Therapeutic LAMA2 payloads for the treatment of congenital muscular dystrophies

Abstract

The present invention relates to a composition comprising: (a) a first protein comprising or consisting of a site-specific DNA-binding protein capable of binding to and cleaving a target nucleic acid sequence, or a nucleic acid construct encoding said first protein; (b) a second protein comprising or consisting of a transposase, or a nucleic acid construct encoding said second protein; and (c) a nucleic acid construct comprising a transgene encoding a laminin-α2 protein or a functional variant or fragment thereof. The present invention also relates to therapeutic uses of this composition to integrate the Lama2 transgene into a specific site in the genome of a cell, particularly for the treatment of congenital muscular dystrophy.

Description

[Technical Field]

[0001] Technical field The present invention relates to a fusion protein comprising a site-specific DNA-binding protein fused to a transposase for incorporating the LAMA2 transgene into a specific site in the genome of a cell, and a composition comprising the LAMA2 transgene, as well as its therapeutic use, particularly for the treatment of congenital muscular dystrophy. [Background technology]

[0002] background Congenital muscular dystrophy (CMD) is a class of severe, early-onset muscular dystrophy that affects the skeletal muscle / cardiac muscle and the central nervous system.

[0003] Among the CMDs, laminin α2 chain deficiency congenital muscular dystrophy (LAMA 2MD), also known as merosin-deficient congenital dystrophy type 1A (MDC1A), is characterized by severe hypotonia, muscle weakness, skeletal deformities, non-walking, and respiratory failure. There is a milder form of the disease with a later onset, which has similar symptoms but exhibits more wild-type phenotypic variation.

[0004] Currently, there are no available treatments for MDC1A. Current clinic strategies focus on management (feeding support, non-invasive ventilatory support for respiratory failure, and physiotherapy for joint contractures, spinal defects, and other problems) (Nguyen et al., 2019. Appl Clin Genet. 12:113-130).

[0005] MDC1A is caused by loss-of-function recessive mutations in both copies of the LAMA2 gene, which encodes the laminin-α2 chain, one of the subunits of laminin-211. Laminin-211 is an extracellular matrix protein that functions to stabilize the basement membrane and muscle fibers during contraction. Since the laminin-α2 chain protein is 3122 amino acid residues long, its coding DNA sequence (CDS) is approximately 9.3 kb long. Therefore, like many other muscular dystrophy conditions, MDC1A is caused by very large gene mutations that exceed the cargo capacity of AAV or lentiviral vectors. Furthermore, dozens of different mutations have been reported in the entire LAMA2 gene to date, ranging from single-point missense, splice site or in-frame mutations to multi-kilobase region deletions (Oliveira et al., 2018. Hum Mutat. 39(10):1314-1337), making radical gene therapy approaches difficult and gene replacement strategies currently unavailable.

[0006] Other gene-based modification strategies for MDC1A have been tested. An exon skipping approach has been used, which generally refers to using synthetic antisense oligonucleotides to inhibit splice enhancer sites and prevent specific exons from participating in splicing (Aoki et al., 2013. Biomed Res Int. 2013:402369). However, this exon skipping approach is not panacea and can only be used in some patients with the deletion, and the read frame can be restored by skipping additional exons adjacent to the deletion (Shieh, 2018. Neurotherapeutics. 15(4):840-848). Exon skipping restores translation of the cleaved but partially functional protein, but does not replicate the benefits of the full-length laminin-α2 protein.

[0007] CRISPR / Cas9 was also used to correct LAMA2 mutations, specifically by excising the LAMA2 intron 2 region containing the splice site mutation and creating a functional donor splice site via non-homologous end joining (NHEJ), which led to the inclusion of exon 2 of the LAMA2 transcript and the restoration of the full-length laminin-α2 chain protein (Kemaladewi et al., 2017. Nat Med. 23(8):984-989). As an exon skipping strategy, this approach is not universally applicable and can only be used in some patients.

[0008] However, NHEJ is not well-suited for integrating large transgenes into the genome, and gene modification relying on homologous recombination repair (HDR), while theoretically capable of correcting most pathogenic point mutations, has proven to be "extremely inefficient," as described by Kemaladewi & Cohn (2019. Emerg Top Life Sci. 3(1):11-18). In conclusion, Kemaladewi & Cohn called for the development of alternative strategies to correct mutations that cause muscular dystrophy.

[0009] Finally, CRISPR / Cas9 has also been used to induce gene expression by enabling increased expression of structurally similar laminin-α1 chain proteins using a catalytically inactive Cas9 (SadCas9) fused to a transcriptional activation domain such as VP64 targeting the promoter of the LAMA1 gene (Kemaladewi et al., 2019. Nature. 572(7767):125-130). However, this approach requires sustained expression of the transgene.

