Targeted DNA integration using lentiviral vectors and uses thereof

Abstract

The present invention relates to a lentiviral particle composition comprising a mutated integrase and an RNA-guided nuclease, for use in targeted gene insertion.

Description

Field of the Invention

[0001] The present invention relates to the field of gene editing and gene therapy, in particular to targeted gene insertion. Background of the Invention

[0003] Pathological gene defects can range from a few bases to large deletions. Targeted DNA integration of gene-sized fragments in mammalian genomes can enable precise addition of therapeutic information and new functions. Precise gene delivery methods based on non-homologous end joining (NHEJ) have been developed, such as homology-independent targeted integration (HITI). This method has been shown to insert several thousand bases, but it is still inefficient for very large edits. Although HITI may be able to deliver exons, its efficiency may not be enough to robustly deliver genes such as cDNAs for DMD (~14kb) or ABCA4 (~6.8kb). Recently, HITI has been expanded to increase DNA efficiency by fusing DNA binding domains. In bacteria, precise gene delivery using CRISPR programmable transposons has been demonstrated, but this technology has not yet been used in mammalian cells.

[0004] The development of new methods for genome editing has allowed for the expansion of human gene therapy (HGT) applications. Many HGT strategies are based on the incorporation of a DNA payload that can complement a genetic defect and / or provide synthetic properties to the recipient cell. Traditionally, delivery of therapeutic payloads has been based on viral and non-viral vectors that integrate uncontrolled into the genome. Although precise delivery technologies are emerging, uncontrolled delivery mediated by viral vectors has benefited a large number of patients. Insertional mutagenesis events have been observed that led to the first attempts at gene therapy trials being halted after leukemia cases were associated with retroviral-based SCID-X1 gene therapy. Vector-driven clonal expansion has recently been observed again in CAR-T therapy and in leukemia cases associated with the retroviral-based drug Strimvelis. rAAV can cause insertional mutagenesis and induce clonal expansion. Viral-free technologies such as PiggyBac transposase (PB) have been associated with oncogenic induction. Lentiviral vectors are used therapeutically for the uncontrolled insertion of transgenes for both ex vivo and in vivo applications. We observed that by mutating the integrase protein and incorporating the SpCas9 nuclease in the lentiviral particles, lentiviral vectors could be reprogrammed to precisely integrate transgenic DNA.

[0005] Discovering and implementing precise delivery strategies using tools such as ZFNs, TALENs, and CRISPR Cas9 has the potential to avoid unwanted gene disruption or activation and enable simultaneous gene inactivation and entry, a critical feature for therapeutic genome modulation. Such technologies have also raised safety-related concerns. It has been reported that integration of the payload can be imprecise, yielding different unintended integration outcomes, such as integration of the donor at off-target cleavage sites and genomic rearrangements such as inversions or translocations.

[0006] Therefore, there is an unmet need for precise and safe gene delivery strategies, especially for the delivery of very large sequences. Summary of the invention

[0007] The present invention relates to a composition comprising:

[0008] (i) a nucleic acid encoding a lentiviral vector having impaired integration activity, optionally comprising an aptamer binding protein,

[0009] (ii) a nucleic acid encoding a polypeptide or protein comprising an RNA-guided nuclease or nickase,

[0010] (iii) a nucleic acid encoding a guide RNA (gRNA) fused to an aptamer,

[0011] (iv) a nucleic acid encoding a transgene of interest, and

[0012] (v) optionally, a nucleic acid encoding a fusion protein comprising:

[0013] - a first protein comprising or consisting of a GAG polyprotein,

[0014] - a second protein comprising or consisting of an aptamer binding protein, and

[0015] - Optionally, a third protein comprises or consists of a POL polyprotein.

[0016] In some embodiments, the aptamer binding protein is an MS2 bacteriophage capsid protein (MCP), preferably at least 75% identical to SEQ ID NO:61; and the aptamer is an MS2 RNA tetraloop binding sequence, preferably at least 75% identical to SEQ ID NO:63.

[0017] In some embodiments, the RNA-guided nuclease or nickase is not fused to an integrase.

[0018] In some embodiments, the RNA-guided nuclease or nickase is a Cas protein.

[0019] In some embodiments, an RNA-guided nuclease or nickase is combined with a mutant hyperactive PiggyBac transposase.

[0020] In some embodiments, the integrase of (i) and / or the integrase contained in the POL polyprotein of (v), when present, comprises at least one amino acid mutation at a position selected from: 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 at a corresponding position in another integrase.

[0021] 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, T 124R, A124D, A124E, A124R, A124K, A128T, E152A, E152D, D164N, Q168L, Q168A, E170G, F185K, K186E, R231G, R231K, R231D, R231E, R231S, K264R, K266R, and K273R, or the corresponding positions in another integrase.

[0022] In some embodiments, the integration activity of the lentiviral vector of (i) is reduced or eliminated by at least one mutation in the integrase sequence, the mutation selected from: D116N substitution, D164N substitution, insertion of a premature stop codon, and deletion (ΔIN) of the integrase gene, numbering based on the HIV-1 integrase of SEQ ID NO: 1.

[0023] The present invention also relates to a method for producing lentiviral particles, comprising the following steps:

[0024] (i) transfecting lentivirus production cells cultured in a suitable medium with the composition of the present invention, and

[0025] (ii) collecting the lentiviral particles in the culture medium of the lentiviral producer cells.

[0026] The present invention also relates to a population of lentiviral particles obtainable by the method of producing lentiviral particles of the present invention.

[0027] The present invention also relates to a population of lentiviral particles, comprising lentiviral particles, wherein the lentiviral particles comprise or consist of:

[0028] a. lentiviral proteins and / or genes or parts thereof, wherein the lentiviral integrase has impaired integration activity,

[0029] b. an RNA-guided nuclease or nickase, preferably a Cas9 protein, or a nucleic acid sequence encoding the same,

[0030] c. a guide RNA fused to an aptamer, preferably to at least one MS2 RNA tetraloop binding sequence, and

[0031] d. Optionally, 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, or a nucleic acid sequence encoding the same.

[0032] The present invention also relates to an in vitro method for site-specific integration of a target transgene into the genome of a cell, the method comprising infecting the cell with a population of lentiviral particles of the present invention, wherein the population of lentiviral particles comprises the target transgene, or wherein the target transgene is delivered to the cell before, simultaneously with or after the population of lentiviral particles.

[0033] The present invention also relates to a population of lentiviral particles according to the present invention for use as a medicament.

[0034] The present invention also relates to the lentiviral particle population of the present invention, for site-specific integration of a target transgene into the genome of a cell, wherein the lentiviral particle population comprises the target transgene, or wherein the target transgene is delivered to the cell before, simultaneously with, or after the lentiviral particle population.

[0035] The present invention also relates to a population of lentiviral particles of the present invention for use in treating a disease, preferably a genetic disease, in a subject in need thereof, wherein the population of lentiviral particles comprises a transgene of interest, or wherein the transgene of interest is delivered to a cell before, simultaneously with or after the population of lentiviral particles.

[0036] definition

[0037] In this disclosure, any use of the singular form "a", "an" or "the" includes singular reference as well as plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a single agent and a plurality of such agents.

[0038] "Nucleic acid (sequence)" and "nucleotide sequence" are used interchangeably to refer to any molecule composed of or containing monomeric nucleotides. A nucleic acid can be an oligonucleotide or a polynucleotide; it can be DNA, RNA, or a mixture of the two. It can be chemically modified or artificial; for example, it includes peptide nucleic acids (PNA), morpholinos, and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA) and threose nucleic acids (TNA). These nucleic acids are distinguished from naturally occurring DNA or RNA by changes in the molecular skeleton. In addition, thiophosphate nucleotides can be used. Other deoxynucleotide analogs include, but are not limited to, methylphosphonates, phosphoramides, dithiophosphates, N3'P5'phosphoramides, and oligoribonucleotide thiophosphates and their 2'O-allyl analogs and 2'O-methylribonucleotide methylphosphonates, which can be used in the nucleic acids of the present disclosure.

[0039] "Polypeptide," "peptide," "protein," and "amino acid sequence" are used interchangeably to refer to a polymer of amino acid residues. Unless otherwise indicated, a polymer of amino acid residues may be of any length. The term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of corresponding naturally occurring amino acids.

[0040] "Nuclease" refers to an enzyme that catalyzes the hydrolysis of nucleic acids within a nucleic acid sequence. Nuclease activity can result in the breakage of single-stranded or double-stranded nucleic acid molecules, where the nucleic acid molecule can be DNA or RNA.

[0041] "Cas9" or "Cas9 nuclease" refers to an RNA-guided nuclease comprising a Cas9 protein or a fragment thereof (e.g., a protein comprising an active or inactive DNA cleavage domain of Cas9 and / or a gRNA binding domain of Cas9). Cas9 nucleases are sometimes also referred to as casn1 nucleases or CRISPR (clustered regularly interspaced short palindromic repeats) binding nucleases. CRISPR is an adaptive immune system that generates protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain spacers (sequences complementary to previous mobile elements) and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, the correct processing of pre-crRNA requires trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9 protein. tracrRNA can serve as a guide for ribonuclease 3-assisted pre-crRNA processing. Subsequently, the Cas9 / crRNA / tracrRNA complex cuts the linear or circular dsDNA target complementary to the spacer by endonuclease. The target chain that is not complementary to crRNA is first cut by endonuclease, and then 3'-5' trimmed by exonuclease. In nature, DNA binding and cutting generally require protein and two kinds of RNA. However, single guide RNA ("sgRNA" or "gRNA" for short) can be engineered to integrate various aspects of both crRNA and tracrRNA into a single RNA molecule.

[0042] Cas9 recognizes short motifs (PAM or protospacer adjacent motifs) in CRISPR repeat sequences to help distinguish self from non-self. Cas9 nuclease sequences and structures are well known to those skilled in the art. Cas9 orthologs have been described in various species, including but not limited to Streptococcus pyogenes (S. pyogenes) and Streptococcus thermophilus (S. thermophilus). Based on this disclosure, other suitable Cas9 nucleases and sequences will be clear to those skilled in the art, and such Cas9 nucleases and sequences include Cas9 sequences from organisms and loci disclosed in Chylinski et al., 2013 (RNA Biol. 10 (5): 726-37) (the entire contents of which are incorporated herein by reference).

[0043] In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain. Nuclease-inactivated Cas9 proteins can be interchangeably referred to as "dCas9" proteins (referring to nuclease "dead" Cas9). Methods for generating Cas9 proteins (or fragments thereof) with inactive DNA cleavage domains are known in the art (see, e.g., Jinek et al., 2012. Science. 337 (6096): 816-821; Qi et al., 2013. Cell. 152 (5): 1173-83, the entire contents of which are incorporated herein by reference).

[0044] "Exogenous" refers to any molecule that does not naturally occur in the target cell or organism, but can be introduced into it by one or more genetic, biochemical or other methods. The natural occurrence of a molecule in a cell or organism can also be determined based on its specific developmental stage and environmental conditions. Thus, for example, a molecule that only exists during muscle embryonic development is an exogenous molecule for an adult muscle cell. Similarly, a molecule induced by heat shock is an exogenous molecule for a cell that is not 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 specific developmental stage under specific environmental conditions.

[0045] "Fusion protein" refers to a single-chain hybrid polypeptide comprising two or more amino acid sequences (i.e., from two or more different proteins and / or peptides) fused together. The two or more amino acid sequences can be fused together by direct peptide bonds or indirectly fused together by peptide linkers. The fusion protein can specifically be completely encoded by a single nucleic acid sequence.

[0046] "Gene" generally refers to a region of DNA that encodes a protein (i.e., a coding region). The term may also include regions of DNA that do not encode a protein per se (i.e., non-coding regions). The latter include, for example, regions that are transcribed into functional non-coding RNA molecules (e.g., transfer RNA, ribosomal RNA, regulatory RNA, etc.). Other non-coding regions regulate the transcription and translation of the coding region (i.e., regulatory elements), or serve as structural elements (e.g., scaffold / matrix attachment regions), DNA replication origins, centromeres, or telomeres, etc. Regulatory elements include, but are not limited to, promoter sequences, terminators, translation regulatory sequences (e.g., ribosome binding sites [RBS] and internal ribosome entry sites [IRES]), enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions.

[0047] "Eukaryotic" refers to cells or organisms composed of such cells that are not bacterial cells (prokaryotic cells). Eukaryotic cells include, but are not limited to, fungal cells, plant cells, and animal cells (eg, mammalian cells and human cells).

[0048] "Transfection" and any of its variations refer to the introduction of one or more nucleic acid molecules (DNA and / or RNA) into one or more cells by non-viral means, whether in vitro or in vivo. Transfection methods are well known in the art and include, for example, lipofection and electroporation.

[0049] "Transduction" and any of its variations refer to the introduction of one or more nucleic acid molecules (DNA and / or RNA) into one or more cells using a viral vector, such as a virus, viral particle or viral vector, including but not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses (AAV) and vectors derived therefrom.

[0050] "Cleavage" refers to the breakage 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 can be caused by two different single-strand cleavage events. DNA cleavage can result in the production of blunt ends or staggered ends.

[0051] "Specificity" refers to the ability to selectively bind to sequences that have a certain sequence identity with a selected sequence.

[0052] "Insertion" and "integration" refer to the addition of a nucleic acid sequence into a second nucleic acid sequence or a genome or portion thereof. Insertion can be "specific," "site-specific," "targeted," or "on-targeted": these adjectives define the insertion of a nucleic acid into a specific site of a second nucleic acid or a specific site of a genome or portion thereof (i.e., a site deliberately selected for insertion). In contrast, the adjectives "random," "non-targeted," or "off-targeted" refer to the non-specific and / or unintended insertion of a nucleic acid into an undesired site. The term "total" or "total" refers to the total number of insertions.

[0053] "Mutation" refers to the substitution of one residue within a sequence (e.g., a nucleic acid or amino acid sequence) with another residue; and / or refers to the deletion or insertion of one or more residues within a nucleic acid or amino acid sequence. Mutations are generally described herein by indicating the original residue, followed by the position of the residue within the sequence, and then indicating the newly substituted residue. The various methods provided herein for making amino acid substitutions (mutations) are well known in the art and are described, for example, by Green & Sambrook, 2012 (Molecular cloning: alaboratory manual (4th Ed.).Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0054] "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 host cell genome. In contrast to transiently expressed nucleic acid sequences (e.g., transfected plasmids), integrated nucleic acid sequences are stably expressed by the cell regardless of mitotic events and replicated along with the rest of the genome.

[0055] "Transposase" refers to an enzyme that binds to the ends of a transposon and catalyzes its movement to another part of the genome by either a cut-and-paste mechanism or a copy-transposition mechanism.

[0056] "Mutated" refers to a sequence (e.g., an amino acid sequence or a nucleic acid sequence) that is different from a reference sequence (e.g., a wild-type sequence). Typically, the mutant sequence comprises at least one of the following: a substitution, addition, or deletion of one or more residues compared to a reference sequence (e.g., a corresponding wild-type sequence).

[0057] "Linker" refers to a chemical group or molecule that connects two adjacent molecules or moieties.

[0058] "Identity" or "identical" when used in reference to sequence relationships 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, as determined by the number of matches between chains of two or more amino acid residues or nucleic acid residues. "Identity" measures the percentage of identical matches between the smaller of two or more sequences, where gap comparisons (if any) are resolved by a particular mathematical model or computer program (i.e., an "algorithm"). The identity of related amino acid sequences or nucleic acid sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in Lesk AM (1988). Computational molecular biology: Sources and methods for sequence analysis. New York, NY: Oxford University Press; Smith DW (1993). Biocomputing: Informatics and genome projects. San Diego, CA: Academic Press; Griffin AM & Griffin HG (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 MR & Devereux J. (1991). Sequence analysis primer. New York, NY: Stockton Press; Carillo et al., 1988. SIAM J Appl Math. 48 (5): 1073-82. Preferred methods for determining identity are designed to provide the largest match between the sequences measured. Methods to determine identity are described in publicly available computer programs.Preferred computer program methods for determining the identity between two sequences include the GCG program package, including GAP (Genetics Computer Group, University of Wisconsin, Madison, WI; 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 / NIHBethesda, Md. 20894). The well-known Smith Waterman algorithm can also be used to determine identity.

