). PILV enabled accurate on-target integration compared to WT-LV, and it was considered an optimal system for therapeutic applications.

[0325] Example 2: Strategies for the incorporation of Cas9 into lentiviral particles To incorporate Cas9 nuclease into virus-like particles (VLPs), several nuclease expression methods in lentivirus-producing cells can be employed. In addition to overexpression and passive loading in the cytoplasm of the producing cells (Figure 5A), six other strategies for Cas9 loading were investigated. These strategies (Figure 5B) include: 1) Cas9 overexpression, 2) fusion to VPR (viral capsid protein), 3) fusion to wild-type viral integrase protein, 4) fusion to mutant viral integrase protein, 5) fusion of Cas9 to both VPR and mutant viral integrase, 6) fusion to the GAP-POL viral gene, or 7) fusion to human endogenous p75 protein.

[0326] Materials and Methods Cell Culture and Plasmid Cloning HEK293T cells (ATCC CRL-3216) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with high glucose (Gibco, Thermo Fisher), 10% fetal bovine serum, 2 mM glutamine, and 100 U penicillin, 0.1 mg / mL streptomycin.

[0327] K-562 cells (ATCC CRL-3343) and Jurkat-T cells (Clone E6-1, ATCC IB-152) were grown in RPMI 1640 medium (Gibco) supplemented with 10% FBS, 1% penicillin-streptomycin (Gibco), and 1% GlutaMAX (100×) (Gibco).

[0328] All cell lines were grown at 37°C in 95% air and 5% CO 2 2.

[0329] Additional vectors not included in Example 1 were constructed by Golden Gate Assembly using Esp3I and T4 ligase. Plasmid pRRL_Cas9_Insynth containing the codon-optimized IN of SEQ ID NO: 79 and the fusion of Cas9, and pRRL_VPR-IN186opt-Cas9 containing VPR of SEQ ID NO: 80, the fusion of mutant IN and Cas9 were used.

[0330] Lentivirus production Lentiviral vectors were produced according to the protocol available on the Addgene website at https: / / www.addgene.org / protocols / lentivirus-production / and replicated as follows. Introduction Using this protocol, lentiviruses can be produced from lentiviral vectors transfected into Lenti-X 293T cells using a polyethyleneimine (PEI) transfection protocol. This procedure can be modified for other packaging cell lines or transfection reagents. Once produced, the lentiviruses can be used for various downstream applications such as the production of stable cell lines. Work schedule Day 0: Seed 293T packaging cells Day 1 (pm): Transfect packaging cells Day 2 (am): 18 hours after transfection. Remove the medium and replace it with fresh medium Days 3 - 4 (am): Harvest the virus Reagent preparation 1. DMEM Complete: 10% v / v FBS and 4 mM L-alanyl-L-glutamine Add 55 mL of heat-inactivated FBS and 11 mL of L-alanyl-L-glutamine (200 mM) to a 500 mL bottle of high-glucose DMEM. Store at 4°C. 2. Chloroquine diphosphate (25 mM) Dissolve 0.129 g of chloroquine diphosphate in 10 mL of sterile water. Filter-sterilize with a 0.22 μm filter. Aliquot into 50 - 100 μL aliquots and store at -20 °C. Before use, thaw an aliquot and it can be stored at 4 °C. The thawed aliquot must be discarded after 1 - 2 months. 3.1 mg / mL PEI, linear MW 25,000 Da Dissolve 100 mg of the powder in 100 mL of deionized water. While stirring, slowly add hydrochloric acid until the solution becomes clear. Check the pH of the solution. Adjust the pH to 7.0 using hydrochloric acid or sodium hydroxide. Typically this solution is basic and initial adjustment with hydrochloric acid is required. Mix this solution for 10 minutes and then re-check the pH to ensure it has not changed. Filter this solution through a 0.22 μm membrane. Aliquot into 500 - 1000 μL aliquots into sterile tubes. Store these tubes at -80 °C. After thawing, this solution can be stored at 4 °C for up to 2 months. After 2 months, discard this tube and thaw a new working stock. The optimal DNA mass:PEI mass ratio needs to be determined empirically for each new batch of 1 mg / mL PEI and for each cell line. Procedure Seed 293T packaging cells at 3.8×10 6 cells per plate in DMEM Complete in a 10 cm tissue culture plate. Incubate the cells at 37 °C in 5% CO 2 for approximately 20 hours. Gently aspirate the medium and add 10 mL of fresh DMEM Complete containing 25 μM chloroquine diphosphate and incubate for approximately 5 hours. For 10 mL of DMEM Complete, add 10 μL of chloroquine diphosphate (25 mM). Prepare a mixture of three types of transfection plasmids. · psPAX2: 1.3 pmol · pMD2.G: 0.72 pmol · Transfer plasmid: 1.64 pmol · OptiPro SFM until a total volume of 500 μL Dilute the above 500 μL mixture with sufficient PEI in 500 μL of PEI - OptiPro SFM so that the ratio of DNA (μg):PEI (μg) is 1:3 (1000 μL per 10 cm dish). When using the transfer plasmid pHAGE TRE dCas9 - KRAB (27.8 μg total μg of plasmid DNA), this results in 83.4 μL (1 mg / mL) of PEI in 416.6 μL of OptiPro SFM per 10 cm dish. Gently add the diluted PEI to the diluted DNA. While gently flicking the diluted DNA tube with your finger, add the diluted PEI dropwise. Incubate the mixture at room temperature for 15 - 20 minutes. Carefully transfer the transfection mixture to Lenti - X 293T packaging cells. Add the transfection mixture dropwise, taking care not to push the cells away. Incubate the cells for 18 hours or until the next morning. The next morning, carefully aspirate the medium. Replace the medium with 15 mL of DMEM Complete. Incubate the cells. Virus can be harvested 48, 72, and 96 hours after transfection, either as individual harvests or as a single harvest in which all individual harvests are pooled. When pooling harvests, transfer the harvested medium to a polypropylene storage tube and store at 4 °C during collection. Centrifuge the virus supernatant at approximately 500 g for 5 minutes to pellet any packaging cells collected during harvest. Filter the supernatant through a 0.45 μm PES filter. The virus supernatant can be stored at 4 °C for several hours, but in order to avoid loss of titer, it must be aliquoted into equal volumes as soon as possible, snap-frozen in liquid nitrogen, and stored at -80 °C.