[0010] Therefore, there remains a need to develop a universal, long-lasting, and efficient strategy for treating MDC1A that is not limited to specific patients with particular mutations. [Overview of the project] [Problems that the invention aims to solve]

[0011] Genetic replacement of the LAMA2 gene has promising therapeutic value. However, this has been difficult to date due to cargo size constraints. The inventors have incorporated a normal full-length copy of the LAMA2 gene into a specific site in the cell genome using a fusion protein containing a site-specific DNA-binding protein (e.g., Cas9) fused to a transposase (e.g., highly active PiggyBac transposase). This has enabled the inventors to provide a therapeutic payload with a large LAMA2 expression cassette (9.3kb of CDS) for ex vivo and in vivo delivery, compatible with different gene delivery technologies. [Means for solving the problem]

[0012] Accordingly, the present invention relates to a composition comprising: a) a first protein comprising or consisting of a site-specific DNA-binding protein capable of binding to and cleaving a target nucleic acid sequence; or a nucleic acid construct encoding the first protein; b) a second protein comprising or consisting of a transposase; or a nucleic acid construct encoding the second protein; and c) preferably a nucleic acid construct comprising a transgene encoding the laminin-α2 protein of Sequence ID No. 74, or a functional variant or fragment thereof.

[0013] In one embodiment, the first and second proteins a) and b) are optionally fused together via a linker.

[0014] In one embodiment, the nucleic acid construct of c) includes a promoter selected from the group consisting of the CMV promoter of SEQ ID NO: 76, the CAG promoter of SEQ ID NO: 77, the EF1-alpha promoter of SEQ ID NO: 78, the SV-40 promoter of SEQ ID NO: 79, and the EalbAAT promoter of SEQ ID NO: 80. In one embodiment, the nucleic acid construct of c) includes the splice acceptor of SEQ ID NO: 81. In one embodiment, the nucleic acid construct of c) includes a poly(A) signal sequence preferably selected from the group consisting of SEQ ID NOs: 83 to 85, and / or an insulator element preferably selected from the group consisting of SEQ ID NOs: 86 to 87. In one embodiment, the nucleic acid construct of c) is adjacent to the inverse end sequences (ITRs) of SEQ ID NOs: 88 and 89, preferably 5'-ITR and 3'-ITR.

[0015] In one embodiment, the nucleic acid construct of c) is contained in a vector selected from the group consisting of plasmid vectors, minicircle vectors, doggybone DNA donor vectors, lentiviral vectors, and retroviral vectors.

[0016] In one embodiment, the site-specific DNA-binding protein is an RNA-induced nuclease containing a Cas protein, and the composition further comprises a guide RNA containing a sequence complementary to a target nucleic acid sequence for incorporating the LAMA2 transgene into a specific site in the cell genome, preferably the Cas protein being the Streptococcus pyogenes (S. pyogenes) Cas9 protein.

[0017] In one embodiment, the guide RNA includes one of sequence numbers 90 to 97.

[0018] In one embodiment, the transposase is a modified high-activity Piggybac transposase or Sleepy Beauty transposase, preferably a modified high-activity Piggybac transposase containing one or more amino acid mutations that increase excision activity compared to unmodified high-activity Piggybac, and one or more amino acid mutations that decrease DNA binding activity compared to unmodified high-activity Piggybac.

[0019] In one embodiment, the highly active PiggyBac transposase is preferably V34, T43, Y177, M194, R202, S230, R245, R275, R277, G325, N347, S351, E377, D450, R372 and K375, E377, T560, S564, S573, M589, S592, F594, including mutations in V34, T43, Y177, M194, R202, S230, R245, R275. This modified high-activity PiggyBac transposase contains at least one mutation in an amino acid selected from the group consisting of R277, G325, S351, N347, R372, K375, R376, E377, E380, A411, D450, T560, S564, S573, M589, S592, and F594, where the position numbers correspond to the amino acid numbers of the unmodified high-activity PiggyBac of Sequence ID No. 9. In one embodiment, the highly active PiggyBac transposase is preferably a modified highly active PiggyBac transposase comprising at least one mutation of an amino acid selected from the group consisting of M194, R245, R275, R277, G325, R372, K375, R376, E377, E380, D450, and S573, including mutations in amino acids D450, R372, and R375, wherein the position number corresponds to the amino acid number of the unmodified highly active PiggyBac of Sequence ID No. 9.

[0020] In one embodiment, the transposase is fused to the N-terminus of the site-specific DNA-binding protein by a linker, preferably a peptide linker containing GGS, XTEN, or FOKI, more preferably XTEN of SEQ ID NO: 53.

[0021] In one embodiment, the composition is packaged within the nanoparticles.

[0022] The present invention also relates to an in vitro method for integrating a LAMA2 transgene into a target nucleic acid sequence in the genome of a cell, the method comprising introducing the composition of the present invention into the cell.

[0023] The present invention also relates to an engineered cell obtained by the in vitro method of the present invention, the engineered cell comprising a nucleic acid integrated into its genome, the nucleic acid comprising a transgene encoding laminin-α2 protein adjacent to an engineered sequence for integrase- and / or transposase-mediated gene insertion.