[0059] "Subject" refers to a mammal, preferably a human. A subject may be a "patient", i.e., a warm-blooded animal, more preferably a human, who is awaiting medical care or is receiving medical care or was / is / will be the subject of a medical procedure, or is being monitored for the development of a disease. The term "mammal" herein refers to any mammal, including humans, domestic and farm animals, and zoo, sport or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is a primate, more preferably a human.

[0060] "Treatment," "relief," "cure," and any of their variations refer to therapeutic treatment and do not include prophylactic or preventative measures; the purpose of which is to slow, alleviate, stop, or even reverse (partially or completely) the development of the target pathological condition or disorder. Those in need of treatment include those already suffering from the disorder as well as those suspected of suffering from the disorder. If, after receiving treatment, the subject shows an observable and / or measurable reduction or disappearance of one or more symptoms associated with the target pathological condition or disorder; a certain degree of relief; a decrease in morbidity and / or mortality; and / or an improvement in quality of life issues, the subject is successfully "treated" for the pathological condition or disorder. The above parameters for evaluating successful treatment and improvement of the disease can be easily measured by routine procedures with which doctors are familiar.

[0061] "Prevention" and any of its conjugations refer to preventative or prophylactic measures, wherein the objective is to reduce the chance of a subject developing a given pathological condition or disorder within a given period of time. Such reduction may be reflected, for example, in a delayed onset of at least one symptom of the subject's pathological condition or disorder. DETAILED DESCRIPTION OF THE INVENTION

[0063] The present invention relates to a composition comprising:

[0064] (i) a nucleic acid encoding a lentiviral vector having impaired integration activity, the lentiviral vector optionally comprising an aptamer binding protein,

[0065] (ii) a nucleic acid encoding a polypeptide or protein comprising an RNA-guided nuclease or nickase,

[0066] (iii) a nucleic acid encoding a guide RNA (gRNA) fused to an aptamer,

[0067] (iv) a nucleic acid encoding a transgene of interest, and

[0068] (v) optionally, a nucleic acid encoding a fusion protein comprising:

[0069] - a first protein comprising or consisting of a GAG polyprotein,

[0070] - a second protein comprising or consisting of an aptamer binding protein; and

[0071] - Optionally, a third protein which comprises or consists of the POL polyprotein.

[0072] In some embodiments, the nucleic acid in the composition is deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a combination thereof. In some embodiments, the nucleic acid in the composition is DNA. In some embodiments, the nucleic acid in the composition is RNA.

[0073] In some embodiments, the nucleic acid of the composition comprises natural nucleotides, non-natural nucleotides or a combination thereof, or consists of the above. In some embodiments, the nucleic acid of the composition comprises natural nucleotides or consists of natural nucleotides. In some embodiments, natural nucleotides are selected from the group comprising or consisting of adenine, guanine, cytosine, thymine and uracil. In some embodiments, natural nucleotides are selected from the group comprising or consisting of adenine, guanine, cytosine and thymine. In some embodiments, natural nucleotides are selected from the group comprising or consisting of adenine, guanine, cytosine and uracil. In some embodiments, natural nucleotides are selected from the group comprising or consisting of adenine, guanine, cytosine and uracil. In some embodiments, the nucleic acid of the composition comprises non-natural nucleotides (e.g., chemically modified) or consists of.

[0074] In some embodiments, the nucleic acid in the composition is single-stranded, double-stranded, or a combination thereof. In some embodiments, the nucleic acid in the composition is single-stranded. In some embodiments, the nucleic acid in the composition is double-stranded.

[0075] In some embodiments, the nucleic acid in the composition is selected from the group comprising or consisting of: 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 a combination thereof. In some embodiments, the nucleic acid in the composition is a plasmid, mRNA or a combination thereof. In some embodiments, the nucleic acid in the composition is a plasmid. In some embodiments, the nucleic acid in the composition is an mRNA.

[0076] In some embodiments, each nucleic acid in the composition is contained in its own vector (e.g., plasmid, cosmid, fosmid, chromosome, etc.). Alternatively, the two nucleic acids in the composition may be contained in 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 in a single vector. Alternatively, the three nucleic acids in the composition may be contained in 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 in a single vector. Alternatively, the four nucleic acids of the composition may be contained in 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), or (ii) and (iii) and (iv) and (v) may be contained in a single vector. Alternatively, the five nucleic acids of the composition may be contained in a single vector.

[0077] In some embodiments, the nucleic acids in the composition comprise at least one coding sequence, and optionally at least one non-coding sequence, such as a regulatory sequence or an origin of replication sequence.

[0078] In some embodiments, the nucleic acid in the composition encodes a protein (including post-translationally cleaved and / or modified polyproteins and propolypeptides), a nucleic acid, or a combination thereof. In other words, the nucleic acid in the composition comprises at least one expression cassette.

[0079] Further described herein are proteins or nucleic acids encoded by the nucleic acids in the compositions.

[0080] According to the present invention, the composition comprises a lentiviral vector with impaired integration activity.

[0081] As used herein, the expression "impaired integration activity" means that the integration activity of the integrase of the lentiviral vector is reduced or eliminated compared to the wild-type lentiviral vector. Preferably, the integration activity of the lentiviral vector is eliminated compared to the wild-type lentiviral vector. In order to achieve the reduction or elimination of integration activity, the integrase of the lentiviral vector is genetically modified, for example, by changing its integration activity using one or several amino acid substitutions, or by partially or completely deleting the integrase coding sequence.

[0082] In some embodiments, the integrase of the lentiviral vector comprises at least one amino acid mutation compared to a wild-type (WT) integrase (e.g., compared to a 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, and the like.

[0083] In the following, unless otherwise indicated, the amino acid residue numbering is based on the sequence of HIV-1 integrase having SEQ ID NO: 1. A person skilled in the art can easily determine the corresponding positions in the sequences of other integrases, for example by sequence alignment.

[0084]

[0085] 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, 11, 13, 64, 94, 116, 117, 119, 120, 122, 124, 128, 152, 164, 168, 170, 185, 186, 231, 264, 266, and 273.

[0086] In some embodiments, the integrase of the lentiviral vector comprises at least one amino acid mutation selected from 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, N120 D. N120E, N120R, N120K, T122K, T122I, T122V, T122A, T122R, A124D, A124E, A124R, A124K, A128T, E152V, E152A, E1 52D, D164N, Q168L, Q168A, E170G, F185K, K186Q, K186E, R231G, R231K, R231D, R231E, R231S, K264R, K266R and K273R.

[0087] 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, 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: 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.

[0088] In some embodiments, the integrase of the lentiviral vector comprises at least one amino acid mutation selected from or consisting of E10K, E13K, D64V, D116N, S119G, E152V, D164N, K186Q, and R231G.

[0089] In some embodiments, the integrase of the lentiviral vector comprises at least one mutation that disrupts the interaction with p75. In some embodiments, the integrase of the lentiviral vector comprises at least one amino acid mutation selected from a group comprising or consisting of double mutations E10K / E13K and K186Q. In some embodiments, the integrase of the lentiviral vector comprises amino acid mutations E10K and E13K, and generally the integrase of the lentiviral vector has an amino acid sequence of SEQ ID NO: 2. In some embodiments, the integrase of the lentiviral vector comprises an amino acid mutation K186Q, and generally the integrase of the lentiviral vector has an amino acid sequence of SEQ ID NO: 8.

[0090] 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 comprising D64V, D116N and E152V or consisting thereof. In some embodiments, the integrase of the lentiviral vector comprises the amino acid mutation D64V, and the integrase of the lentiviral vector generally 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 the integrase of the lentiviral vector generally 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 the integrase of the lentiviral vector generally has the amino acid sequence of SEQ ID NO: 6.

[0091] In some embodiments, the integrase of the lentiviral vector comprises at least one mutation that relocates 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 a group comprising S119G and R231G or consisting thereof. 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.

[0092] 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: 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 comprising or consisting of 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.

[0093] 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: 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: K264R, K266R, and K273R.

[0094] 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.

[0095] 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.

[0096] In some embodiments, the integrase of the lentiviral vector comprises at least one amino acid mutation selected from the group comprising or consisting of a D116N substitution, a D164N substitution, an insertion of a premature stop codon in its coding sequence, and a deletion of its coding sequence (ΔIN).

[0097] 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.

[0098] In some embodiments, the nucleic acid sequence encoding the lentiviral vector integrase 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 a quality control system of the cell.

[0099] In some embodiments, the nucleic acid sequence encoding integrase is deleted from the pol gene of the lentiviral vector, referred to as "ΔIN".

[0100] In some embodiments, the lentiviral vector encodes at least one lentiviral polyprotein selected from POL, GAG, and ENV. In some embodiments, the lentiviral vector encodes GAG and POL.

[0101] In some embodiments, the lentiviral vector comprises a lentiviral vector genome comprising at least one gene selected from gag and pol. In some embodiments, the lentiviral vector comprises a lentiviral vector genome comprising at least one gene selected from gag, pol and vpr. In some embodiments, the lentiviral vector comprises a lentiviral vector genome comprising at least one gene selected from gag, pol, env and vpr. In some embodiments, the lentiviral vector comprises a lentiviral vector genome comprising at least one gene selected from gag, pol, env, vpr, vpu, vif, nef, tat and rev.

[0102] 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.

[0103] In some embodiments, the lentiviral vector further comprises at least one non-lentiviral protein, or a nucleic acid encoding the same, in particular at least one non-lentiviral surface protein. In some embodiments, the non-lentiviral protein is capable of binding to a surface protein of a eukaryotic cell, preferably a mammalian cell, more preferably a human cell.

[0104] According to the present invention, the lentiviral vector with impaired integration activity optionally comprises an aptamer binding protein.

[0105] 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 fused to the N-terminus of the GAG ​​polyprotein, optionally through a linker. In some embodiments, the N-terminus of the aptamer binding protein is fused to the C-terminus of the GAG ​​polyprotein, optionally through a linker.

[0106] 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 fused to the N-terminus of the POL polyprotein, optionally through a linker. In some embodiments, the N-terminus of the aptamer binding protein is fused to the C-terminus of the POL polyprotein, optionally through a linker.

[0107] In some embodiments, the aptamer binding protein is selected from the group comprising or consisting of: MS2 bacteriophage capsid protein (MCP), PP7 capsid protein (PCP), λN22 peptide, and COM.

[0108] In some embodiments, the aptamer binding protein is MS2 bacteriophage capsid protein (MCP).

[0109] In some embodiments, the MCP is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:61 (e.g., encoded by a nucleic acid sequence having SEQ ID NO:60).

[0110]

[0111]

[0112] In some embodiments, the aptamer binding protein is an MS2 bacteriophage capsid protein (MCP), preferably at least 75% identical to SEQ ID NO:61, and the aptamer is an MS2 RNA tetraloop binding sequence, preferably at least 75% identical to SEQ ID NO:63.

[0113] According to the invention, the composition comprises a nucleic acid encoding a polypeptide or protein comprising an RNA-guided nuclease or nickase.

[0114] 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.

[0115] 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), or Campylobacter jejuni Cas9 (CjCas9), or variants thereof, nickase Cas9 (nCas9), dead Cas9 (dCas9), Cas12a protein, Cas12b protein, Cas12f protein, Cpf1 protein, or CasX protein, including variants and functional fragments thereof.

[0116] In some embodiments, the RNA-guided nuclease or nickase is a Cas9 protein, including variants and functional fragments thereof.

[0117] In some embodiments, the RNA-guided nuclease or nickase is a Cas9 protein.

[0118] In some embodiments, the Cas9 protein has at least 80%, 90%, 95%, 99%, or at least 100% amino acid sequence identity to a 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.

[0119] In some embodiments, the Cas9 protein has at least 80%, 90%, 95%, 99%, or at least 100% amino acid sequence identity to a 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.

[0120] 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.

[0121] In some embodiments, the Cas9 variant comprises an amino acid sequence of a Cas9 protein having one or more amino acid substitutions.

[0122] In some embodiments, the RNA-guided nuclease or nickase is a variant or functional fragment of the Cas9 protein.

[0123] In some embodiments, the Cas9 variant is a humanized Cas9 (hCas9) or a functional fragment thereof. The term "humanized Cas9" or "hCas9" as used herein refers to a Cas9 protein that is sequence-optimized for human cells. In some embodiments, the hCas9 protein has an amino acid sequence with 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 to SEQ ID NO: 40.

[0124] 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 to SEQ ID NO: 41.

[0125] In some embodiments, the RNA-guided nuclease or nickase is a dead Cas9 protein. In some embodiments, the dead Cas9 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 to SEQ ID NO: 42.

[0126] 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 to SEQ ID NO: 43.

[0127] In some embodiments, the RNA-guided nuclease or nickase is a Cas12f protein. In some embodiments, the Cas12f protein is from the bacterium Acidibacillus sulfuroxidans (AsCas12f). In some embodiments, Cas12f has an amino acid sequence with 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 to SEQ ID NO: 78.

[0128] In some embodiments, an RNA-guided nuclease or nickase is combined with a transposase.

[0129] In some embodiments, the RNA-guided nuclease or nickase binds to a mutant hyperactive PiggyBac transposase (hyPB).In some embodiments, the RNA-guided nuclease or nickase binds to a hyperactive PiggyBac transposase having at least 75% amino acid sequence identity to SEQ ID NO:10.

[0130]

[0131] As used herein, the term "mutated hyPB" refers to a transposase comprising one or more amino acid substitutions, typically no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, compared to the hyPB of the amino acid sequence SEQ ID NO: 10. More specifically, the mutant hyPB comprises (i) one or more amino acid substitutions to increase the cleavage activity compared to the hyPB of the amino acid sequence SEQ ID NO: 10, and / or (ii) one or more amino acid substitutions to decrease the DNA binding activity compared to the hyPB of the amino acid sequence SEQ ID NO: 10. In some embodiments, the mutant hyPB 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 shown in SEQ ID NO: 10.

[0132] In some embodiments, the mutant hyperactive PiggyBac comprises one or more amino acid mutations to increase cleavage activity.

[0133] In some embodiments, the mutated high-activity PiggyBac comprises one or more amino acid mutations to increase cleavage activity, wherein the amino acid mutations are selected from amino acid mutations within the 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, and the position numbers correspond to the amino acid numbers of the unmutated high-activity PiggyBac of SEQ ID NO: 10.

[0134] In some embodiments, the mutated high-activity PiggyBac comprises one or more amino acid mutations to increase cleavage activity, wherein the amino acid mutations are selected from amino acid mutations at positions 450, 560, 564, 573, 589, 592 and / or 594, and the position numbers correspond to the amino acid numbers of the unmutated high-activity PiggyBac of SEQ ID NO: 10.

[0135] In some embodiments, the mutated high-activity PiggyBac comprises one or more amino acid mutations to increase the cleavage activity, wherein the amino acid mutations are selected from amino acid mutations at positions M194 and / or D450, and the position numbers correspond to the amino acid numbers of the unmutated high-activity PiggyBac of SEQ ID NO: 10, preferably selected from amino acid substitutions of M194V and / or D450N.

[0136] In some embodiments, the mutant hyperactive PiggyBac comprises one or more amino acid mutations to reduce DNA binding activity.