[0331] One day before transfection, 0.72 pmol of pCMV-VSV-G envelope plasmid, 1.64 pmol of pSICO or pRRL dual payload (transfer plasmid), and 1.30 pmol of psPAX2 plasmid (wild-type or containing pol mutations) were used to produce cells in a 10 cm dish seeded with 4.9×10 6 HEK293T cells.

[0332] For nuclease packaging, 0.65 pmol of psPAX2 and 0.65 pmol of psMCP-GAG or psMCP-GAGPOL plasmid were used together with 0.65 pmol of nuclease plasmid.

[0333] For transfection, the plasmids were mixed in 500 μL of Optimem and 100 mg of polyethyleneimine (PEI). Two days after plasmid transfection, the supernatant was collected, filtered, and centrifuged overnight at 4000 g and 4 °C. The supernatant was discarded and the lentiviral particles were resuspended to achieve a 100× vector concentration.

[0334] Determination of nuclease activity HEK293T or other cell line models were infected using lentiviral particles produced using different strategies for loading Cas9 and gRNA. Three days after addition of the nuclease to the viral particles, the cells were pelleted and the DNA was extracted by KAPPA rapid extraction. PCR amplification of each target site was performed and sequencing was carried out by Illumina sequencing on a synthetic MiSeq (trademark) platform using the MiSeq Reagent Kit v2 (300 cycles, 2×150 configuration).

[0335] Determination of nuclease activity Lentiviral particles were lysed in RIPA buffer for proteins. Proteins were analyzed by Western blot according to standard methods. Briefly, proteins were loaded onto an SDS-PAGE gel (Invitrogen) and electrophoresed, and then transferred to a nitrocellulose blot for protein detection using anti-Cas9 and anti-p24 antibodies (AbCam) as well as an HRP-conjugated secondary antibody. Proteins were detected using luminol as a substrate and quantified by the generation of chemiluminescence.

[0336] Results The presence of Cas9 could be achieved by most of the seven tested strategies (Figure 6A), but nuclease activity could not be detected using these methods (Figure 6B).

[0337] Next, the present inventors tested several other loading strategies using the MS2-MCP RNA-protein system to utilize the gRNA interaction with Cas9. In one of these strategies, the present inventors co-expressed tetravalent MS2 in the gRNA together with a modified GAG-POL viral gene fused to the MCP protein for passive loading of Cas9 into viral particles and loading of the gRNA for proper targeting and cleavage of target DNA in infected cells (Figure 6A).

[0338] This method showed not only an increase in the presence of Cas9 (Figure 7B) when using the MCP-MS2 system but also a recovery of nuclease activity in infected cells, and surprisingly, a complete recovery of nuclease activity in infected cells (Figure 6B) when using the MCP-MS2 system.