[0024] In one embodiment, the engineered sequences adjacent to the transgene comprise or consist of SEQ ID NOs: 88 and 89.

[0025] In one embodiment, the size between the ITRs is at least 300 bp.

[0026] In one embodiment, the transgene encodes the full-length laminin-α2 protein.

[0027] The present invention also relates to a pharmaceutical composition comprising the composition of the present invention, or the engineered cell of the present invention, optionally in combination with one or more pharmaceutically acceptable excipients.

[0028] The present invention also relates to the composition of the present invention, the engineered cell of the present invention or the pharmaceutical composition of the present invention for use in a method of treatment, particularly for the treatment of merosin-deficient congenital muscular dystrophy type 1A (MDC1A) in a subject that needs it.

[0029] Definitions As used herein, the singular forms “a,” “an,” and “it” include singular and plural references unless the context explicitly indicates otherwise. For example, a reference to “drug” includes a single drug and multiple such drugs.

[0030] The terms “nucleic acid sequence” and “nucleotide sequence” may be used interchangeably to refer to any molecule composed of or containing monomeric nucleotides. Nucleic acids may be oligonucleotides or polynucleotides. Nucleic acid sequences may be DNA, RNA, or mixtures thereof. Nucleic acid sequences may be chemically modified or artificial. Nucleic acid sequences include peptide nucleic acids (PNA), morpholino and roch nucleic acids (LNA), as well as glycol nucleic acids (GNA) and threose nucleic acids (TNA). Each of these sequences is distinguished from naturally occurring DNA or RNA by alterations to the molecular backbone. Phosphothioate nucleotides may also be used. Other deoxyribonucleotide analogs, but not limited to, methylphosphonates, phosphoramidates, phosphorodithioates, N3'P5'-phosphoamidates and oligoribonucleotide phosphorothioates, as well as their 2'-O-allyl analogs and 2'-O-methylribonucleotide methylphosphonates, which may be used in the nucleotides of this disclosure.

[0031] The term “transgene” refers to an exogenous nucleic acid sequence, particularly exogenous DNA or cDNA that encodes a gene product. The gene product may be RNA, a peptide, or a protein. In addition to the coding region (CDS) of the gene product, a transgene may include, or associate with, one or more operational sequences for promoting or enhancing expression, such as promoters, enhancers, response elements, reporter elements, insulator elements, polyadenylation signals, and / or other functional elements. Embodiments of this disclosure may utilize any known suitable promoter, enhancer, response element, reporter element, insulator element, polyadenylation signals, and / or other functional elements unless otherwise specified. Suitable elements and sequences are well known to those skilled in the art.

[0032] The term "nucleic acid construct" refers to an artificial nucleic acid molecule resulting from the use of recombinant DNA technology. A nucleic acid construct is either a single-stranded or double-stranded nucleic acid molecule that has been modified to contain segments of nucleic acid sequences that are combined and juxtaposed in a way that would not otherwise occur in nature. A nucleic acid construct is typically a "vector," i.e., a nucleic acid molecule used to deliver the constructed DNA to a host cell from the outside.

[0033] The term "vector" or "expression vector" may refer to either an autonomous replicating vector, i.e., a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome, which may contain any means to ensure self-replication; or an embedded vector, i.e., a vector that, when introduced into a host cell, is integrated into its genome and replicates along with the chromosome(s) in which it is integrated.

[0034] The term "sequence identity" or "identity" refers to the number (%) of identical amino acid residues or nucleotides in the alignment of two polypeptide or polynucleotide sequences. Sequence identity is determined by comparing sequences that have been aligned to maximize overlap while minimizing sequence gaps. In particular, sequence identity can be determined using one of a number of mathematical global or local alignment algorithms, depending on the lengths of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm that optimally aligns the sequences along their entire length (e.g., Needleman and Wunsch algorithm [Needleman & Wunsch, 1970. J Mol Biol. 48(3):443-53]), while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith and Waterman algorithm [Smith & Waterman, 1981. J Mol Biol. 147(1):195-197] or the Altschul algorithm [Altschul et al., 1997. Nucleic Acids Res. 25(17):3389-3402; Altschul et al., 2005. FEBS J. 272(20):5101-9]). Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the scope of the art using publicly available computer software, for example, available on internet websites such as http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / . A person skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm necessary to achieve the greatest possible alignment over the entire length of the sequences being compared.For the purposes of this specification, the %nucleotide or amino acid sequence identity value refers to the value generated using the pairwise sequence alignment program EMBOSS Needle, which uses the Needleman-Wunsch algorithm to create the optimal global alignment of two sequences, with all search parameters set to their default values: score matrix = BLOSUM62, gap open = 10, gap extend = 0.5, end gap penalty = false, end gap open = 10, and end gap extend = 0.5.

[0035] The term "fusion" refers to a molecule in which two or more subunit molecules are linked together. In some embodiments, the link between the two is a covalent bond; or, the link between the two is a non-covalent bond, which may depend, for example, on intermolecular interactions. The subunit molecules may be molecules of the same chemical type, or they may be molecules of different chemical types.