[0137] In some embodiments, the mutated hyperactive PiggyBac comprises one or more amino acid mutations to reduce DNA binding activity, wherein the amino acid mutations are selected from amino acid mutations at positions 254, 275, 277, 347, 372, 375 and / or 465, and the position numbers correspond to the amino acid numbers of the unmutated hyperactive PiggyBac of SEQ ID NO: 10.

[0138] In some embodiments, the mutated high-activity PiggyBac comprises one or more amino acid mutations to reduce DNA binding activity, wherein the amino acid mutations are selected from R275, N347, R372, K375, R376, E377 and E380, and the position numbers correspond to the amino acid numbers of the unmutated high-activity PiggyBac of SEQ ID NO:10.

[0139] In some embodiments, the mutated high-activity PiggyBac comprises one or more amino acid mutations to reduce DNA binding activity, wherein the amino acid mutations are selected from R372, K375, R376, E377 and E380, and the position numbers correspond to the amino acid numbers of the unmutated high-activity PiggyBac of SEQ ID NO: 10, preferably selected from amino acid substitutions R372A, K375A, R376A, E377A and / or E380A.

[0140] In some embodiments, the mutated high-activity PiggyBac comprises one or more amino acid mutations to reduce DNA binding activity, wherein the amino acid mutations are selected from N347, R372 and K375, and the position numbers correspond to the amino acid numbers of the unmutated high-activity PiggyBac of SEQ ID NO:10, preferably selected from amino acid substitutions N347S, N347A, R372A, K375A, and more preferably selected from amino acid substitutions N347S, N347A.

[0141] In some embodiments, the mutated hyperactive PiggyBac comprises one or more amino acid mutations as described above to increase cleavage activity; and one or more amino acid mutations as described above to reduce DNA binding activity.

[0142] In some embodiments, the mutated high-activity PiggyBac comprises at least one amino acid substitution at position D450 to increase cleavage activity, and at least two amino acid substitutions at positions N347, R372, and K375 to reduce DNA binding activity, preferably, the mutated high-activity PiggyBac transposase comprises double mutations N347S and D450N or triple mutations D450N, R372A, and K375A, and the position numbers correspond to the amino acid numbers of the unmutated high-activity PiggyBac of SEQ ID NO: 10. In a more preferred embodiment, the mutated high-activity PiggyBac transposase comprises double mutations N347S and D450N, and the position numbers correspond to the amino acid numbers of the unmutated high-activity PiggyBac of SEQ ID NO: 10.

[0143] In some embodiments, the mutated hyperactive PiggyBac as disclosed in the previous embodiments further comprises at least one mutation in the region defined by amino acid position numbers [158-169], e.g., A166S; and / or at least one mutation at positions Y527, R518, K525, N463.

[0144] Typically, the mutated hyperactive PiggyBac comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% identical, or 100% identical to the mutated hyperactive PiggyBac of SEQ ID NO:1.

[0145] In some embodiments, the mutated high-activity PiggyBac is a variant of the high-activity PiggyBac of SEQ ID NO: 10, having one or more amino acid substitutions compared to SEQ ID NO: 10, typically having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions.

[0146] In some embodiments, the mutated hyperactive PiggyBac further comprises one or more of the following amino acid mutations: 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 the position numbers correspond to the amino acid numbers of the hyperactive PiggyBac sequence (SEQ ID NO: 10).

[0147] In some embodiments, the mutant hyperactive PiggyBac comprises the following mutations or mutation combinations: 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.

[0148] In some embodiments, the mutated hyperactive PiggyBac comprises the following amino acid substitutions or a combination of amino acid substitutions:

[0149] -R372A / K375A / D450N,

[0150] -R372A / K375A / R376A / D450N,

[0151] -K375A / R376A / E377A / E380A / D450N,

[0152] -R372A / K375A / R376A / E377A / E380A / D450N,

[0153] -M194V,

[0154] -M194V / R372A / K375A,

[0155] -S351A / R372A / K375A / R388A / D450N / W465A / S573A / M589V / S592G / F594L,

[0156] -R245A / R275A / R277A / R372A / W465A / M589V,

[0157] -R275A / 325A / R372A / T560A,

[0158] -N347A / D450N,

[0159] -N347S / D450N / T560A / S573A / F594L,

[0160] -R202K / R275A / N347S / R372A / D450N / T560A / F594L, R275A / N347S / K375A / D450N / S592G,

[0161] -R275A / N347S / R372A / D450N / T560A / F594L,

[0162] -R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L,

[0163] -R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G,

[0164] -R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L,

[0165] -V34M / R275A / G325A / N347S / S351A / R372A / K375A / D450N / T560A / S564P,

[0166] -G325A / N347S / K375A / D450N / S573A / M589V / S592G,

[0167] -S230N / R277A / N347S / K375A / D450N,

[0168] -T43I / R372A / K375A / A411T / D450N,

[0169] -G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, and

[0170] -Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G,

[0171] The position numbers correspond to the amino acid numbers of the hyperactive PiggyBac sequence (SEQ ID NO: 10).

[0172] Preferred mutant hyperactive PiggyBac transposases used in the present disclosure include mutant hyperactive PiggyBacs comprising the following amino acid substitution combinations:

[0173] -R372A / K375A / D450N,

[0174] -S351A / R372A / K375A / R388A / D450N / W465A / S573A / M589V / S592G / F594L,

[0175] -R245A / R275A / R277A / R372A / W465A / M589V,

[0176] -N347A / D450N,

[0177] -N347S / D450N / T560A / S573A / F594L,

[0178] -R202K / R275A / N347S / R372A / D450N / T560A / F594L,

[0179] -R275A / N347S / K375A / D450N / S592G,

[0180] -R275A / N347S / R372A / D450N / T560A / F594L,

[0181] -R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L,

[0182] -R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G,

[0183] -R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L,

[0184] -V34M / R275A / G325A / N347S / S351A / R372A / K375A / D450N / T560A / S564P,

[0185] -G325A / N347S / K375A / D450N / S573A / M589V / S592G,

[0186] -S230N / R277A / N347S / K375A / D450N,

[0187] -T43I / R372A / K375A / A411T / D450N,

[0188] -G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G,

[0189] -Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G, and

[0190] -R275A / 325A / R372A / T560A,

[0191] The position numbers correspond to the amino acid numbers of the hyperactive PiggyBac sequence (SEQ ID NO: 10).

[0192] In some embodiments, the mutated hyperactive PiggyBac comprises the following amino acid substitutions or a combination of amino acid substitutions:

[0193] -R245A / R275A / R277A / R372A / W465A / M589V,

[0194] -R275A / 325A / R372A / T560A,

[0195] -N347A / D450N,

[0196] -N347S / D450N / T560A / S573A / F594L,

[0197] -R202K / R275A / N347S / R372A / D450N / T560A / F594L,

[0198] -R275A / N347S / K375A / D450N / S592G,

[0199] -R275A / N347S / R372A / D450N / T560A / F594L,

[0200] -R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L,

[0201] -R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G,

[0202] -R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L,

[0203] -G325A / N347S / K375A / D450N / S573A / M589V / S592G,

[0204] -S230N / R277A / N347S / K375A / D450N,

[0205] -G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, and

[0206] -Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G,

[0207] The position numbers correspond to the amino acid numbers of the hyperactive PiggyBac sequence (SEQ ID NO: 10).

[0208] In a more preferred embodiment, the mutated high-activity PiggyBac comprises the following amino acid substitution combinations:

[0209] -N347A / D450N,

[0210] -N347S / D450N / T560A / S573A / F594L,

[0211] -R202K / R275A / N347S / R372A / D450N / T560A / F594L,

[0212] -R275A / N347S / K375A / D450N / S592G,

[0213] -R275A / N347S / R372A / D450N / T560A / F594L,

[0214] -R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L,

[0215] -R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G,

[0216] -R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L,

[0217] -G325A / N347S / K375A / D450N / S573A / M589V / S592G,

[0218] -S230N / R277A / N347S / K375A / D450N,

[0219] -G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G,

[0220] -Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G,

[0221] The position numbers correspond to the amino acid numbers of the hyperactive PiggyBac sequence (SEQ ID NO: 10).

[0222] In some embodiments, the mutant transposase comprises the following amino acid substitution combination: R372A / K375A / D450N, the position numbers correspond to the amino acid numbers of the unmutated hyperactive PiggyBac of SEQ ID NO: 10. In some embodiments, the mutant transposase has the amino acid sequence of SEQ ID NO:11.

[0223] In some embodiments, the mutant transposase has an amino acid sequence selected from any one of SEQ ID NOs: 12-33.

[0224] In some embodiments, the mutant transposase has an amino acid sequence selected from any one of SEQ ID NO: 12 to SEQ ID NO:20.

[0225] In some embodiments, the mutant transposase has an amino acid sequence selected from any one of SEQ ID NO:21 to SEQ ID NO:33.

[0226] In some embodiments, the mutant transposase can comprise one or more mutations relative to hyPB that involve a conserved catalytic triad, such as at amino acids 268 and / or 346 corresponding to the amino acid numbering of SEQ ID NO: 10 (eg, D268N and / or D346N).

[0227] In some embodiments, the mutant transposase can comprise one or more mutations relative to hyPB that are critical for cleavage, such as at amino acids 287, 287 / 290, and / or 460 / 461 corresponding to the amino acid numbering of SEQ ID NO: 10 (e.g., K287A, K287A / K290A, and / or R460A / K461A).

[0228] In some embodiments, the mutant transposase can comprise one or more mutations relative to hyPB that are involved in target joining, such as at amino acids 351, 356, and / or 379 corresponding to the amino acid numbering of SEQ ID NO: 10 (e.g., S351E, S351P, S351A, and / or K356E).

[0229] In some embodiments, the mutant transposase can comprise one or more mutations relative to hyPB that are critical for integration, such as at amino acids 560, 564, 571, 573, 589, 592, and / or 594 corresponding to the amino acid numbering of SEQ ID NO: 10 (e.g., T560A, S564P, S571N, S573A, M589V, S592G, and / or F594L).

[0230] In some embodiments, the mutant transposase can comprise one or more mutations relative to hyPB that are implicated in the alignment, e.g., 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).

[0231] In some embodiments, the mutant transposase can comprise one or more mutations relative to hyPB that are well conserved, such as at amino acids 576 and / or 587 corresponding to the amino acid numbering of SEQ ID NO: 10 (eg, K576A and / or I587A).

[0232] In some embodiments, the mutant transposase may comprise one or more mutations relative to hyPB that are related to Zn2 + Binds, for example, to amino acid number 586 corresponding to SEQ ID NO: 10 (eg, H586A).

[0233] In some embodiments, the programmable transposase can comprise one or more mutations relative to hyPB that are involved in integration, such as 315, 341, 372, and / or 375 corresponding to amino acid numbering of SEQ ID NO: 10 (e.g., R315A, R341A, R372A, and / or K375A).

[0234] In some embodiments, the mutated 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 shown in SEQ ID NO: 10. In some embodiments, the mutated hyperactive PiggyBac is selected for its high specificity for integrating DNA into the genome compared to the hyperactive PiggyBac. In some embodiments, the mutated hyperactive PiggyBac comprises an amino acid sequence having one or more modifications disclosed herein relative to SEQ ID NO: 10, and retains a sequence that is 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% identical to the sequence shown in SEQ ID NO: 12 to SEQ ID NO: 33.

[0235] In some embodiments, the mutated hyperactive PiggyBac transposase can comprise a mutation in one or more amino acids selected from the following 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.

[0236] In some embodiments, the mutant hyperactive PiggyBac transposase mutation can comprise one or more amino acid modifications selected from the group consisting of 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.

[0237] In one embodiment, the mutated hyperactive PiggyBac transposase comprises the amino acid modification D450N, corresponding to the amino acid numbering of SEQ ID NO:10.

[0238] In one embodiment, the mutated hyperactive PiggyBac transposase comprises amino acid modifications R245A and D450, corresponding to the amino acid numbering of SEQ ID NO:10.

[0239] In one embodiment, the mutated hyperactive PiggyBac transposase comprises amino acid modifications R245A, G325A and S573P, corresponding to the amino acid numbering of SEQ ID NO:10.

[0240] In one embodiment, the mutated hyperactive PiggyBac transposase comprises amino acid modifications R245A, G325A, D450 and S573P, corresponding to the amino acid numbering of SEQ ID NO:10.

[0241] In one embodiment, the mutated hyperactive PiggyBac transposase comprises the amino acid modification N347S or N347A, corresponding to the amino acid numbering of SEQ ID NO:10.

[0242] In one embodiment, the mutated hyperactive PiggyBac transposase comprises amino acid modifications N347S and D450N, corresponding to the amino acid numbering of SEQ ID NO:10.

[0243] In another embodiment, the mutated hyperactive PiggyBac transposase comprises amino acid modifications N347A and D450N, corresponding to the amino acid numbering of SEQ ID NO: 10. In some embodiments, the mutated hyperactive PiggyBac transposase comprises the amino acid sequence of SEQ ID NO:21.

[0244] In some embodiments, the mutant hyperactive PiggyBac transposase comprises the amino acid sequence of SEQ ID NO: 10, wherein:

[0245] - the amino acid residue at position 34 is V or M,

[0246] - the amino acid residue at position 43 is T or I,

[0247] - the amino acid residue at position 177 is Y or H,

[0248] - the amino acid residue at position 202 is R or K,

[0249] - the amino acid residue at position 230 is S or N,

[0250] - the amino acid residue at position 245 is A,

[0251] - the amino acid residue at position 268 is D or N,

[0252] - the amino acid residue at position 277 is R or A,

[0253] - the amino acid residue at position 275 is R or A,

[0254] - the amino acid residue at position 277 is R or A,

[0255] - the amino acid residue at position 325 is A or G,

[0256] - the amino acid residue at position 347 is S or A,

[0257] - the amino acid residue at position 351 is E, P or A,

[0258] - the amino acid residue at position 372 is R or A,

[0259] - the amino acid residue at position 375 is K or A,

[0260] - the amino acid residue at position 388 is R or A,

[0261] - the amino acid residue at position 409 is K or A,

[0262] - the amino acid residue at position 411 is A or T,

[0263] - the amino acid residue at position 412 is K or A,

[0264] - the amino acid residue at position 450 is D or N,

[0265] - the amino acid residue at position 460 is R or A,

[0266] - the amino acid residue at position 465 is W or A,

[0267] - the amino acid residue at position 517 is S or A,

[0268] - the amino acid residue at position 560 is T or A,

[0269] - the amino acid residue at position 564 is P or S,

[0270] - the amino acid residue at position 571 is S or N,

[0271] - the amino acid residue at position 573 is S or A,

[0272] - the amino acid residue at position 576 is K or A,

[0273] - the amino acid residue at position 586 is H or A,

[0274] - the amino acid residue at position 587 is I or A,

[0275] - the amino acid residue at position 589 is M or V,

[0276] - the amino acid residue at position 592 is G or S, and / or, - the amino acid residue at position 594 is L or F.

[0277] In some embodiments, the mutated 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 comprising or consisting of 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.

[0278] In some embodiments, the mutated transposase is not a Himar1C9 mutant.

[0279] In some embodiments, the RNA-guided nuclease or nickase and hyPB are combined together in the form of a fusion protein. In some embodiments, the RNA-guided nuclease or nickase and hyPB are contained in a fusion protein. In some embodiments, the fusion protein further comprises a linker.

[0280] In some embodiments, the RNA-guided nuclease / nickase-hyPB fusion protein comprises or consists of:

[0281] (i) a first protein comprising or consisting of an RNA-guided nuclease or nickase, as described above,

[0282] (ii) optionally, a linker, and

[0283] (iii) a second protein comprising or consisting of a transposase, said transposase being a mutant hyPB comprising one or more amino acid mutations compared to the hyPB of SEQ ID NO: 10, as described above.