[0339] Example 3: Verification of the best-performing Cas9-loading PILV prototype Materials and methods Using different strategies for loading Cas9 and gRNA, HEK293T reporter cells or K562 cells were infected with lentiviral particles produced by different loading strategies according to the same method as in Examples 1 and 2. The infected cells were analyzed as follows.

[0340] Determination of nuclease activity Three days after adding the nuclease to the viral particles, the cells were pelleted and DNA was extracted by KAPPA rapid extraction. PCR amplification of each target site was performed and sequenced by Illumina sequencing on the synthetic MiSeq (trademark) platform using the MiSeq Reagent Kit v2 (300 cycles, 2×150 configuration).

[0341] Determination of on-target integration A reporter cell line (Hershey) was constructed to evaluate target integration, where a fluorescent signal (GFP) measurable by reconstitution of the fluorescent protein ORF upon on-target integration was generated.

[0342] A viral payload plasmid containing 1 / 2 Emerald GFP (emGFP) and 1 / 2 intron was packaged into a lentiviral vector using the corresponding packaging system and / or integrase mutant. A reporter cell line was constructed by randomly inserting the C-terminal (C-t) half without the promoter of EmGFP preceding the splicing acceptor into the genome of HEK293T cells. The "target site" was added upstream of C-temGFP.

[0343] For the experiment, 200,000 HEK293T Hershey reporter cells were seeded in 12-well / plates and infected with lentiviral particles the next day simultaneously with transfection that caused emGFP reconstitution upon addition of the payload plasmid.

[0344] Lentivirus was produced using the standard protocol. Four days after infection, the emGFP fluorescence signal was measured by FACS (BD LSR Fortessa instrument; blue 488 nm laser equipped with a 530 / 30 filter, yellow-green 561 nm laser equipped with a 610 / 20 filter).

[0345] For K562 cells, genomic DNA was extracted five days after infection, and junction PCR of the target site and viral payload was amplified by semi-quantitative standard method (SYBRgreen qPCR, Applied Bio Systems) and compared with the total viral copy and endogenous gene copy number.

[0346] Results Once a loading system for both Cas9 and gRNA into the infected donor was established for efficient nuclease activity, several integrase variants were tested in terms of integrase-deficient viral particles to achieve programmable integration.

[0347] To evaluate programmable integration into the target site defined by gRNA, a reporter HEK293T cell line based on split GFP reporter was used (Figure 8A). The efficiency of each packaging system was evaluated, and it was found that the most performant system was the MCP-GAG fusion with gRNA-MS2 and mRNA encapsulation by the MCP-MS2 system instead of mRNA. PILV containing MCP-GAGPOL showed very promising results (Figure 8B). By increasing the production rate of PILV containing MCP-GAGPOL, improvement was achieved in subsequent repeats. Direct fusion with integrase or POL showed the worst results.

[0348] In addition to the cell reporter system for programmable integration, the programmable insertion efficiency of PILV into the endogenous target sites of another cell line model, K562, was tested and verified. The programmable integration efficiency after episome attenuation was evaluated by junction-targeted qPCR, the relative copy numbers were plotted, and compared with the target NHEJ activity (Figure 9). Strategy 1 consisting of the MCP-GAGPOL fusion and the MS2-MCP interaction with gRNA, as well as the MCP-GAG fusion, were found to be the most promising, followed by mRNA encapsulation into virus-like particles instead of RNPs.

[0349] Example 4: Programmable integration of the PILV genome by homology-directed recombination (HDR) Materials and methods Cell culture and plasmid cloning HEK293T cells (ATCC CRL-3216) were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with high glucose (Gibco, Thermo Fisher), 10% fetal bovine serum, 2 mM glutamine and 100 U penicillin, 0.1 mg / mL streptomycin.

[0350] K-562 cells (ATCC CRL-3343) and T cells (isolated and activated from human donors) were grown in RPMI 1640 medium (Gibco) supplemented with 10% FBS, 1% penicillin-streptomycin (Gibco) and 1% GlutaMAX (100×) (Gibco).

[0351] T cells were isolated from the meninges of up to four human donors, PBMCs were purified, and T cells were activated using standard methods for active proliferation before infection.

[0352] All cells were grown at 37 °C in 95% air and 5% CO 2 2.