[0036] The term “fusion protein” refers to a hybrid polypeptide comprising protein domains derived from at least two different proteins. For example, one protein domain may be located at the amino-terminal (N-terminal) or carboxy-terminal (C-terminal) protein of the fusion protein, thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein,” respectively. In a preferred embodiment, the fusion protein is a single-stranded polypeptide that can be fully encoded by a nucleic acid sequence and comprises at least two protein domains directly covalently linked by peptide bonds, or optionally covalently linked via a peptide linker.

[0037] The term "linker" refers to a chemical group or molecule that connects two adjacent molecules or parts.

[0038] The term "binding protein" refers to a protein that can bind to another molecule non-covalently. Binding proteins can, for example, bind to DNA molecules (DNA-binding proteins), RNA molecules (RNA-binding proteins), and / or protein molecules (protein-binding proteins). In the case of protein-binding proteins, they can bind to one or more molecules of the same protein to form homodimers, homotrimers, etc., and / or to one or more molecules of different proteins or multiple different proteins. Binding proteins can possess two or more binding activities. For example, zinc finger proteins possess DNA-binding activity, RNA-binding activity, and protein-binding activity.

[0039] The term "Cas9" or "Cas9 nuclease" refers to an RNA-inducible nuclease containing the Cas9 protein, or a fragment thereof (e.g., a protein containing the active or inactive DNA cleavage domain of Cas9, and / or the gRNA-binding domain of Cas9). Cas9 nucleases are sometimes also called cason1 nucleases or CRISPR (clustered regularly interspaced short palindromic repeat)-associated nucleases. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposition elements, and conjugation plasmids). A CRISPR cluster contains a spacer, a sequence complementary to the preceding mobile element, and a target entry nucleic acid. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In the type II CRISPR system, precise processing of precrRNA requires transcoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. TracrRNA acts as a guide for the processing of precrRNA by ribonuclease 3. Cas9 / crRNA / tracrRNA then cleaves linear or circular dsDNA targets complementary to the spacer with an endonuclease. Target strands not complementary to the crRNA are first cleaved with an endonuclease and then trimmed with a 3'-5' exonuclease. In nature, DNA binding and cleavage typically require both proteins and RNAs. However, a single guide RNA ("sgRNA" or simply "gNRA") can be manipulated to incorporate both aspects of crRNA and tracrRNA into a single RNA species.

[0040] Cas9 recognizes short motifs within CRISPR repeat sequences (PAM or protospacer-adjacent motifs) that help distinguish between self and non-self. The sequences and structures of Cas9 nucleases are well known to those skilled in the art. Cas9 orthologs have been described in various species, including but not limited to Streptococcus pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on this disclosure, and such Cas9 nucleases and sequences include those of organisms and loci-derived Cas9 sequences disclosed in Chylinski et al., 2013 (RNA Biol. 10(5):726-37), the entirety of which is incorporated herein by reference.

[0041] In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain. The nuclease-inactivated Cas9 protein can be interchangeably referred to as the "dCas9" protein (nuclease "dead" Cas9). Methods for producing Cas9 proteins (or fragments thereof) having an inactive DNA cleavage domain are known in the art (see, for example, 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).

[0042] The term "zinc finger protein" refers to a protein that binds to DNA in a sequence-specific manner via one or more zinc fingers, which are amino acid sequences within the binding domain of a zinc finger protein whose structure is stabilized by the coordination of zinc ions, or a domain within a larger protein. The term "zinc finger protein" is often abbreviated as "ZFP".

[0043] The term "zinc finger nuclease" refers to an artificial restriction enzyme created by fusing a zinc finger DNA-binding domain to a DNA-cleaving domain. The zinc finger domain can be manipulated to target specific, desired DNA sequences, allowing zinc finger nucleases to target unique sequences within complex genomes. "Zinc finger nuclease" is often abbreviated as "ZFN" or "ZNP".

[0044] The term "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 and double-strand breaks are possible, and a double-strand break can result from two different single-strand break events. DNA breaks can produce either blunt or adherent ends. In certain embodiments, fusion polypeptides are used for targeted double-strand DNA breaks.

[0045] The term "specificity" refers to the ability to selectively bind sequences that share a certain degree of sequence identity with a selected sequence.

[0046] The terms “insertion” and “integration” refer to the addition of a nucleic acid sequence to a second nucleic acid sequence or to a genome or part thereof. The terms “specific,” “site-specific,” “targeted,” and “on-target” related to insertion or integration are used interchangeably herein to refer to the insertion of a nucleic acid to a specific site in a second nucleic acid or to a specific site in a genome or part thereof. Conversely, the terms “random,” “untargeted,” and “off-target” refer to nonspecific and unintended insertions of nucleic acids to undesirable sites. The term “total” or “whole” refers to the total number of insertions.