[0284] In some embodiments, the RNA-guided nuclease or nickase contained in the RNA-guided nuclease / nickase-hyPB fusion protein is a Cas9 protein as described above.

[0285] In some embodiments, the RNA-guided nuclease / nickase-hyPB fusion protein comprises or consists of:

[0286] (i) a first protein comprising or consisting of a Cas9 protein or a variant thereof, as described above,

[0287] (ii) optionally, a linker, and

[0288] (iii) a second protein comprising or consisting of a transposase, said transposase being a mutant hyPB comprising one or more amino acid mutations compared to the hyPB of SEQ ID NO: 10, as described above.

[0289] In some embodiments, the first protein and the second protein can be arranged in any order in the fusion protein.

[0290] In some embodiments, the N-terminus of the RNA-guided nuclease or nickase is fused directly to the C-terminus of hyPB or indirectly through a linker. In another embodiment, the N-terminus of hyPB is fused directly to the C-terminus of the RNA-guided nuclease or nickase or indirectly through a linker.

[0291] In some embodiments, the RNA-guided nuclease or nickase (eg, Cas9 protein) is fused to a lentiviral protein, preferably a structural lentiviral protein (eg, capsid or matrix protein), to facilitate its packaging in lentiviral particles.

[0292] In some embodiments, the RNA-guided nuclease or nickase binds to a viral protein R (VPR). In some embodiments, the VPR has an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 67.

[0293]

[0294] In some embodiments, the VPR-RNA-guided nuclease / nickase fusion protein comprises or consists of:

[0295] (i) a first protein comprising or consisting of VPR or a variant thereof,

[0296] (ii) optionally, a linker, and

[0297] (iii) a second protein comprising or consisting of an RNA-guided nuclease or nickase, as described above.

[0298] In some embodiments, the RNA-guided nuclease or nickase and VPR are combined together in the form of a fusion protein. In some embodiments, the RNA-guided nuclease or nickase contained in the fusion protein is a Cas9 protein as described above.

[0299] In some embodiments, the VPR-Cas9 fusion protein comprises or consists of:

[0300] (i) a first protein comprising or consisting of VPR or a variant thereof,

[0301] (ii) optionally, a linker, and

[0302] (iii) a second protein, which comprises or consists of a Cas9 protein or a variant thereof, as described above.

[0303] In some embodiments, the first protein and the second protein can be arranged in any order in the fusion protein.

[0304] In some embodiments, the N-terminus of Cas9 is fused directly to the C-terminus of VPR or indirectly fused through a linker. 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 to SEQ ID NO:66.

[0305] In some embodiments, the C-terminus of Cas9 is fused to the N-terminus of VPR directly or indirectly through a linker.

[0306] In some embodiments, the VPR-Cas9 fusion protein is further fused to an RNA-guided nuclease or nickase, as described above.

[0307] In some embodiments, the RNA-guided nuclease or nickase is combined with Lens Epithelium Derived Growth Factor (LEDGF, used interchangeably with P75).

[0308] In some embodiments, the P75-RNA-guided nuclease / nickase fusion protein comprises or consists of:

[0309] (i) a first protein comprising or consisting of P75 or a variant thereof,

[0310] (ii) optionally, a linker, and

[0311] (iii) a second protein comprising or consisting of an RNA-guided nuclease or nickase, as described above.

[0312] In some embodiments, the RNA-guided nuclease or nickase and P75 are combined together in the form of a fusion protein. In some embodiments, the RNA-guided nuclease or nickase contained in the fusion protein is a Cas9 protein as described above.

[0313] In some embodiments, the P75-Cas9 fusion protein comprises or consists of:

[0314] (i) a first protein comprising or consisting of P75 or a variant thereof,

[0315] (ii) optionally, a linker, and

[0316] (iii) a second protein, which comprises or consists of a Cas9 protein or a variant thereof, as described above.

[0317] In some embodiments, the first protein and the second protein can be arranged in any order in the fusion protein.

[0318] In some embodiments, the N-terminus of Cas9 is fused directly to the C-terminus of P75 or indirectly fused through a linker. In another embodiment, the C-terminus of Cas9 is fused directly to the N-terminus of P75 or indirectly fused through a linker.

[0319] 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 to 73. In some embodiments, the P75-Cas9 fusion protein has an amino acid sequence selected from the group comprising or consisting of SEQ ID NOs: 69 to 73. In some embodiments, the P75-Cas9 fusion protein has an amino acid sequence of SEQ ID NO: 69. In some embodiments, the P75-Cas9 fusion protein has an amino acid sequence of SEQ ID NO: 70. In some embodiments, the P75-Cas9 fusion protein has an amino acid sequence of SEQ ID NO: 71. In some embodiments, the P75-Cas9 fusion protein has an amino acid sequence of SEQ ID NO: 72. In some embodiments, the P75-Cas9 fusion protein has an amino acid sequence of SEQ ID NO: 73.

[0320] In some embodiments, the P75-Cas9 fusion protein is further fused to an RNA-guided nuclease or nickase as described above.

[0321] 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 an integrase of the lentiviral vector of (i). In some embodiments, the RNA-guided nuclease or nickase is not fused to an integrase (if present) of a fusion protein encoded by the nucleic acid of (v).

[0322] In some embodiments, the RNA-guided nuclease or nickase is combined with an integrase or a mutant thereof as defined herein.

[0323] In some embodiments, the integrase-RNA-guided nuclease / nickase fusion protein comprises or consists of:

[0324] (i) a first protein comprising or consisting of an integrase as defined herein or a mutant thereof,

[0325] (ii) optionally, a linker, and

[0326] (iii) a second protein comprising or consisting of an RNA-guided nuclease or nickase, as described above.

[0327] In some embodiments, the RNA-guided nuclease or nickase defined herein and the integrase or its mutant are combined together in the form of a fusion protein. In some embodiments, the RNA-guided nuclease or nickase contained in the fusion protein is a Cas9 protein as described above.

[0328] In some embodiments, the integrase-Cas9 fusion protein comprises or consists of:

[0329] (i) a first protein comprising or consisting of an integrase as defined herein or a mutant thereof, or a variant thereof,

[0330] (ii) optionally, a linker, and

[0331] (iii) a second protein, which comprises or consists of a Cas9 protein or a variant thereof, as described above.

[0332] In some embodiments, the first protein and the second protein can be arranged in any order in the fusion protein.

[0333] In some embodiments, the N-terminus of Cas9 is directly fused to the C-terminus of an integrase or a mutant thereof as defined herein or is indirectly fused through a linker. In another embodiment, the C-terminus of Cas9 is directly fused to the N-terminus of an integrase or a mutant thereof as defined herein or is indirectly fused through a linker.

[0334] In some embodiments, the integrase-Cas9 fusion protein is further fused to an RNA-guided nuclease or nickase as described above.

[0335] In some embodiments, the RNA-guided nuclease or nickase binds to GAG and / or POL.

[0336] In some embodiments, the GAG ​​and / or POL-RNA-guided nuclease / nickase fusion protein comprises or consists of:

[0337] (i) a first protein comprising or consisting of GAG and / or POL or a variant thereof,

[0338] (ii) optionally, a linker, and

[0339] (iii) a second protein comprising or consisting of an RNA-guided nuclease or nickase, as described above.

[0340] In some embodiments, the RNA-guided nuclease or nickase is combined with GAG and / or POL in the form of a fusion protein. In some embodiments, the RNA-guided nuclease or nickase contained in the fusion protein is a Cas9 protein as described above.

[0341] In some embodiments, the GAG ​​and / or POL-Cas9 fusion protein comprises or consists of:

[0342] (i) a first protein comprising or consisting of GAG and / or POL or a variant thereof,

[0343] (ii) optionally, a linker, and

[0344] (iii) a second protein, which comprises or consists of a Cas9 protein or a variant thereof, as described above.

[0345] In some embodiments, the first protein and the second protein can be arranged in any order in the fusion protein.

[0346] The N-terminus of Cas9 is fused directly to the C-terminus of GAG and / or POL or indirectly fused through a linker. In another embodiment, the C-terminus of Cas9 is fused directly to the N-terminus of GAG and / or POL or indirectly fused through a linker.

[0347] In some embodiments, the GAG ​​and / or POL-Cas9 fusion protein is further fused to an RNA-guided nuclease or nickase as described above.

[0348] According to the invention, the composition comprises a nucleic acid encoding a guide RNA (gRNA) fused to at least one aptamer.

[0349] As used herein, the term "at least one" means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0350] 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".

[0351] In some embodiments, the aptamer binding proteins described herein are capable of binding at least one aptamer.

[0352] In some embodiments, the at least one tetraloop is a MS2 RNA tetraloop binding sequence. In some embodiments, the at least one MS2 RNA tetraloop binding sequence is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 63 (e.g., encoded by a DNA sequence of SEQ ID NO: 62).

[0353]

[0354] In some embodiments, the gRNA is capable of forming a complex with an RNA-guided nuclease or nickase described above, such as a Cas9 protein or a fusion protein comprising Cas9.

[0355] In some embodiments, gRNA is capable of targeting the RNA-guided nuclease or nickase described above 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 to the Cas9 protein.

[0356] In some embodiments, the specific sequence targeted by gRNA may be located in a safe harbor locus in the cell genome. "Safe harbor locus" refers to a region of the cell genome where the integrated material can be fully expressed without disrupting endogenous gene structure or function. Safe harbor loci include, but are not limited to, AAVS1 (intron 1 of PPP1R12C), HPRT, HI 1, hRosa26, albumin, and FA regions. 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 may be selected from: exon 1, intron 1, or exon 2 of PPP1R12C, exon 1, intron 1, or exon 2 of HPRT, exon 1, intron 1, or exon 2 of hRosa26, and intron 1 of the albumin gene. The safe harbor locus may also include a genomic region lacking an endogenous gene and having open chromatin that allows expression of an inserted transgene without disrupting genomic structure or function.

[0357] In some embodiments, the specific sequence targeted by the gRNA may be located within or near a gene in the cell genome. For example, it can be contemplated that the specific sequence targeted by the gRNA is located within or near a gene that requires gene repair or gene modification (by integrating an exogenous copy of the gene, which is brought by the nucleic acid encoding the target transgene in (iv)).

[0358] According to the present invention, the composition comprises a nucleic acid encoding a transgene of interest.

[0359] The term "target transgene" as used herein refers to an exogenous nucleic acid sequence to be inserted into the genome of a cell (preferably a eukaryotic cell, more preferably a mammalian cell), which comprises at least one open reading frame. Typically, the target transgene is a coding sequence, i.e., it encodes at least one protein or polypeptide. However, the target transgene may also be a non-coding sequence.

[0360] In some embodiments, the transgene of interest comprises at least one gene and at least one non-coding sequence, such as a regulatory element or promoter.

[0361] In some embodiments, the transgene 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, wherein the allele is not endogenous to the genome of the cell.

[0362] In some embodiments, the transgene of interest comprises at least one gene from the same species as the cell. In another embodiment, the transgene of interest comprises at least one gene from a different species than the cell.

[0363] In some embodiments, the transgene of interest is at least 500 base pairs (bp) in length. As used herein, at least 500 means 500, 600, 700, 800, 900, 1,000, 10,000, 20,000 bp or more.

[0364] In some embodiments, the transgene of interest is a DNA molecule, an RNA molecule, or a combination thereof. In some embodiments, the transgene of interest is a DNA molecule. In some embodiments, the transgene of interest is an RNA molecule.

[0365] In some embodiments, the transgene of interest may be flanked by inverted terminal repeat (ITR) sequences. This is particularly desirable when an RNA-guided nuclease or nickase is combined with a transposase as described above.

[0366] 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.

[0367] The term "GAG" as used herein refers to a retroviral polyprotein. The GAG ​​polyprotein is usually processed into MA (matrix), CA (capsid) and NC (nucleocapsid) parts by the viral protease (PR) after translation, and sometimes into more parts. For example, the GAG ​​polyprotein of the HIV-1 virus is processed into matrix protein p17, capsid protein p24, nucleocapsid protein p7 and p6-gag after translation. In some embodiments, the fusion protein contains a protein comprising HIV-1GAG polyprotein or a protein consisting of HIV-1GAG polyprotein.

[0368] The term "POL" as used herein refers to a retroviral polyprotein. The POL polyprotein is usually processed by a viral protease (PR) into PR (protease), RT (reverse transcriptase), p15 (ribonuclease H) and INT (integrase) parts after translation. The protease itself is released by autocatalytic cleavage. In some embodiments, the fusion protein contains a protein comprising HIV-1 POL polyprotein or contains a protein consisting of HIV-1 POL polyprotein.

[0369] In some embodiments, the fusion protein comprises a POL polyprotein, and thus the composition may comprise nucleic acid molecules encoding two different integrases: one integrase with impaired integration activity in the lentiviral vector of (i); and another integrase in the fusion protein of (v).

[0370] In some embodiments, the aptamer binding protein is fused to the GAG ​​polyprotein. In some embodiments, the C-terminus of the aptamer binding protein is fused to the N-terminus of the GAG ​​polyprotein, optionally through a linker. In some embodiments, the N-terminus of the aptamer binding protein is fused to the C-terminus of the GAG ​​polyprotein, optionally through a linker.

[0371] In some embodiments, the C-terminus of the aptamer binding protein is fused to the N-terminus of the polyprotein GAG, optionally via a linker. Typically, the fusion protein has the amino acid sequence of SEQ ID NO:64.

[0372]

[0373] In some embodiments, the aptamer binding protein is fused to the POL polyprotein (if present). In some embodiments, the C-terminus of the aptamer binding protein is fused to the N-terminus of the POL polyprotein (preferably GAG-POL polyprotein), optionally via a linker. In some embodiments, the N-terminus of the aptamer binding protein is fused to the C-terminus of the POL polyprotein (preferably GAG-POL polyprotein), optionally via a linker.

[0374] In some embodiments, the C-terminus of the aptamer binding protein is fused to the N-terminus of the multimeric protein GAG-POL, optionally via a linker. Typically, the fusion protein has the amino acid sequence of SEQ ID NO:65.

[0375]

[0376]

[0377] Aptamer binding proteins have been described above, and the description thereof applies here mutatis mutandis.

[0378] In some embodiments, when the fusion protein contains a third protein comprising a POL polyprotein or contains a third protein consisting of a POL polyprotein, the integrase of the POL polyprotein may have impaired integration activity.

[0379] Mutations (including substitutions and deletions) that impair the integration activity of integrase have been described above, and the description thereof also applies here with appropriate modifications.

[0380] 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).

[0381] In some embodiments, the fusion protein comprises one or several linkers.

[0382] In some embodiments, the linker is a peptide linker. In one embodiment, the peptide linker is selected from the group comprising or consisting of: (GGS) n 、(GGGGS) n 、(G) n 、(EAAAK) n , XTEN connector and (XP) n motifs, and any combination of these, wherein n is independently an integer between 1 and 50.

[0383] In some embodiments, the length of the joint is 1 to 24 amino acids, or is encoded by a nucleic acid sequence of 3 to 72 nucleotides in length. In one embodiment, the length of the joint is 1 to 12 amino acids, or is encoded by a nucleic acid sequence of 3 to 36 nucleotides in length. In one embodiment, the length of the joint is 1 to 6 amino acids, or is encoded by a nucleic acid sequence of 3 to 18 nucleotides in length.

[0384] In some embodiments, the linker is an XTEN linker or (GGS) n Connector.

[0385] In some embodiments, the linker is selected from the linkers shown in Table 1.

[0386] Table 1: Connectors

[0387]

[0388]

[0389] In some embodiments, the linker comprises 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 or any combination thereof; encoded by the exemplary nucleic acid sequences 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, respectively.

[0390] 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.