[0353] Additional vectors not included in Examples 1 and 2 were constructed by Golden Gate Assembly using Esp3I and T4 ligase. Plasmid pLV RAB11A-HA-GFP-HA, a lentiviral payload donor with a GFP tag for targeted integration at the RAB11A site of SEQ ID NO: 81, pLV RAB11A-HA-GFPinv-HA, a lentiviral payload donor with a GFP tag in the reverse direction for targeted integration at the RAB11A site of SEQ ID NO: 82, pLV FBL-HA-mGFP-HA, a lentiviral payload donor with a GFP tag for targeted integration at the FBL site of SEQ ID NO: 83, pLV GFP-TRAC-HA, a lentiviral payload donor with a GFP tag for targeted integration at the TRAC site of SEQ ID NO: 84, pLV eGFP-TRAC-HA, a lentiviral payload donor with an eGFP tag for targeted integration at the TRAC site of SEQ ID NO: 85 were used.

[0354] Determination of on-target integration Primary T cells, namely HEK293T and K562 cell line models, were infected with virus particles produced in the same manner as in Examples 1 and 2 with an HDR gene trap reporter in which a GFP tag was added to the protein and was on the endogenous gene for site-specific insertion detection.

[0355] For the experiment, 200,000 HEK293T or K562 or 500,000 activated T cells were infected with lentiviral particles. The GFP fluorescence signal was measured by FACS (BD LSR Fortessa instrument; blue 488 nm laser equipped with a 530 / 30 filter, yellow-green 561 nm laser equipped with a 610 / 20 filter) at different time points to track the signal.

[0356] Results The virus genome was produced for programmable insertion by homologous arms into the target site of interest, improving the cargo donor to enhance efficiency against actively dividing cells. Three other PILV cargos were designed and generated (Figure 10A). In HEK293T cells (Figure 10B), high levels of non-specific signal were generated by the RAB11A reporter construct, but this promoter in the reverse orientation dramatically reduced the noise signal and increased fluorescence under PILV conditions consistent with programmable insertion of the payload. Figure 10C shows a modest increase in the PILV signal for both cell lines consistent with programmable insertion of the HDR payload.

[0357] Also, the FBL reporter showed similar results with up to 10% specific GFP signal to PILV in HEK293T cells (Figure 10B). For HDR payloads in the cell models K562 and JurcaTs, similar results were observed by the presence of episomal leaky expression in both payloads and the increase in the PILV signal (Figure 10C).

[0358] To verify the therapeutic potential of programmable integration of lentiviral vectors from the perspective of cell manipulation, a TRAC-specific gene trap was designed and constructed to produce T cells containing a therapeutic cargo inserted into the TRAC locus (Figure 11A). Programmable insertion of the HDR-based TRAC-specific PILV payload was tested in primary T cells from two different PBMC donors. Transduction efficiencies of over 50% were obtained when using the programmable insertion vector, whereas nothing was obtained with the non-integrating control in contrast (Figure 11B).

[0359] From these results, the feasibility of introducing PILV as a safe and controllable method for T cell and other primary cell manipulations for therapeutic purposes is confirmed.

[0360] Conclusion Targeted DNA integration of gene-sized fragments into the mammalian genome can enable the precise addition of therapeutic messages and new functions. Lentiviral vectors have been therapeutically used for the uncontrolled insertion of transgenes for both ex vivo applications for advanced cell therapy and in vivo applications for gene therapy. The present inventors point out that by mutating the integrase protein and incorporating SpCas9 nuclease into lentiviral particles, the lentiviral vector can be programmed to precisely integrate transgenic DNA.

[0361] Precise DNA integration was consistent in immortalized cell models and primary cells. PILV was also able to deliver the transgene to the ROSA26 locus of the mouse liver. The present inventors also detected PILV cis-integration during co-delivery of two transgenes, which provides a robust method for multiple KO-KI in ex vivo models. By changing two packaging vectors during lentivirus production, the lentiviral vector can be made programmable, enabling targeted DNA addition by applications in ex vivo cell manufacturing, in vivo gene replacement, and mammalian genome engineering.

Claims

1. (i) Nucleic acids encoding lentiviral vectors with reduced integration activity, (ii) nucleic acids encoding polypeptides or proteins, including RNA-induced nucleases or nicasses. (iii) Nucleic acid encoding guide RNA (gRNA) fused to an aptamer, (iv) The nucleic acid encoding the target transgene, and (v) Below: - A first protein containing or consisting of GAG polyprotein, and - A second protein containing or consisting of an aptamer-binding protein. nucleic acids encoding fusion proteins containing A composition containing the following:

2. The composition according to claim 1, wherein the nucleic acid encoding the lentiviral vector having reduced integration activity according to (i) further comprises an aptamer-binding protein.