[0047] The term "mutation" refers to the substitution of a residue in a sequence, for example, a nucleic acid or amino acid sequence, with another residue; and / or the deletion or insertion of one or more residues in a nucleic acid or amino acid sequence. Mutations are usually described herein by identifying the original residue, followed by the position of the residue in the sequence, and then the identity of the newly substituted residue. Various methods for making amino acid substitutions (mutations) provided herein are well known in the art and are provided, for example, in Green & Sambrook, 2012 (Molecular cloning: a laboratory manual (4th edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). In preferred embodiments, the term mutation in a protein refers to an amino acid substitution.

[0048] The term "transposase" refers to an enzyme that binds to the end of a transposon and catalyzes its movement to another part of the genome via a cut-and-paste mechanism or a replication-transposition mechanism.

[0049] The term "modified" refers to a protein or nucleic acid sequence that is different from the corresponding unmodified protein or nucleic acid sequence.

[0050] The term "linker" refers to a chemical group or molecule that joins two adjacent molecules or parts together. [Modes for carrying out the invention]

[0051] Detailed explanation The inventors used a fusion protein containing a site-specific DNA-binding protein fused to a transposase to incorporate the LAMA2 transgene into a specific site within the cell genome.

[0052] Therefore, the present invention is a) A first protein comprising or consisting of a site-specific DNA-binding protein capable of binding to and cleaving a target nucleic acid sequence; or a nucleic acid construct, preferably cDNA or mRNA, encoding the first protein; (b) A second protein comprising or consisting of a transposase; or a nucleic acid construct encoding the second protein, preferably cDNA or mRNA; c) A nucleic acid construct comprising or consisting of laminin-α2 protein, a transgene encoding a functional variant or fragment thereof. This relates to compositions containing or consisting of the same.

[0053] In one embodiment, the composition is (a) a first protein comprising or consisting of (i) a site-specific DNA-binding protein capable of binding to and cleaving a target nucleic acid sequence, and (ii) a fusion protein comprising or consisting of a second protein comprising or consisting of a transposase; or a nucleic acid construct encoding the fusion protein, preferably cDNA or mRNA; and b) A nucleic acid construct comprising or consisting of a transgene encoding the laminin-α2 protein, a functional variant or fragment thereof. It includes or consists of.

[0054] According to the present invention, the first protein comprises or consists of a site-specific DNA-binding protein capable of binding to and cleaving a target nucleic acid sequence.

[0055] Current genome engineering tools, including manipulated zinc finger proteins (ZFPs), transcription activator-like effector nucleases (TALENs), and more recently, RNA-induced DNA nucleases such as Cas9, result in sequence-specific DNA breaks within the genome. These programmable breaks can result in DNA mutation at the break site via non-homologous end joining (NHEJ) or DNA replacement around the break site via homologous recombination repair (HDR).

[0056] In one embodiment, the site-specific DNA-binding protein is selected from the group comprising or consisting of RNA-induced DNA nucleases, zinc finger proteins, and transcription activator-like effector nucleases.

[0057] In one embodiment, the site-directed DNA-binding protein is selected from the group comprising or consisting of RNA-induced DNA nucleases and zinc finger proteins.

[0058] In one embodiment, the site-specific DNA-binding protein is an RNA-induced nuclease.

[0059] In one embodiment, the site-specific DNA-binding protein is a Cas9 protein (e.g., Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), or Campylobacter jejuni Cas9 (CjCas9); several other suitable examples are described below), or a variant thereof (e.g., nickase Cas9 (nCas9) or dead Cas9 (dCas9)), or a Cas12a protein, Cas12b protein, Cpf1 protein, or CasX protein containing these variants and functional fragments.

[0060] In one embodiment, the site-directed DNA-binding protein is a Cas9 protein, including its variants and functional fragments.

[0061] The CRISPR-Cas9 system is a highly effective tool for inactivating or modifying genes via sequence-specific double-strand breaks (DSBs). These DSBs are recognized by the cell's DNA damage response mechanism and can be repaired by the endogenous DSB repair pathway. The primary repair pathway is non-homologous end joining (NHEJ), which often results in small insertions and / or deletions that cause frameshift mutations and disrupt gene function. This pathway can be used to create gene knockout mutations. Alternatively, in the presence of a repair template (e.g., a nucleic acid construct containing or consisting of a laminin-α2 protein, a transgene encoding a functional variant or fragment thereof), damage can be seamlessly repaired by homologous recombination repair (HDR). However, despite remarkable progress, genome editing via HDR to introduce precise gene modifications is far less efficient than gene disruption via NHEJ. Furthermore, large multi-kb substitutions via the HDR pathway are difficult and require selection and / or large-scale population cell sorting. As a result, the primary use of the HDR pathway is currently limited to local substitutions of critical regions within genes, rather than large-scale full-length gene substitutions. As described above, the present invention addresses this limitation.

[0062] In one embodiment, the Cas9 protein comprises (i) an active DNA cleavage domain and (ii) a guide RNA binding domain.