[0391] 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 fused to the C-terminus of the mutant integrase, optionally via a linker. In some embodiments, the C-terminus of the aptamer binding protein is fused to the N-terminus of the mutant integrase, optionally via a linker.

[0392] In some embodiments, the fusion protein further comprises a 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 the VPR. In some embodiments, the 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 the VPR. In some embodiments, the aptamer binding protein is fused to the GAG-POL polyprotein, and the GAG-POL polyprotein is further fused to the VPR.

[0393] In some embodiments, the N-terminus of the integrase of the POL polyprotein is fused to the C-terminus of the VPR, optionally via a linker, and the N-terminus of the aptamer binding protein is fused to the C-terminus of the integrase of the POL polyprotein, optionally via a linker. In some embodiments, the N-terminus of the integrase of the POL polyprotein is fused to the C-terminus of the aptamer binding protein, optionally via a linker, and the N-terminus of the VPR is fused to the C-terminus of the integrase of the -POL polyprotein, optionally via a linker.

[0394] In some embodiments, the N-terminus of the GAG ​​polyprotein is fused to the C-terminus of the VPR, optionally through a linker, and the N-terminus of the aptamer binding protein is fused to the C-terminus of the GAG ​​polyprotein, optionally through a linker. In some embodiments, the N-terminus of the GAG ​​polyprotein is fused to the C-terminus of the aptamer binding protein, optionally through a linker, and the N-terminus of the VPR is fused to the C-terminus of the GAG ​​polyprotein, optionally through a linker.

[0395] In some embodiments, the N-terminus of the GAG-POL polyprotein is fused to the C-terminus of the VPR, optionally through a linker, and the N-terminus of the aptamer binding protein is fused to the C-terminus of the GAG-POL polyprotein, optionally through a linker. In some embodiments, the N-terminus of the GAG-POL polyprotein is fused to the C-terminus of the aptamer binding protein, optionally through a linker, and the N-terminus of the VPR is fused to the C-terminus of the GAG-POL polyprotein, optionally through a linker.

[0396] The present invention also relates to a population of lentiviral particles obtained by the above method for producing lentiviral particles.

[0397] In some embodiments, the population of lentiviral particles comprises lentiviral particles comprising or consisting of:

[0398] (i) lentiviral proteins and / or genes or parts thereof, wherein the lentiviral integrase has an impaired integration activity, as described above,

[0399] (ii) an RNA-guided nuclease or nickase, preferably a Cas9 protein, 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, as described above, or a nucleic acid sequence encoding the same,

[0400] (iii) a guide RNA (gRNA) fused to an aptamer, preferably to at least one MS2 RNA tetraloop binding sequence, as described above, and

[0401] (iv) Optionally, 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, as described above, or a nucleic acid sequence encoding the fusion protein.

[0402] In some embodiments, the lentiviral particles in the population of lentiviral particles further comprise a transgene of interest, as described above.

[0403] In some embodiments, the lentiviral particles are suspended and stored in a suitable solvent, preferably a biocompatible solvent (eg, biological buffer, physiological serum, etc.).

[0404] 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 of 4°C to -200°C. As used herein, the expression "4°C to -200°C" encompasses 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 of 4°C to -80°C. In some embodiments, the lentiviral particles are stored at a temperature of 4°C to -20°C. In some embodiments, the lentiviral particles are stored at a temperature of 4°C to 0°C. In some embodiments, the lentiviral particles are stored at a temperature of 0°C to -200°C. In some embodiments, the lentiviral particles are stored at a temperature of -20°C to -200°C. In some embodiments, the lentiviral particles are stored at a temperature of -80°C to -200°C. In some embodiments, the lentiviral particles are stored at a temperature of -196°C.

[0405] In some embodiments, the lentiviral particles are substantially pure.

[0406] In some embodiments, the lentiviral particle further comprises at least one therapeutic agent.

[0407] In some embodiments, when the lentiviral particle comprises a fusion protein comprising a GAG polyprotein, an aptamer binding protein, and optionally a POL polyprotein, the fusion protein (or a nucleic acid encoding it) and the RNA-guided nuclease or nickase (or a nucleic acid encoding it) can be contained in different lentiviral particles.

[0408] In some embodiments, when the lentiviral particles contain a fusion protein comprising a GAG polyprotein, an aptamer binding protein, and optionally a POL polyprotein, the fusion protein (or a nucleic acid encoding it) and the RNA-guided nuclease or nickase (or a nucleic acid encoding it) are contained in two different lentiviral particles.

[0409] In some embodiments, the population of lentiviral particles comprises:

[0410] A first lentiviral particle comprising or consisting of:

[0411] (i) lentiviral proteins and / or genes or parts thereof, wherein the lentiviral integrase has an impaired integration activity, as described above,

[0412] (ii) an RNA-guided nuclease or nickase, preferably a Cas9 protein, 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, as described above, or a nucleic acid sequence encoding the same, and

[0413] (iii) a guide RNA (gRNA) fused to an aptamer, preferably to at least one MS2 RNA tetraloop binding sequence, as described above; and

[0414] A second lentiviral particle comprising or consisting of:

[0415] (i) lentiviral proteins and / or genes or parts thereof, wherein the lentiviral integrase has an impaired integration activity, as described above, and

[0416] (ii) 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, as described above, or a nucleic acid sequence encoding the fusion protein.

[0417] In some embodiments, the first lentiviral particle, the second lentiviral particle, or both comprise a transgene of interest, as described above.

[0418] In some embodiments, the population of lentiviral particles comprises:

[0419] A first lentiviral particle comprising or consisting of:

[0420] (i) lentiviral proteins and / or genes or parts thereof, wherein the lentiviral integrase has an impaired integration activity, as described above, and

[0421] (ii) an RNA-guided nuclease or nickase, preferably a Cas9 protein, 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, as described above, or a nucleic acid sequence encoding the same; and

[0422] A second lentiviral particle comprising or consisting of:

[0423] (i) lentiviral proteins and / or genes or parts thereof, wherein the lentiviral integrase has an impaired integration activity, as described above,

[0424] (ii) 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, as described above, or a nucleic acid sequence encoding said fusion protein, and

[0425] (iii) a guide RNA (gRNA) fused to an aptamer, preferably to at least one MS2 RNA tetraloop binding sequence, as described above.

[0426] In some embodiments, the first lentiviral particle, the second lentiviral particle, or both comprise a transgene of interest, as described above.

[0427] In some embodiments, when the lentiviral particles contain a fusion protein comprising a GAG polyprotein, an aptamer binding protein, and optionally a POL polyprotein, the fusion protein (or a nucleic acid encoding it) and an RNA-guided nuclease or nickase (or a nucleic acid encoding it) are contained in three different lentiviral particles.

[0428] In some embodiments, the population of lentiviral particles comprises:

[0429] A first lentiviral particle comprising or consisting of:

[0430] (i) lentiviral proteins and / or genes or parts thereof, wherein the lentiviral integrase has an impaired integration activity, as described above, and

[0431] (ii) an RNA-guided nuclease or nickase, preferably a Cas9 protein, 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, as described above, or a nucleic acid sequence encoding the same; and

[0432] A second lentiviral particle comprising or consisting of:

[0433] (i) lentiviral proteins and / or genes or parts thereof, wherein the lentiviral integrase has an impaired integration activity, as described above, and

[0434] (ii) 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, as described above, or a nucleic acid sequence encoding said fusion protein; and

[0435] A third lentiviral particle, comprising or consisting of:

[0436] (i) lentiviral proteins and / or genes or parts thereof, wherein the lentiviral integrase has an impaired integration activity, as described above, and

[0437] (ii) a guide RNA (gRNA) fused to an aptamer, preferably to at least one MS2 RNA tetraloop binding sequence, as described above.

[0438] In some embodiments, the first lentiviral particle, the second lentiviral particle, the third lentiviral particle, or two or all three thereof comprise a transgene of interest as described above.

[0439] In some embodiments, when the lentiviral particles contain a fusion protein comprising a GAG polyprotein, an aptamer binding protein, and optionally a POL polyprotein, the fusion protein (or a nucleic acid encoding it) and an RNA-guided nuclease or nickase (or a nucleic acid encoding it) are contained in four different lentiviral particles.

[0440] In some embodiments, the population of lentiviral particles comprises:

[0441] A first lentiviral particle comprising or consisting of:

[0442] (i) lentiviral proteins and / or genes or parts thereof, wherein the lentiviral integrase has an impaired integration activity, as described above, and

[0443] (ii) an RNA-guided nuclease or nickase, preferably a Cas9 protein, 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, as described above, or a nucleic acid sequence encoding the same; and

[0444] A second lentiviral particle comprising or consisting of:

[0445] (i) lentiviral proteins and / or genes or parts thereof, wherein the lentiviral integrase has an impaired integration activity, as described above, and

[0446] (ii) 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, as described above, or a nucleic acid sequence encoding said fusion protein; and

[0447] A third lentiviral particle, comprising or consisting of:

[0448] (i) lentiviral proteins and / or genes or parts thereof, wherein the lentiviral integrase has an impaired integration activity, as described above, and

[0449] (ii) a guide RNA (gRNA) fused to an aptamer, preferably to at least one MS2 RNA tetraloop binding sequence, as described above; and

[0450] A fourth lentiviral particle, comprising or consisting of:

[0451] (i) lentiviral proteins and / or genes or parts thereof, wherein the lentiviral integrase has an impaired integration activity, as described above, and

[0452] (ii) A transgene of interest as described above.

[0453] The present invention also relates to a method for producing lentiviral particles, comprising the following steps:

[0454] (i) transfecting lentiviral production cells cultured in a suitable culture medium with the composition of the present invention, and

[0455] (ii) collecting the lentiviral particles in the culture medium of the lentiviral producer cells.

[0456] In some embodiments, the lentiviral production cells are transfected with 1, 2, 3, 4 or 5 nucleic acids of the above composition. For example, it can be contemplated that different batches of lentiviral production cells are transfected with different nucleic acids of the above composition to produce different lentiviral particles.

[0457] Cells can be transfected by methods known in the art; non-limiting examples include lipofection, electroporation, sonoporation, gene gun, microinjection, or chemical-based methods, such as calcium phosphate.

[0458] In some embodiments, cells are transfected with nucleic acid molecules of the composition simultaneously and / or separately. In some embodiments, cells are transfected with nucleic acid molecules of the composition simultaneously. In some embodiments, cells are transfected with nucleic acid molecules of the composition separately.

[0459] In some embodiments, lentiviral particles can be obtained by a split system, e.g., a trans-complementation system (vector / packaging system), by in vitro transfection of permissive cells (e.g., HEK 293T cells) with a plasmid containing the lentiviral vector genomic components, wherein the lentiviral vector genomic components include gag, pol, and env sequences encoding the polyproteins GAG, POL, and envelope polypeptides, or a portion of these polypeptides sufficient to form retroviral particles.

[0460] Cells suitable for producing 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 by 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, as well as insect cells (e.g., Spodoptera fugiperda, Sf), or fungal cells (e.g., Saccharomyces, Pichia, and Schizosaccharomyces).

[0461] In some embodiments, the lentiviral particles are secreted by the lentiviral producer cells into their culture medium. In some embodiments, the lentiviral particles are collected in the culture medium of the lentiviral producer cells.

[0462] In some embodiments, the lentiviral particles are collected 12 hours to one week after transfection, preferably 24 to 72 hours after transfection.

[0463] In some embodiments, the lentiviral particles are purified from the culture medium of the lentiviral production cells. The lentiviral particles can be purified by any suitable method to increase the concentration, such as by density gradient purification (e.g., cesium chloride (CsCl)), by chromatographic techniques (e.g., column chromatography or batch chromatography) or by ultracentrifugation. For example, the lentiviral particles of the present invention can be subjected to two or three CsCl density gradient purification steps. It is desirable to use a method comprising lysing cells, applying the lysate to a chromatographic resin, eluting lentiviral particles from the chromatographic resin, and collecting fractions containing lentiviral particles to purify lentiviral particles from infected cells.

[0464] The present invention also relates to a production cell comprising (ie, transfected with) a composition of the present invention.

[0465] In some embodiments, a producer cell comprising a composition of the invention produces lentiviral particles. Typically, lentiviral particles bud from the plasma membrane of the cell.

[0466] In some embodiments, the production cells are from immortalized cell lines. In some embodiments, the production cells are from primary cell cultures.

[0467] In some embodiments, the production cell is a eukaryotic cell. In some embodiments, the production cell is an animal cell or a plant cell. In some embodiments, the production cell is an animal cell. In some embodiments, the production cell is a vertebrate cell or an insect cell. In some embodiments, the production cell is a vertebrate cell. In some embodiments, the production cell is a mammalian cell.

[0468] In some embodiments, the production cell is a non-human mammalian cell (e.g., non-human primate, mouse, rat). In some embodiments, the production cell is a human cell. Cultured cell lines known in the art (e.g., HeLa cells, HEK293 cells, HAP-1 cells, etc.). In some embodiments, the production cell is from a HEK293 cell line. In some embodiments, the production cell is from a HEK293T cell line.

[0469] In some embodiments, the production cell comprises one or more mutations compared to the cell line from which it is derived. In some embodiments, the production cell is not modified compared to the cell line from which it is derived.

[0470] Methods for maintaining cell culture are well known in the art. In some embodiments, the production cells are maintained in a suitable culture medium, such as Dulbecco's Modified Eagle's Medium (DMEM). In some embodiments, the production cells are typically maintained at 37°C, 95% air and 5% CO 2 Next cultivation.

[0471] The present invention also relates to a method for site-specifically integrating a target transgene into a target cell genome, the method comprising infecting the cell with the above-mentioned lentiviral particle group.

[0472] In some embodiments, the method is an in vitro method.

[0473] In some embodiments, the lentiviral particle comprises a transgene of interest. In some embodiments, some of the lentiviral particles in the population of lentiviral particles comprise a transgene of interest.

[0474] In some embodiments, the target transgene is contained in one or more or all of the lentiviral particles of the lentiviral particle group. In some embodiments, the lentiviral particles do not contain the target transgene, and the target transgene is delivered to the target cell by a suitable method, such as by transfection (e.g., lipofection, electroporation, etc.). In some embodiments, the target transgene is delivered to the target cell before the lentiviral particles. In some embodiments, the target transgene is delivered to the target cell simultaneously with the lentiviral particles. In some embodiments, the target transgene is delivered to the target cell after the lentiviral particles.

[0475] In some embodiments, the methods are directed to editing the genome of a target cell, in particular to performing targeted insertion of a transgene of interest.

[0476] In some embodiments, the target cell can be infected by the lentiviral particles of the present invention. In some embodiments, the target cell expresses at least one protein or part thereof or protein complex on its extracellular surface that can be recognized by the lentiviral particles, in particular, recognized by the surface proteins of the lentiviral particles. In some embodiments, the at least one protein or protein complex allows the lentiviral particles to bind (or anchor) and / or enter the target cell.

[0477] In some embodiments, the target cell expresses CD4, CCR5 and / or CXCR4 surface proteins. Typically, CD4, CCR5 and / or CXCR4 interact with HIV-1 surface proteins Gp120 and / or Gp41.

[0478] In some embodiments, the target cells are primary cells or cells from a cell line suitable for lentiviral infection, for example, HEK 293T 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.

[0479] In some embodiments, the target cell artificially expresses at least one surface protein that allows for binding and / or entry of lentiviral particles. "Artificial expression" means that the cell does not endogenously express the surface protein, but has been modified or engineered to transiently or stably express the surface protein.

[0480] In some embodiments, the target cell is a cell isolated from an organism, preferably a mammalian organism, more preferably a human organism. In some embodiments, the target cell is a bone marrow cell. In some embodiments, the target cell is a lymphocyte.