3. The composition according to claim 1, wherein the nucleic acid encoding the fusion protein of (v) further comprises a third protein comprising or consisting of a POL polyprotein.

4. - The aptamer-binding protein is preferably an MS2 bacteriophage coat protein (MCP) that shares at least 75% identity with SEQ ID NO: 61, and The aptamer is preferably an MS2 RNA tetraloop binding sequence that shares at least 75% identity with SEQ ID NO:

63. The composition according to claim 1.

5. The composition according to claim 4, wherein the MS2 bacteriophage coat protein (MCP) shares at least 75% identity with SEQ ID NO: 61, and the MS2 RNA tetraloop binding sequence shares at least 75% identity with SEQ ID NO:

63.

6. The composition according to claim 1, wherein the RNA-induced nuclease or nicasse is not fused to the integrase.

7. The composition according to claim 1, wherein the RNA-induced nuclease or nicasse is a Cas protein.

8. The composition according to claim 1, wherein the RNA-inducible nuclease or nickas is bound to a mutant, highly active PiggyBac transposase.

9. The composition according to claim 1, wherein the integrase contained in (i) and / or (v) the POL polyprotein, if present, contains at least one amino acid mutation at a position selected from the group consisting of 10, 11, 13, 64, 94, 116, 117, 119, 120, 122, 124, 128, 152, 164, 168, 170, 185, 186, 231, 264, 266 in HIV-1 integrase of SEQ ID NO: 1 or at a corresponding position in another integrase.

10. (i) and / or (v) integrases are D10K, E11K, E13K, D64A, D64E, G94D, G94E, G94R, G94K, D116A, D116E, N117D, N117E, N117R, N117K, S119A, S119P, S119T, S119G, S119D, S119E, S119R, S119K, N120D, N120E, N120R, N120K, T122K, T122I, T122V, The composition according to claim 1, comprising at least one amino acid mutation selected from the group consisting of T122A, T122R, A124D, A124E, A124R, A124K, A128T, E152A, E152D, D164N, Q168L, Q168A, E170G, F185K, K186E, R231G, R231K, R231D, R231E, R231S, K264R, K266R and K273R or an amino acid mutation at a corresponding position in another integrase.

11. The composition according to claim 1, wherein the integration activity of the lentiviral vector (i) is reduced or lost by at least one mutation in the integrase sequence selected from the group consisting of D116N substitution in the numbering based on SEQ ID NO: 1, D164N substitution in the numbering based on SEQ ID NO: 1, insertion of an immature stop codon, and deletion of the integrase gene (ΔIN).

12. (i) A step of transfecting lentivirus-producing cells cultured in a suitable culture medium with the composition described in claim 1, (ii) A step of recovering lentiviral particles in the culture medium of the lentiviral-producing cells A method for producing lentiviral particles, including [a specific type of virus].

13. A group of lentiviral particles obtained by the method described in claim 12.

14. a. Lentiviral proteins and / or genes or parts thereof that have reduced integration activity of the lentiviral integrase, b. RNA-induced nucleases or nicasses, or nucleic acid sequences encoding them. c. Guide RNA fused to the aptamer, and d. A fusion protein comprising (1) a first protein containing or consisting of a GAG polyprotein, and (2) a second protein containing or consisting of an aptamer-binding protein, or a nucleic acid sequence encoding such a fusion protein. A collection of lentiviral particles, including lentiviral particles that contain or consist of such particles.

15. The lentiviral particle population according to claim 14, wherein the RNA-induced nuclease or nickase is the Cas9 protein.

16. The population of lentiviral particles according to claim 14, wherein the aptamer is at least one MS2 RNA tetraloop binding sequence.

17. The lentiviral particle collection according to claim 14, wherein the fusion protein of d. further comprises a third protein comprising or consisting of a POL polyprotein.

18. An in vitro method for site-specific integration of a target transgene into the genome of a cell, comprising the step of infecting the cell with a population of lentiviral particles as described in claim 13 or 14, wherein the population of lentiviral particles contains the target transgene, or the target transgene is delivered to the cell before, simultaneously with, or after the population of lentiviral particles.

19. A pharmaceutical composition for use in the treatment of a disease in a subject requiring the use thereof, comprising a population of lentiviral particles according to claim 13 or 14, wherein the population of lentiviral particles comprises a target transgene, or the target transgene is delivered to the cells before, simultaneously with, or after the population of lentiviral particles.

20. The pharmaceutical composition for use according to claim 19, wherein the disease is a hereditary disease.

21. Use of a population of lentiviral particles according to claim 13 or 14 for drug preparation.

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