[0063] Among known Cas9 proteins, the Streptococcus pyogenes Cas9 protein is widely used as a tool in genome engineering. This Cas9 protein is a large multi-domain protein containing two different nuclease domains.

[0064] In one embodiment, the Cas9 protein is associated with Corynebacterium ulcerans (SEQ ID NO: 19, NCBI reference: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (SEQ ID NO: 20, NCBI reference: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (SEQ ID NO: 21, NCBI reference: NC_021284.1); Prevotella intermedia (SEQ ID NO: 22, NCBI reference: NC_017861.1); Spiroplasma taiwanense (SEQ ID NO: 23, NCBI reference: NC_021846.1); Streptococcus iniae (SEQ ID NO: 24, NCBI reference: NC_021314.1); and Bellilla baltica (SEQ ID NO: 25). Psychroflexus baltica (NCBI reference: NC_018010.1); Sequence ID 26 Selected from the group including or consisting of Cas9 proteins derived from: torquisi) (NCBI reference: NC_018721.1); Streptococcus thermophilus (SEQ ID NO: 27) (NCBI reference: YP_820832.1); Listeria inocure (SEQ ID NO: 28) (NCBI reference: NP_472073.1); Campylobacter jejuni (CjCas9) (SEQ ID NO: 29) (NCBI reference: YP_002344900.1) (encoded in SEQ ID NO: 63); Neisseria meningitidis (SEQ ID NO: 30) (NCBI reference: YP_002342100.1); Staphylococcus aureus (SaCas9) (SEQ ID NO: 60) (encoded in SEQ ID NO: 68); and Streptococcus pyogenes (SpCas9) (SEQ ID NO: 31) (NCBI reference: NC_017053.1).

[0065] In one embodiment, when the wild-type Cas9 protein is referred to herein, unless otherwise specified, the wild-type Cas9 protein corresponds to the Streptococcus pyogenes-derived Cas9 (spCas9) of Sequence ID No. 31.

[0066] In one embodiment, the Cas9 protein may be a “Cas9 variant.” When used herein, a “Cas9 variant” is a protein that shares homology with the Cas9 protein described herein, and includes fragments thereof.

[0067] In one embodiment, the Cas9 variant may be at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% identical to the wild-type Cas9 protein of SEQ ID NO: 31, or any other Cas9 protein of SEQ ID NOs: 19-30 or 68.

[0068] In one embodiment, a Cas9 variant includes an amino acid sequence of a Cas9 protein having one or more amino acid substitutions. For example, the DNA cleavage domain of Cas9 is known to include two subdomains: an HNH nuclease subdomain and a RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand.

[0069] Mutations within these subdomains can silence the nuclease activity of Cas9. For example, substitutions D10A and H841A are known to completely inactivate the nuclease activity of the Streptococcus pyogenes Cas9 protein of SEQ ID NO: 31, resulting in dead Cas9 (dCas9) that still retains the ability to bind to DNA in an sgRNA-programmed manner. In principle, when fused to another protein or domain, dCas9 can target virtually any DNA sequence simply by co-expressing it with a suitable sgRNA. In one embodiment, the dCas9 protein is encoded by a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 59. In one embodiment, the dCas9 protein comprises or consists of an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 67.

[0070] With respect to Cas9 nickase (nCas9), this is a variant of Cas9 nuclease with a point mutation (D10A) in the RuvC nuclease domain that cannot cleave DNA but can introduce nicks. In one embodiment, the nCas9 protein is encoded by a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 57. In one embodiment, the nCas9 protein contains or consists of an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 65. In some embodiments, SaCas9 nickase (SanCas9) is encoded by a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 58. In some embodiments, SaCas9 nickase (SanCas9) includes an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 66.

[0071] In one embodiment, the Cas9 variant comprises a fragment of Cas9, the fragment being at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% identical to the wild-type Cas9 protein of SEQ ID NO: 31, or the corresponding fragment of any other Cas9 protein of SEQ ID NOs: 19-30 or 68.

[0072] In one embodiment, the Cas9 variant includes only one of either a DNA cleavage domain or a guide RNA binding domain.

[0073] In one embodiment, the exemplary Cas9 variant is humanized Cas9 (hCas9) or a variant or functional fragment thereof. As used herein, the terms “humanized Cas9” or “hCas9” refer to a Cas9 protein sequence-optimized for human cells.

[0074] In one embodiment, the hCas9 protein is encoded by a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 56. In one embodiment, the hCas9 protein comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 64.

[0075] In one embodiment, the site-specific DNA-binding protein is the cpf1 protein. In one embodiment, the cpf1 protein is encoded by a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 61. In one embodiment, the cpf1 protein contains an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 69.

[0076] In one embodiment, the site-specific DNA-binding protein is the CasX protein. In one embodiment, CasX is encoded by a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 62. In one embodiment, CasX comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 70.