[0481] In some embodiments, the method is used to perform targeted gene insertion in a target cell population that is extracted from an organism, cultured in vitro, and reintroduced into the organism. According to this embodiment, the method is referred to as an ex vivo method.

[0482] It is understood that after the contents of the lentiviral particles are delivered into the target cells, the RNA-guided nuclease or nickase (e.g., Cas9) in complex with the gRNA comprising at least one aptamer (e.g., MS2 RNA tetraloop binding sequence) will interact with the aptamer binding protein (e.g., MCP) fused to the integrase (from the lentiviral vector with impaired integration activity and / or from the fusion protein comprising a third protein, the third protein comprising or consisting of the POL polyprotein (when present), as described above). Specifically, this interaction occurs through an aptamer / aptamer binding protein interaction (e.g., MS2 / MCP).

[0483] In some embodiments, the site specificity of the gRNA directs the RNA-guided nuclease or nickase / integrase complex activity to a specific region or site in the target cell genome (e.g., to a safe harbor locus or within or near a gene in the target cell genome).

[0484] In some embodiments, the target transgene is inserted into a site specifically recognized by the gRNA. In some embodiments, the target transgene is inserted into a position 1 to 20 nucleotides away from the sequence specifically recognized by the gRNA, wherein "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.

[0485] In some embodiments, the mutated integrase activity results in targeted genetic integration of the transgene of interest, also known as site-specific integration.

[0486] In some embodiments, the transgene of interest is not inserted into the coding sequence and / or regulatory sequence of the genome of the target cell. In some embodiments, the targeted gene insertion produced using the methods of the invention does not disrupt, alter, or reduce the expression of one or more genes.

[0487] In some embodiments, the transgene of interest is inserted into a coding sequence and / or a regulatory sequence of the genome of the target cell, resulting in partial or complete inactivation of the coding sequence.

[0488] In some embodiments, the transgene of interest is inserted by nonhomologous end joining (NHEJ). In some embodiments, the transgene of interest is inserted by homologous recombination (HR).

[0489] In some embodiments, the inserted transgene of interest is expressed by the cell. In some embodiments, the inserted transgene of interest is stably expressed by the cell. In some embodiments, expression of the inserted transgene of interest is controlled by a promoter or regulatory element.

[0490] In some embodiments, site-specific integration of the transgene of interest is controlled by sequencing the genomic region surrounding the target site (ie, the site recognized by the gRNA).

[0491] In some embodiments, the ratio of the number of lentiviral particles to the number of cells (multiplicity of infection, MOI) is between MOI 0.5 and MOI 40.

[0492] The present invention also relates to a population of lentiviral particles as described above (plus an optional transgene of interest, when said transgene of interest is not comprised in the lentiviral particles of the population of lentiviral particles), for use as a medicament.

[0493] The present invention also relates to a population of lentiviral particles as described above (plus an optional transgene of interest, when the transgene of interest is not contained in the lentiviral particles of the population of lentiviral particles), for site-specific integration of the transgene of interest into the genome of a target cell.

[0494] The present invention also relates to a lentiviral particle population as described above (plus an optional target transgene, when the target transgene is not contained in the lentiviral particles of the lentiviral particle population), for use in treating a disease in a subject in need thereof. 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 lentiviral particle population as described above (plus an optional target transgene, when the target transgene is not contained in the lentiviral particles of the lentiviral particle population).

[0495] In some embodiments, treatment of a disease is achieved by site-specific integration of a transgene of interest into the genome of cells of a subject in need thereof.

[0496] In some embodiments, the disease is a genetic disease. In some embodiments, the genetic disease is characterized by at least one gene being partially or completely silenced, inactivated, or dysfunctional. In some embodiments, the genetic disease is characterized by at least one gene in the cells of a subject in need encoding at least one non-functional protein. In some embodiments, the genetic disease is characterized by at least one gene in the cells of a subject in need encoding at least one protein or polypeptide that has a deleterious effect on the cell.

[0497] In some embodiments, the disease is cancer. In some embodiments, the cancer is a blood cancer (e.g., leukemia).

[0498] Typically, targeted gene insertion restores normal expression and function of the at least one gene associated with the disease.

[0499] In some embodiments, the population of lentiviral particles will be administered prior to, simultaneously with, or after the second therapeutic agent.

[0500] BRIEF DESCRIPTION OF THE DRAWINGS

[0501] Figure 1A -D involves several packaging strategies to achieve programmable insertion. Figure 1A It was shown that simultaneous loading of nuclease and cargo DNA (co-packaging) was more efficient in targeted integration than adding components separately (editor + DNA). Figure 1B Targeted integration of LV cargo with aptamer binding protein (MCP) fused to polyprotein GAG or polyprotein GAG-POL is shown. Figure 1C Targeted integration of LV cargo with different packaging strategies is shown: co-packaging with NLS (NLS-Cas9), integrase fused to viral protein R (VPRIN-Cas9), LEDGF (p75-LEDGF-Cas9), and Gag-Pol (GAGPOL-Cas9). Figure 1D Shown is how integrase mutant lentiviral proteins determine the efficiency of on-target integration levels following simultaneous SpCas9 nuclease RNP delivery and LV vector infection.

[0502] Figure 2 Targeted DNA integration using different RNA-guided nucleases was shown: SpCas9, SaCas9, Cas12f, and TnpB were effectively used for targeted delivery.

[0503] Figure 3 Shown is the percentage of NHEJ-activated cells after delivery of Cas9 nuclease or Cas9 fused to a programmable transposase (FiCAT) as DNA, RNA, or protein in lentiviral particles using the MS2 system. Loading was measured by NHEJ in a reporter cell line.

[0504] Figure 4A -C involves integration junction sequencing. Figure 4A Shown is a comparison of targeted integration titers (TU / mL) obtained using wild-type lentiviral vectors (WT-LV) and co-delivery of mutant integrase fused to MCP, Cas9 protein, and gRNA containing MS2 (PILV) in HEK293T cells, comparing on-target and off-target integration. Figure 4B Insertion events detected on each chromosome of HEK293T cells are shown; the larger band on chromosome 19 corresponds to on-target integration. Figure 4C Integration junction sequencing is illustrated.

[0505] Figure 5A and 5B The loading strategy for the introduction of Cas9 into lentiviral particles is shown. Figure 5A An overview of virus production and corresponding genome components are shown. Figure 5BSeven (1 to 7) loading strategies for Cas9 loading into VLPs are illustrated. Row 1) overexpression of Cas9; Row 2) fusion with VPR (viral capsid protein); Row 3) fusion with wild-type viral integrase protein; Row 4) fusion with mutant viral integrase protein; Row 5) fusion of Cas9 with both VPR and mutant viral integrase; Row 6) fusion with GAP-POL viral gene; or Row 7) fusion with human endogenous p75 protein.

[0506] Fig. 6A and 6B The loading and activity of Cas9 in VLPs are shown. Fig. 6A Western blot results show the relative loading efficiency of Cas9 in the seven loading strategies 1 to 7 above; condition 0 represents a non-integrated control without Cas9. Figure 6B Editing activity measured by NHEJ reads in next-generation Illumina targeted sequencing of cells infected with viral particles relying on the seven packaging strategies described above is shown using conventional RNA (negative) and gRNA with a tetravalent MS2 domain (for MCP-specific binding).

[0507] Fig. 7A and 7B The MS2-MCP system and activity for Cas9 loading in VLPs are demonstrated. Fig. 7A Schematic diagram of the MS2-MCP system as an aptamer in gRNA for efficient loading of Cas9 in VLPs. Figure 7B The results of Western blots show the relative Cas9 loading efficiency in the seven loading strategies 1 to 7 above; condition 0 represents a non-integrated control without the presence of Cas9. In all cases, the gRNA expressed in the generated cells was the gRNA-MS2 variant.

[0508] Fig. 8A and 8B Shown is the performance of PILV (Programmable Insertion Lentiviral Vector) in a Programmable Insertion Reporter cell line. Fig. 8A Schematic representation of the split GFP reporter system for programmable integration detection. The reporter cargo is cloned into a lentiviral vector that uses LTRs instead of ITRs. Figure 8B The programmable insertion (GFP) and overall insertion (RFP) capacities of different packaging systems tested in the Hershey reporter system are illustrated. The efficiency was measured and compared by flow cytometry analysis.

[0509] Fig. 9Demonstrated performance of PILVs for programmable insertion into endogenous targets in a cell line model. Following infection of HEK293T cells with the PILV packaging strategy and controls, targeted integration measured by junctional qPCR and targeted NHEJ activity measured by Illumina sequencing were plotted to visualize associations. The packaging strategies are as follows: “Clone”, a clonal cell line with a known targeted insertion event as a positive control; “NI”, a non-integrated negative control; “PILV”, a packaging strategy using MCP-GAGPOL, MS2-gRNA and Cas9 overexpression; “MCP-GAG”, MCP is fused to GAG protein, and gRNA-MS2 is used to package Cas9; “Cas9_Insynthetic”, strategy 3) fused to the wild-type viral integrase protein; “Cas9_IND64Vsynth”, strategy 4) fused to mutant viral integrase protein; “Cas9_Ms2_mRNA”, MCP is fused to GAG, expressing Cas9 with an MS2 tag to package mRNA instead of RNP; “VPRIN_Cas9-186+11”, strategy 5) Cas9 is fused to both VPR and mutant viral integrase; “GAGPOL-NLS-Cas9”, strategy 6) fused to the GAP-POL viral gene.

[0510] Fig. 10A , 10B and 10C show improved PILV performance of HDR donors. Fig. 10A Schematic representation of HDR design donors for PILV and programmable integration mechanisms using HDR. Fig. 10B GFP signals of non-integrated and PILV vectors of all three donors in HEK293T cells are shown. Fig. 10C Shown are GFP signals 7 days after transduction of HDR cargo using non-integrating lentiviral particles (NI) or PILV.

[0511] Fig.11A and 11B Improved HDR donor PILV performance for T cell engineering is shown. Fig.11A is a schematic representation of the HDR design donor for PILV of the TRAC locus. Fig. 11B Represents the GFP signal of a non-integrating GFP control and PILV vectors with TRAC programmable insertion in primary T cells from two alternative PBMC donors. Example

[0512] The present invention is further illustrated by the following examples.

[0513] Example 1: Targeted DNA integration using lentiviral vectors

[0514] Materials and methods

[0515] Plasmid construction

[0516] The following plasmids were obtained from Addgene:

[0517] -hCas9 (plasmid #41815), pcDNA TM 3.3-TOPO TM plasmid, expressing human codon-optimized Cas9 nuclease under the control of the CMV promoter;

[0518] -psPAX2 (plasmid #12260), an empty second-generation lentiviral packaging plasmid containing gag, pol, tat, and rev genes;

[0519] - pCMV-VSV-G (plasmid #8454), a plasmid expressing the G glycoprotein of vesicular stomatitis virus under the control of the CMV promoter;

[0520] -pMDLg / pRRE (plasmid #12251), a third-generation lentiviral packaging plasmid, expressing HIV-1 Gag (encoding the major structural protein of the virion), HIV-1 Pol (encoding HIV-1 protease, HIV-1 reverse transcriptase, and HIV-1 integrase), and HIV-1 Rev response element (RRE, a binding site for the Rev protein that promotes RNA export from the cell nucleus);

[0521] -pRSV Rev (plasmid #12253), a third-generation lentiviral packaging plasmid, expressing Rev protein.

[0522] Integrase mutations were obtained 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.

[0523] All other vectors were constructed by Golden Gate assembly using Esp3I and T4 ligase. Plasmids pSICO of SEQ ID NO: 74 (Addgene reference plasmid #41815; a universal vector containing a transgene of interest), pRRL dual of SEQ ID NO: 75 (a universal vector containing a transgene of interest), psMCP-GAG of SEQ ID NO: 76, and psMCP-GAGPOL of SEQ ID NO: 77 were used.

[0524] Cell culture

[0525] 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.

[0526] 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 (100x) (Gibco).

[0527] All cells were maintained at 37°C in 95% air and 5% CO 2 Down growth.

[0528] Lentiviral production

[0529] Lentiviral plasmids were produced according to the protocol provided on the Addgene website https: / / www.addgene.org / protocols / lentivirusproduction / and are recapitulated as follows:

[0530] introduce

[0531] This protocol can be used to transfect lentiviral vectors into Lenti-X293T cells using a polyethyleneimine (PEI) transfection protocol to produce lentivirus. This procedure can be modified to use other packaging cell lines or transfection reagents. Once produced, lentivirus can be used in a variety of downstream applications, such as the production of stable cell lines.

[0532] Workflow Timeline

[0533] Day 0: Inoculation of 293T packaging cells

[0534] Day 1 (afternoon): Transfection of packaging cells

[0535] Day 2 (morning): 18 hours after transfection. Remove the culture medium and replace with fresh medium

[0536] Day 3-4 (Morning): Harvest the virus

[0537] Reagent preparation

[0538] 1. DMEM complete medium: 10% v / v FBS and 4 mM L-alanyl-L-glutamine

[0539] Add 55 mL of heat-inactivated FBS and 11 mL of 200 mM L-alanyl-L-glutamine to a 500 mL bottle of DMEM high glucose medium and store at 4°C.

[0540] 2.25 mM Chloroquine diphosphate

[0541] Dissolve 0.129 g of chloroquine diphosphate in 10 mL of sterile water.

[0542] Sterilize by filtration through a 0.22 μm filter.

[0543] Aliquot into 50-100 μL portions and store at -20°C.

[0544] Aliquots may be thawed and stored at 4°C prior to use. Thawed aliquots should be discarded after 1-2 months.

[0545] 3.1mg / mL PEI, linear MW 25,000Da

[0546] 100 mg of powder was dissolved in 100 mL of deionized water.

[0547] While stirring, slowly add hydrochloric acid until the solution becomes clear.

[0548] Check the pH of the solution.

[0549] Adjust the pH to 7.0 using hydrochloric acid or sodium hydroxide. Usually the solution is alkaline and needs to be adjusted with hydrochloric acid first.

[0550] Allow the solution to mix for 10 minutes, then recheck the pH to make sure it has not fluctuated.

[0551] The solution was filtered through a 0.22 μm membrane.

[0552] Aliquot 500 to 1000 μL into sterile tubes.

[0553] The tubes were stored at -80°C.

[0554] Once thawed, the solution can be stored at 4°C for up to 2 months. After 2 months, discard the tube and thaw a new working stock solution.

[0555] The optimal DNA mass:PEI mass ratio needs to be determined empirically for each new batch of 1 mg / mL PEI and each cell line.

[0556] program

[0557] 293T packaging cells were cultured at 3.8 × 10 6 Cells / plate were seeded in 10 cm tissue culture plates in complete DMEM medium.

[0558] The cells were incubated at 37°C, 5% CO2 for approximately 20 hours.

[0559] The culture medium was gently aspirated, and 10 mL of fresh DMEM complete medium containing 25 μM chloroquine diphosphate was added and incubated for about 5 hours.

[0560] To 10 mL of DMEM complete medium, add 10 μL of 25 mM chloroquine diphosphate.

[0561] Prepare a mixture of 3 plasmids for transfection:

[0562] psPAX2: 1.3 pmol

[0563] pMD2.G: 0.72 pmol

[0564] Transfer plasmid: 1.64 pmol

[0565] OptiPro SFM to a total volume of 500 μL

[0566] 500 μL of the above mixture was diluted into 500 μL PEI-OptiPro SFM with enough PEI to give a μg DNA:μg PEI ratio of 1:3 (1000 μL total per 10 cm dish).

[0567] Using the transfer plasmid pHAGE TRE dCas9-KRAB (total μg of plasmid DNA is 27.8 μg), 416.6 μL of OptiPro SFM per 10 cm dish will contain 83.4 μL of 1 mg / mL PEI.