[0077] As will be further detailed below, certain aspects of this disclosure relate to vectors or plasmids (e.g., expression vectors, packaging vectors, etc.) comprising a nucleic acid construct encoding a site-specific DNA-binding protein, in particular an RNA-induced nuclease, in particular one of the Cas9 proteins described herein, said vector or plasmid being preferably suitable for expression in host cells, such as mammalian cells, yeast cells, insect cells, plant cells, fungal cells, or algal cells.

[0078] In one embodiment, the site-specific DNA-binding protein is a zinc finger protein (ZFP).

[0079] Zinc finger proteins are proteins capable of sequence-specific binding to DNA. ZFPs are heterogeneously distributed in eukaryotes. ZFPs have been identified as being involved in DNA recognition, RNA binding, and protein binding. The specific classification of zinc finger proteins is based on the “fold group,” considering the overall shape of the protein backbone in the folded domain. The most common “fold groups” of zinc fingers are C2H2 or Cys2His2-like (“classic zinc finger”), treble clef, and zinc ribbon. Representative motifs characterizing these proteins are disclosed in Table 1 of Li & Liu, 2020 (Int J Mol Sci. 21(4):1361), which is incorporated herein by reference.

[0080] A ZFP can be any ZFP, a variant thereof, or a functional fragment that can bind to a specific genomic DNA sequence in the genome. Non-limiting examples of ZFPs include those containing a folded group or zinc finger motif selected from C2H2, gag knuckle, treble clef, zinc ribbon, Zn2 / Cys6-like, or TAZ2 domain-like, or any combination thereof. In one embodiment, the ZFP is a C2H2 zinc finger protein.

[0081] In one embodiment, the ZFP is an engineered ZFP. A zinc finger nuclease can be produced by fusing the engineered zinc finger array to a DNA cleavage domain (usually the FokI cleavage domain). Such zinc finger-FokI fusions are useful reagents for manipulating genomes.

[0082] A ZFP may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more zinc finger domains. A ZFP may contain 2-12, 2-10, 2-8, 3-8, 4-8, or 5-8 zinc finger domains. In one embodiment, a ZFP contains 6 zinc finger domains.

[0083] A typical modular assembly process involves combining separate zinc fingers, each capable of recognizing a 3-base pair DNA sequence, to create 3-finger, 4-, 5-, or 6-finger arrays that recognize target sites ranging in length from 9 to 18 base pairs. Alternatively, a 2-finger module can be used to create zinc finger arrays with up to six individual zinc fingers.

[0084] In one embodiment, the binding domain of a ZFP can be manipulated to bind to a target sequence. The manipulated zinc finger binding domain may have improved binding specificity compared to naturally occurring ZFPs.

[0085] In one embodiment, the exemplary nucleic acid sequence encoding the ZFP includes or comprises SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38. In one embodiment, the exemplary amino acid sequence encoded by these sequences includes or comprises SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39.

[0086] In one embodiment, the ZFP comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with any one of sequence numbers 33, 35, 37, or 39.

[0087] In one embodiment, the ZFP does not have a Gal4 DNA-binding domain. Gal4 is CGG-N 11- It binds to CCG, and N can be any base. This protein is a positive regulator of gene expression of galactose-inducible genes such as GAL1, GAL2, GAL7, GAL10, and MEL1, which encode enzymes used to convert galactose to glucose. It recognizes a 17-base pair sequence in the upstream activation sequence (UAS-G) of these genes. Therefore, Gal4 recognizes short, very frequent sequences in the genome and is therefore not site-specific. In one embodiment, the ZFP has a Gal4 DNA-binding domain that has been engineered to be site-specific.

[0088] As will be further detailed below, certain aspects of this disclosure relate to vectors or plasmids (e.g., expression vectors, packaging vectors, etc.) comprising site-specific DNA-binding proteins, particularly nucleic acid constructs encoding ZFPs as described herein, said vectors or plasmids, which are preferably suitable for expression in host cells, such as mammalian cells, yeast cells, insect cells, plant cells, fungal cells, or algal cells.

[0089] According to the present invention, the second protein includes or consists of a transposase.

[0090] A transposon is a chromosomal segment that can be transposed, for example, a DNA molecule that can be translocated as a whole when a complementary sequence is not present in the host DNA. Transposons can be used to perform large-scale DNA manipulation in human cells. Common transposon systems used in mammalian cells include, but are not limited to, Sleeping Beauty (SB), which is reconstructed from an inactive transposon, and PiggyBac (PB), which is isolated from the moth nettle. PiggyBac has higher transposition activity than SB and can be excised without scarring.

[0091] Native DNA transposons typically contain a single gene encoding a transposase protein, flanked by an inverted terminal sequence (ITR) containing a transposase binding site. During their transposition, the transposase protein recognizes these ITRs and catalyzes the excision and subsequent reintegration of elements at random other locations. Furthermore, some of these transposons can be adapted for use in gene therapy protocols employing them as a two-component system, the plasmid containing an expression cassette in which a target DNA sequence (e.g., LAMA2 transgene) positioned between the transposon ITRs can be introduced into a host genome directed by a co-transfected plasmid containing a transposase enzyme or a sequence encoding its mRNA synthesized in vitro. According to this disclosure, transposon-based systems are used to efficiently mediate the stable integration and sustained expression of LAMA2 transgenes in cells.