[0568] Gently add the diluted PEI to the diluted DNA. Gently shake the diluted DNA tube while adding the diluted PEI dropwise. Incubate the mixture at room temperature for 15 to 20 minutes.

[0569] Carefully transfer the transfection mixture to the Lenti-X 293T packaging cells. Add the transfection mixture dropwise, taking care not to dislodge the cells.

[0570] Incubate cells for 18 hours, or until the next morning.

[0571] The next morning, carefully aspirate the medium and replace it with 15 mL of complete DMEM.

[0572] Incubate cells.

[0573] Virus can be harvested at 48, 72, and 96 hours post-transfection, either individually or by pooling all individual harvests. If pooling harvests, transfer harvested media to polypropylene storage tubes and store at 4°C between harvests.

[0574] The viral supernatant was centrifuged at -500 g for 5 minutes to pellet any packaging cells collected during the harvest.

[0575] The supernatant was filtered through a 0.45 μm PES filter.

[0576] Viral supernatant can be stored at 4°C for several hours but should be aliquoted and snap-frozen in liquid nitrogen and stored at -80°C as soon as possible to avoid loss of titer.

[0577] One day before transfection, 4.9 × 10 6 HEK293T cells were grown in 10 cm dishes and transfected with 0.72 pmol of pCMV-VSV-G envelope plasmid, 1.64 pmol of pSICO or pRRL dual cargo (transfer plasmid) and 1.30 pmol of psPAX2 plasmid (wild type or pol mutant).

[0578] For nuclease packaging, 0.65 pmol psPAX2 and 0.65 pmol psMCP-GAG or psMCP-GAGPOL plasmid were used together with 0.65 pmol nuclease plasmid.

[0579] For transfection, the plasmid was mixed in 500 μl Optimem and 100 mg polyethyleneimine (PEI). Two days after plasmid transfection, the supernatant was harvested and filtered, centrifuged overnight at 4000 g and 4° C. The supernatant was discarded and the lentiviral particles were resuspended to reach 100× vector concentration.

[0580] Determination of nuclease activity

[0581] For VPR / IN / LEDGF nuclease fusion activity, a traffic light reporter (Certo et al., 2011. Nat Methods. 8(8): 671-676) HEK293T cell line containing an out-of-frame mCherry ORF reconstituted by NHEJ activity after Cas9 targeting was used. Fluorescence was measured by flow cytometry using a BD LSR Fortessa instrument (yellow-green 561 nm laser with 610 / 20 filter).

[0582] The effects of SaCas9, AsCas12f, and TnpB editing and the MS2 scaffold in the gRNA were evaluated in HEK293T cells by transfecting cells with gRNA at 240,000 cells / well in p12 cells. Three days after nuclease addition, cells were pelleted and DNA was extracted using the KAPPA rapid extraction method. The corresponding target sites were PCR amplified and analyzed by synthetic MiSeq TM Platform Sequencing was performed on an Illumina sequencer using the MiSeq kit v2 (300 cycles, 2×150 configuration).

[0583] Determination of on-target integration

[0584] A reporter cell line (Hershey) was established to assess targeted integration, in which reconstitution of a fluorescent protein ORF after on-target integration results in a measurable fluorescent signal (GFP).

[0585] The viral load plasmid containing 1 / 2 Emerald GFP (emGFP) and 1 / 2 intron was packaged into a lentiviral vector with the corresponding packaging system and / or integrase mutant. The C-terminal half (Ct) of the promoter-free emGFP before the splice acceptor was randomly inserted into the HEK293T cell genome to construct a reporter cell line. A "target site" was added upstream of Ct emGFP.

[0586] For experiments, 200 000 HEK293T Hershey reporter cells were seeded in 12 wells / plate and infected with lentiviral particles the following day at the same time as transfection, which resulted in emGFP reconstitution upon addition of the cargo plasmid.

[0587] Lentivirus was produced using standard protocols. Four days after infection, emGFP fluorescence signal was measured by FACS (BD LSRFortessa instrument; blue 488 nm laser with 530 / 30 filter, yellow-green 561 nm laser with 610 / 20 filter).

[0588] Integration junction sequencing

[0589] The INSERTseq method combines targeted amplification of integrated DNA, UMI-based PCR bias correction, and OxfordNanopore long-read sequencing to robustly analyze DNA integration in the genome. INSERT-seq is able to detect events with a frequency of up to 0.1%. INSERT-seq can fully process all insertions, regardless of the size of the repeat.

[0590] Library preparation and sequencing

[0591] DNA was extracted using the Nanobind kit (Circulomics, catalog number NB-900-001-01) and quantified using g-TUBE TM (Covaris, catalog number 520079) was sheared into fragments of about 2 kb. The WGP primer mixture in the Nanopore PCR Barcoding Kit (SQKPBK004) was additionally added to the second PCR. Sequencing was performed in a Flongle R9.4.1 flow cell with a total output of about 300,000 reads. In order to calculate the limit of detection (LOD), monoclonal samples from HEK293T cells (with a true lentiviral insert) were diluted with WT genomic material at a ratio of 1 / 100, 1 / 1 000 and 1 / 10 000. The dilutions were sequenced in a Flongle R9.4.1 flow cell, and sequencing outputs of 500,000, 500,000 and 150,000 reads were obtained, respectively. Analysis was performed after INSERTseq analysis.

[0592] Integration site analysis

[0593] Nanopore raw reads were base-called using Guppy 4.0.11 (provided by ONT through its webpage https: / / community.nanoporetech.com).

[0594] Read quality was assessed 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 (pipeline-umi-amplicon released by ONT, https: / / github.com / nanoporetech / pipeline-umi-amplicon). Clustering was performed by extracting UMI sequences using a Python script, the sequences were clustered using vsearch (Rognes et al., 2016. Peer J. 4: e2584), and the consensus sequence for clustering was obtained by performing two rounds of racon modification (Vaser et al., 2017. Genome Res. 27(5): 737-746) and two rounds of medaka (https: / / github.com / nanoporetech / medaka).

[0595] In order to analyze the insertion, the read is filtered to force the presence of the adapter used, and cutadapt (Martin, 2011.EMBnet j.17 (1): 10-12) is used to trim to remove the adapter and the insertion sequence from the read. Reads are mapped to the reference genome using minimap2 (Li, 2018.Bioinformatics.34 (18): 3094-3100) "map-ont" default parameters, and Python script is used to filter, and the unique mapping reads with a mapping quality higher than 30 are selected. Bedtools (Quinlan & Hall, 2010.Bioinformatics.26 (6): 841-842) is used to return the first output, which is in the form of bed, containing all mapped reads. Afterwards, Python script is used to perform the peak call step, and the peak is filtered by shape. When the residual sum of squares (RSS) of the peak coverage fitting to the beta distribution is less than 1, it is considered that the peak has passed the shape filter.

[0596] Implementation of the optimized INSERTseq protocol

[0597] Genomic DNA is extracted, fragmented, end-repaired, and A-tailed, and then joined with adapters containing UMIs for read clustering and barcodes for sample demultiplexing in the computational pipeline. Reads are clustered by UMI, integrated sequences and adapters are filtered and trimmed, reads are mapped to the reference genome, and significant peaks are reported and annotated.

[0598] result

[0599] Nuclease packaging strategy

[0600] Given that lentiviral DNA integrates across double-strand breaks (DSBs), it was reasoned that packaging a functional Cas9-gRNA into lentiviral particles should result in targeted integration.

[0601] Six different packaging system configurations were tested to achieve targeted integration by packaging the SpCas9 protein into lentiviruses:

[0602] (i) co-expression of spCas9 during vector production,

[0603] (ii) co-expression of SpCas9 fused to the virion targeting protein Vpr p6-GAG interaction domain as previously described (Indikova & Indik, 2020. Nucleic Acids Res. 48(14):8178-8187),

[0604] (iii) SpCas9 fused to integrase protein,

[0605] (iv) fused to the VPR integrase as previously described (Montagna et al., 2018. Mol Ther Nucleic Acids. 12:453-462),

[0606] (v) fused to the integrase binding domain of the LEDGF p75 chromatin docking factor, as previously described (Hare et al., 2009. PLoS Pathog. 5(1):e1000259), and

[0607] (vi) Direct fusion with GAGPOL polyprotein in packaging plasmid.

[0608] SpCas9 protein was detected in all packaging systems, but no nonhomologous end joining (NHEJ) editing was detected after lentiviral administration to cultured cells. One hypothesis is that the lack of editing may be due to the absence of SpCas9 gRNA, as U6-driven expression localizes the gRNA to the nucleus, whereas cytoplasmic gRNA expression has been shown to rescue gRNA packaging in lentiviral-derived vesicles.

[0609] MCP-GAGPOL was used to package the fusion protein and the MS2 stem-loop containing the SpCas9 gRNA to recruit the gRNA into the lentiviral particles. Using this configuration, editing activity was rescued when transduced with lentiviral particles ( Figure 1A ).

[0610] The targeted integration efficiency was compared between lentiviral particles containing the aptamer binding protein MS2 capsid protein (MCP) fused to the lentiviral polyprotein GAG-POL or the polyprotein GAG alone. Figure 1B ). Both situations resulted in similar targeted integration rates.

[0611] Targeted integration was observed upon simultaneous lentiviral infection and Cas9 RNP delivery in Jurkat-T cells. Programmable lentiviruses (PILVs) were generated using four different packaging strategies: Cas9 co-packaged with NLS (NLS-Cas9), with integrase fused to viral protein R (VPRIN-Cas9), with LEDGF (p75-LEDGF-Cas9), or with Gag-Pol (GAGPOL-Cas9). NLS-Cas9 and VPRIN-Cas9 produced the highest insertion rates ( Figure 1C ).

[0612] Several integrase (IN) mutants were tested, and all showed improved targeted integration performance compared to wild-type integrase ( Figure 1D ).

[0613] Finally, the proportion of NHEJ activated cells was compared when the DNA payload was delivered using the MS2 system using editing tools, either Cas9 nuclease or a fusion protein containing Cas9 and a programmable transposase (PT condition) in lentiviral particles. In addition, editing tools were provided in the form of DNA, RNA, or protein (RNP).

[0614] Compatibility with other nucleases

[0615] Given the large number of programmable nucleases described since SpCas9, we tested a programmable lentiviral packaging system (PILV) with different programmable nucleases (AsCas12 and TnpB). Targeted integration was detected in all systems ( Figure 2 ), indicating that the system is compatible with any nuclease.

[0616] Co-delivery with programmable transposase

[0617] Using the MCP-MS2 system, Cas9 or a fusion protein containing Cas9 and the highly active PiggyBac transposase (FiCAT, with R372A / K375A / D450N substitutions) was loaded into lentiviral particles. Editing activity ( Figure 3 ).

[0618] The results showed that gene editing activity was present in both cases, but better results were obtained when the gene editor was delivered in protein form.

[0619] Measurement of on-target integration

[0620] On-target integration was measured using the INSERTseq method, as Figure 4B and Figure 4C shown.

[0621] like Figure 4A As shown, overall integration and on-target integration activity were compared between wild-type lentiviral particles (WT-LV) and programmable lentiviruses (PILV). PILV was able to achieve precise on-target integration compared to WT LV, making it an optimal system for therapeutic applications.

[0622] Example 2: Strategy for introducing Cas9 into lentiviral particles

[0623] To introduce Cas9 nucleases into virus-like particles (VLPs), several approaches to express nucleases in lentiviral producer cells can be used. In addition to overexpression and passive loading in the cytoplasm of producer cells ( Figure 5A ), six other Cas9 loading strategies were explored. These strategies ( Figure 5B ) include: 1) Cas9 overexpression; 2) fusion with VPR (viral capsid protein); 3) fusion with wild-type viral integrase protein; 4) fusion with mutant viral integrase protein; 5) fusion of Cas9 with both VPR and mutant viral integrase; 6) fusion with GAP-POL viral gene; or 7) fusion with human endogenous p75 protein.

[0624] Materials and methods

[0625] Cell culture and plasmid cloning

[0626] 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.

[0627] 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 (100x) (Gibco).

[0628] All cell lines were maintained at 37°C in 95% air and 5% CO 2 Down growth.

[0629] Other vectors not included in Example 1 were constructed by Golden Gate assembly using Esp3I and T4 ligase. Plasmid pRRL_Cas9_Insynth (SEQ ID NO: 79) containing a fusion of codon-optimized IN and Cas9, and plasmid pRRL_VPR-IN186opt-Cas9 (SEQ ID NO: 80) containing a fusion of VPR, mutant IN and Cas9 were used.

[0630] Lentiviral production

[0631] Lentiviral plasmids were produced according to the protocol provided on the Addgene website https: / / www.addgene.org / protocols / lentivirusproduction / and are recapitulated as follows:

[0632] introduce

[0633] This protocol can be used to produce lentivirus by transfecting lentiviral vectors into Lenti-X293T cells using a polyethyleneimine (PEI) transfection protocol. This procedure can be modified to use other packaging cell lines or transfection reagents. Once produced, lentivirus can be used in a variety of downstream applications, such as the production of stable cell lines.

[0634] Workflow Timeline

[0635] Day 0: Inoculation of 293T packaging cells

[0636] Day 1 (afternoon): Transfection of packaging cells

[0637] Day 2 (morning): 18 hours after transfection. Remove the culture medium and replace with fresh medium

[0638] Day 3-4 (Morning): Harvest the virus

[0639] Reagent preparation

[0640] 1. DMEM complete medium: 10% v / v FBS and 4 mM L-alanyl-L-glutamine

[0641] Add 55 mL of heat-inactivated FBS and 11 mL of 200 mM L-alanyl-L-glutamine to a 500 mL bottle of DMEM high glucose medium. Store at 4°C.

[0642] 2.25 mM Chloroquine diphosphate

[0643] Dissolve 0.129 g of chloroquine diphosphate in 10 mL of sterile water.

[0644] Sterilize by filtration through a 0.22 μm filter.

[0645] Aliquot into 50-100 μL portions and store at -20°C.

[0646] Aliquots may be thawed and stored at 4°C prior to use. Thawed aliquots should be discarded after 1-2 months.

[0647] 3.1mg / mL PEI, linear MW 25,000Da

[0648] 100 mg of powder was dissolved in 100 mL of deionized water.

[0649] While stirring, slowly add hydrochloric acid until the solution becomes clear.

[0650] Check the pH of the solution.

[0651] Adjust the pH to 7.0 using hydrochloric acid or sodium hydroxide. Usually the solution is alkaline and needs to be adjusted with hydrochloric acid first.

[0652] Allow the solution to mix for 10 minutes, then recheck the pH to make sure it has not fluctuated.

[0653] The solution was filtered through a 0.22 μm membrane.

[0654] Aliquot 500 to 1000 μL into sterile tubes.

[0655] The tubes were stored at -80°C.

[0656] Once thawed, the solution can be stored at 4°C for up to 2 months. After 2 months, discard the tube and thaw a new working stock solution.

[0657] The optimal DNA mass:PEI mass ratio needs to be determined empirically for each new batch of 1 mg / mL PEI and each cell line.

[0658] program

[0659] 293T packaging cells were cultured at 3.8 × 10 6 Cells / plate were seeded in 10 cm tissue culture plates in complete DMEM medium.

[0660] The cells were incubated at 37°C, 5% CO2 for approximately 20 hours.

[0661] The culture medium was gently aspirated, and 10 mL of fresh DMEM complete medium containing 25 μM chloroquine diphosphate was added and incubated for about 5 hours.

[0662] To 10 mL of DMEM complete medium, add 10 μL of 25 mM chloroquine diphosphate.

[0663] Prepare a mixture of 3 plasmids for transfection:

[0664] psPAX2: 1.3 pmol

[0665] pMD2.G: 0.72 pmol

[0666] Transfer plasmid: 1.64 pmol

[0667] OptiPro SFM to a total volume of 500 μL

[0668] Dilute 500 μL of the above mixture into 500 μL of PEI-OptiPro SFM with enough PEI to give a μg DNA:μg PEI ratio of 1:3 (1000 μL total per 10 cm dish).

[0669] Using the transfer plasmid pHAGE TRE dCas9-KRAB (total μg of plasmid DNA is 27.8 μg), 416.6 μL of OptiPro SFM per 10 cm dish will contain 83.4 μL of 1 mg / mL PEI.

[0670] Gently add the diluted PEI to the diluted DNA. Gently shake the diluted DNA tube while adding the diluted PEI dropwise. Incubate the mixture at room temperature for 15 to 20 minutes.

[0671] Carefully transfer the transfection mixture to the Lenti-X 293T packaging cells. Add the transfection mixture dropwise, taking care not to dislodge the cells.

[0672] Incubate cells for 18 hours, or until the next morning.

[0673] The next morning, carefully aspirate the medium and replace it with 15 mL of complete DMEM.

[0674] Incubate cells.

[0675] Virus can be harvested at 48, 72, and 96 hours post-transfection, either individually or by pooling all individual harvests. If pooling harvests, transfer harvested media to polypropylene storage tubes and store at 4°C between harvests.

[0676] The viral supernatant was centrifuged at -500 g for 5 minutes to pellet any packaging cells collected during the harvest.

[0677] The supernatant was filtered through a 0.45 μm PES filter.

[0678] Viral supernatant can be stored at 4°C for several hours but should be aliquoted and snap-frozen in liquid nitrogen and stored at -80°C as soon as possible to avoid loss of titer.

[0679] One day before transfection, 4.9 × 10 6 HEK293T cells were grown in 10 cm dishes and transfected with 0.72 pmol of pCMV-VSV-G envelope plasmid, 1.64 pmol of pSICO or pRRL dual cargo (transfer plasmid) and 1.30 pmol of psPAX2 plasmid (wild type or pol mutant).

[0680] For nuclease packaging, 0.65 pmol psPAX2 and 0.65 pmol psMCP-GAG or psMCP-GAGPOL plasmid were used together with 0.65 pmol nuclease plasmid.

[0681] For transfection, the plasmid was mixed in 500 μl Optimem and 100 mg polyethyleneimine (PEI). Two days after plasmid transfection, the supernatant was harvested and filtered, centrifuged overnight at 4000 g and 4° C. The supernatant was discarded and the lentiviral particles were resuspended to reach 100× vector concentration.

[0682] Determination of nuclease activity

[0683] The produced lentiviral particles loaded with Cas9 and gRNA using different strategies were used to infect HEK293T or other cell line models. Three days after the addition of nuclease to the viral particles, the cells were pelleted and DNA was extracted using the KAPPA rapid extraction method. The corresponding target sites were PCR amplified and analyzed by synthetic MiSeq TM Platform Sequencing was performed on an Illumina sequencer using the MiSeq kit v2 (300 cycles, 2×150 configuration).

[0684] Determination of nuclease activity

[0685] Lentiviral particles were lysed in RIPA buffer to obtain proteins. Proteins were analyzed by Western blotting according to standard methods. Briefly, proteins were loaded and run in SDS-PAGE gels (Invitrogen) and then transferred to nitrocellulose blots and protein detection was performed using anti-Cas9 and anti-p24 antibodies (AbCam) and HRP-conjugated secondary antibodies. Proteins were detected and quantified by chemiluminescence using luminol as a substrate.

[0686] result

[0687] Although the presence of Cas9 was achieved by most of the 7 strategies tested ( Fig. 6A ), no nuclease activity could be detected using these methods ( Figure 6B ).

[0688] Next, we tested several alternative loading strategies using the MS2-MCP RNA protein system to exploit the interaction of gRNA with Cas9. In one strategy, we co-expressed a tetravalent MS2 in gRNA and a modified GAG-POL viral gene fused to the MCP protein to actively load Cas9 into viral particles along with gRNA for full targeting and cleavage of target DNA in infected cells ( Fig. 6A ).

[0689] This method showed that the presence of Cas9 increased when the MCP-MS2 system was used ( Figure 7B ), and nuclease activity in infected cells was restored, but surprisingly, nuclease activity in infected cells was fully restored when the MCP-MS2 system was used ( Figure 6B ).

[0690] Example 3: Validation of the best performing Cas9-loaded PILV prototype

[0691] Materials and methods

[0692] Lentiviral particles produced according to different loading strategies (production methods are the same as in Examples 1 and 2, but with different Cas9 and gRNA loading strategies) were used to infect HEK293T reporter cells or K562 cells. The infected cells were analyzed as follows.

[0693] Determination of nuclease activity

[0694] Three days after the addition of nuclease to the viral particles, the cells were pelleted and DNA was extracted using the KAPPA rapid extraction method. The corresponding target sites were PCR amplified and analyzed by synthetic MiSeq TM Platform Sequencing was performed on an Illumina sequencer using the MiSeq kit v2 (300 cycles, 2×150 configuration).

[0695] Determination of on-target integration

[0696] A reporter cell line (Hershey) was established to assess targeted integration, in which reconstitution of a fluorescent protein ORF produces a measurable fluorescent signal (GFP) upon on-target integration.

[0697] The viral load plasmid containing 1 / 2 Emerald GFP (emGFP) and 1 / 2 intron was packaged in a lentiviral vector using the corresponding packaging system and / or integrase mutant. The C-terminal half (Ct) of the promoter-free emGFP before the splice acceptor was randomly inserted into the HEK293T cell genome to construct a reporter cell line. A "target site" was added upstream of Ct emGFP.

[0698] For the experiments, 200 000 HEK293T Hershey reporter cells were seeded in 12 wells / plate and infected with lentiviral particles the next day at the same time as transfection, which resulted in reconstitution of emGFP upon addition of the cargo plasmid.

[0699] Lentivirus was produced using standard protocols. Four days after infection, emGFP fluorescence signal was measured by FACS (BD LSRFortessa instrument; blue 488 nm laser with 530 / 30 filter, yellow-green 561 nm laser with 610 / 20 filter).

[0700] For K562 cells, genomic DNA was extracted 5 days after infection, and junction PCR amplification of target sites and viral load was performed by semiquantitative standard methods (SYBRGreen qPCR, Applied Biosystems) and compared with total viral copy number and endogenous gene copy number.

[0701] result

[0702] After establishing a loading system for Cas9 and gRNA to incorporate efficient nuclease activity into infected donors, several integrase variants were tested in the context of integrase-deficient viral particles to enable programmable integration.

[0703] To evaluate programmable integration of gRNAs at specific target sites, we used a split GFP reporter-based reporter HEK293T cell line ( Fig. 8A ). The efficiency of each packaging system was evaluated, and the results showed that the best performing systems were the fusion of MCP-GAG with gRNA-MS2 and the use of the MCP-MS2 system to encapsulate mRNA without using mRNA. PILV using MCP-GAGPOL showed very promising results ( Figure 8B). PILVs using MCP-GAGPOL were improved in subsequent replicates by increasing productivity. Direct fusions to integrase or Pol gave the worst results.

[0704] In addition to the cellular reporter system for programmable integration, the programmable insertion efficiency of PILVs was tested and validated in the endogenous target site of the alternative cell line model K562. Programmable integration efficiency after episomal decay was assessed by junction-targeted qPCR, and relative copy numbers were plotted and compared to targeted NHEJ activity ( Fig. 9 Strategy 1, involving MCP-GAGPOL fusion and MS2-MCP interaction with gRNA and MCP-GAG fusion, showed the most promising results, followed by mRNA encapsulation in virus-like particles instead of RNPs.

[0705] Example 4: Programmable integration of PILV genomes via homology-dependent recombination (HDR)

[0706] Materials and methods

[0707] Cell culture and plasmid cloning

[0708] 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.

[0709] K-562 cells (ATCC CRL-3343) and T cells (isolated from human donors and activated) were grown in RPMI 1640 medium (Gibco) supplemented with 10% FBS, 1% penicillin-streptomycin (Gibco) and 1% GlutaMAX (100x) (Gibco).

[0710] T cells were isolated from buffy coats of up to 4 human donors; PBMCs were purified and T cells were activated using standard methods for active proliferation prior to infection.

[0711] All cell lines were maintained at 37°C in 95% air and 5% CO 2 Down growth.

[0712] The remaining 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 load donor with a GFP tag, was used for targeted integration at the RAB11A site, SEQ ID NO: 81; plasmid pLV RAB11A-HA-GFPinv-HA, a lentiviral load donor with an inverted GFP tag, was used for targeted integration at the RAB11A site, SEQ ID NO: 82; plasmid pLV FBL-HA-mGFP-HA, a lentiviral load donor with a GFP tag, was used for targeted integration at the FBL site, SEQ ID NO: 83; plasmid pLV GFP-TRAC-HA, a lentiviral load donor with a GFP tag, was used for targeted integration at the TRAC site, SEQ ID NO: 84; plasmid pLV eGFP-TRAC-HA, a lentiviral load donor with an eGFP tag, was used for targeted integration at the TRAC site, SEQ ID NO: 85.

[0713] Determination of on-target integration

[0714] Primary T cells or HEK293T, K562 cell line models were infected with viral particles produced by the same method as in Examples 1 and 2, which carried an HDR gene capture reporter on the endogenous gene, in which a GFP tag was added to the protein for site-specific insertion detection.

[0715] For the experiments, 200 000 HEK293T or K562, or 500 000 activated T cells were infected with lentiviral particles. The GFP fluorescence signal was measured at different time points by FACS (BD LSR Fortessa instrument; blue 488 nm laser with 530 / 30 filter, yellow-green 561 nm laser with 610 / 20 filter) to track the signal.

[0716] result

[0717] The cargo donors were improved by generating viral genomes for programmable insertion with homology arms to the target site of interest to improve efficiency in actively dividing cells. Three alternative PILV cargos were designed and generated ( Fig. 10A In HEK293T cells ( Fig. 10B ), the RAB11A reporter construct produced a high level of nonspecific signal, but the inverted version of this promoter greatly reduced the noise signal and increased fluorescence under PILV conditions, consistent with programmable insertion of the cargo. Fig. 10CThe results showed a slight increase in PILV signal on both cell lines, consistent with programmable insertion of the HDR payload.

[0718] In addition, the FBL reporter gene also showed similar results, with the specific GFP signal of PILV in HEK293T cells reaching 10% ( Fig. 10B Similar HDR loading results were observed in the cell models K562 and JurcáT, with episomal leaky expression and increased PILV signals in both loadings ( Fig. 10C ).

[0719] To verify the therapeutic potential of programmable integration of lentiviral vectors in cell engineering, a TRAC-specific gene trap was designed and constructed to generate T cells with therapeutic cargo inserted into the TRAC locus ( Fig.11A HDR-based programmable insertion of TRAC-specific PILV payloads was tested in primary T cells from two different PBMC donors. The transduction efficiency of the programmable insertion vector exceeded 50% compared to a non-integrated control with no transduction efficiency ( Fig. 11B ).

[0720] These results validate the transfer potential of PILV as a safe and controllable way to engineer T cells and other primary cells for therapeutic purposes.

[0721] Summarize

[0722] Targeted DNA integration of gene-sized fragments into mammalian genomes enables precise addition of therapeutic information and new properties. Lentiviral vectors are used therapeutically for uncontrolled insertion of transgenes for ex vivo applications in advanced cell therapy, as well as in vivo gene therapy. By mutating the integrase protein and incorporating the SpCas9 nuclease into lentiviral particles, we observed that lentiviral vectors can be reprogrammed to precisely integrate transgenic DNA.

[0723] Precise DNA integration was consistent in immortalized cell models and primary cells. PILVs can also deliver transgenes to the ROSA26 locus in mouse liver. We also detected PILV cis integration after simultaneous delivery of two transgenes, which provides a reliable method for multiple KO-KI in ex vivo models. By changing the two packaging vectors during lentiviral production, lentiviral vectors can become programmable to achieve targeted DNA addition for ex vivo cell manufacturing, in vivo gene replacement, and mammalian genome engineering.

Claims

1. A composition comprising: (i) a nucleic acid encoding a lentiviral vector having impaired 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, and (v) a nucleic acid encoding a fusion protein comprising: - 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 which comprises or consists of the POL polyprotein.

2. The composition according to claim 1, wherein: - the aptamer binding protein is a MS2 bacteriophage capsid protein (MCP), preferably having at least 75% identity to SEQ ID NO: 61, and - The aptamer is a MS2 RNA tetraloop binding sequence, preferably having at least 75% identity to SEQ ID NO:

63.

3. The composition of claim 1 or 2, wherein the RNA-guided nuclease or nickase is not fused to an integrase.

4. A composition according to any one of claims 1 to 3, wherein the RNA-guided nuclease or nickase is a Cas protein.

5. The composition of any one of claims 1 to 4, wherein the RNA-guided nuclease or nickase is combined with a mutant hyperactive PiggyBac transposase.

6. A composition according to any one of claims 1 to 5, wherein the integrase of (i) and / or the integrase comprised in the POL polyprotein of (v), when present, comprises 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 of the HIV-1 integrase of SEQ ID NO: 1, or at a corresponding position in another integrase.

7. The composition according to any one of claims 1 to 6, wherein 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, T 122A, T122R, A124D, A124E, A124R, A124K, A128T, E152A, E152D, D164N, Q168L, Q168A, E170G, F185K, K186E, R231G, R231K, R231D, R231E, R231S, K264R, K266R, and K273R, or at the corresponding positions of another integrase.

8. The composition according to any one of claims 1 to 7, wherein the integration activity of the lentiviral vector of (i) is reduced or eliminated by at least one mutation in the integrase sequence, the mutation being selected from: D116N substitution, numbering based on HIV-1 integrase of SEQ ID NO: 1; D164N substitution, numbering based on HIV-1 integrase of SEQ ID NO: 1; premature stop codon insertion; and deletion (ΔIN) of the integrase gene.

9. A method for preparing lentiviral particles, comprising the following steps: (i) transfecting a lentivirus producing cell cultured in a suitable medium with the composition of any one of claims 1 to 8, and (ii) collecting the lentiviral particles in the culture medium of the lentiviral producer cells.

10. A population of lentiviral particles obtainable by the method of claim 9.

11. A population of lentiviral particles comprising lentiviral particles, wherein the lentiviral particles comprise or consist of: a. lentiviral proteins and / or genes or parts thereof, wherein the lentiviral integrase has impaired integration activity, b. an RNA-guided nuclease or nickase, preferably a Cas9 protein, or a nucleic acid sequence encoding the same, c. a guide RNA fused to an aptamer, preferably to at least one MS2 RNA tetraloop binding sequence, and d. 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, or a nucleic acid sequence encoding the fusion protein.

12. An in vitro method for site-specific integration of a target transgene into the genome of a cell, the method comprising infecting the cell with the lentiviral particle population of claim 10 or 11, wherein the lentiviral particle population comprises the target transgene, or wherein the target transgene is delivered to the cell before, simultaneously with, or after the lentiviral particle population.

13. The lentiviral particle population according to claim 10 or 11, for use as a medicament.

14. The lentiviral particle population according to claim 10 or 11, for site-specific integration of a target transgene into the genome of a cell, wherein the lentiviral particle population comprises the target transgene, or wherein the target transgene is to be delivered to the cell before, simultaneously with, or after the lentiviral particle population.

15. The lentiviral particle population according to claim 10 or 11, for use in treating a disease, preferably a genetic disease, in a subject in need thereof, wherein the lentiviral particle population comprises a target transgene, or wherein the target transgene is to be delivered to a cell before, simultaneously with or after the lentiviral particle population.

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