[0092] In one embodiment, the transposase or modified transposase used in the present invention may be any transposase capable of inserting the LAMA2 transgene into a specific site in the genome.

[0093] Non-exclusive examples of transposases include Frog Prince, Sleeping Beauty, highly active Sleeping Beauty, PiggyBac, and highly active PiggyBac.

[0094] In one embodiment, the transposase is highly active PiggyBac transposase (hyPB).

[0095] The wild-type highly active PiggyBac transposase has an amino acid sequence containing or consisting of SEQ ID NO: 9. An exemplary nucleic acid sequence encoding this protein is described in SEQ ID NO: 71.

[0096] In one embodiment, the transposase is a modified, highly active PiggyBac transposase.

[0097] As used herein, “modified high-activity PiggyBac transposase” refers to a transposase comprising one or more amino acid substitutions, typically only one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions, compared to the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9. More specifically, a modified high-activity PiggyBac comprises (i) one or more amino acid substitutions that increase cleavage activity compared to the wild-type high-activity PiggyBac transposase, and / or (ii) one or more amino acid substitutions that decrease DNA binding activity compared to the wild-type high-activity PiggyBac transposase. In one embodiment, a modified high-activity PiggyBac transposase 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 described in SEQ ID NO: 9.

[0098] In some embodiments, the modified high-activity PiggyBac transposase contains one or more amino acid substitutions that increase cleavage activity compared to the wild-type high-activity PiggyBac transposase.

[0099] In one embodiment, the modified high-activity PiggyBac transposase includes one or more amino acid substitutions that increase cleavage activity within the region defined by amino acid positions [194-200], [214-222], [434-442] and / or [446-456], for example, at amino acid positions D198, D201, R202, M212, and / or S213; the numbering is based on the wild-type high-activity PiggyBac transposase of Sequence ID No. 9.

[0100] In one embodiment, the modified high-activity PiggyBac transposase comprises one or more amino acid substitutions that increase cleavage activity at an amino acid position selected from positions 450, 560, 564, 573, 589, 592, and / or 594; the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9.

[0101] In one embodiment, the modified high-activity PiggyBac transposase includes one or more amino acid substitutions that increase cleavage activity at amino acid positions selected from positions M194 and D450; the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9. In another embodiment, one or more amino acid substitutions are selected from M194V and D450N, and the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9.

[0102] In one embodiment, the modified high-activity PiggyBac transposase contains one or more amino acid substitutions that reduce DNA binding activity compared to the wild-type high-activity PiggyBac transposase.

[0103] In one embodiment, the modified high-activity PiggyBac transposase comprises one or more amino acid substitutions that reduce DNA binding activity at amino acid positions selected from positions 254, 275, 277, 347, 372, 375, and / or 465; the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9.

[0104] In one embodiment, the modified high-activity PiggyBac transposase comprises one or more amino acid substitutions that reduce DNA binding activity at amino acid positions selected from positions R275, N347, R372, K375, R376, E377, and / or E380; the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9.

[0105] In one embodiment, the modified high-activity PiggyBac transposase includes one or more amino acid substitutions that reduce DNA binding activity at amino acid positions selected from positions R372, K375, R376, E377, and / or E380; the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9. In another embodiment, one or more amino acid substitutions are selected from R372A, K375A, R376A, E377A, and / or E380A; the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9.

[0106] In one embodiment, the modified high-activity PiggyBac transposase includes one or more amino acid substitutions that reduce DNA binding activity at amino acid positions selected from positions N347, R372, and K375; the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9. In one embodiment, one or more amino acid substitutions are selected from N347S, N347A, R372A, and / or K375A; the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9. In one embodiment, one or more amino acid substitutions are selected from N347S or N347A; the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9.

[0107] In one embodiment, the modified high-activity PiggyBac transposase includes one or more amino acid substitutions that increase the cleavage activity as defined above, and one or more amino acid substitutions that decrease the DNA binding activity as defined above.

[0108] In one embodiment, the modified high-activity PiggyBac transposase includes at least one substitution that increases cleavage activity at position D450, and at least two substitutions that decrease DNA binding activity at positions N372, R372 and K375; the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9; preferably, the modified high-activity PiggyBac transposase includes a double mutation of N347S and D450N, or a triple substitution of D450N, R372A and K375A; the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9. In one embodiment, the modified transposase of high-activity PiggyBac includes a double mutation of N347S and D450N, where the position numbers correspond to the amino acid numbers of the unmodified high-activity PiggyBac of SEQ ID NO: 9.

[0109] In some embodiments, the modified transposase of high-activity PiggyBac does not contain the D450N, R372A and K375A triple mutation, and the positional numbers correspond to the amino acid numbers of the unmodified high-activity PiggyBac of SEQ ID NO: 9.

[0110] In one embodiment, the modified high-activity PiggyBac transposase disclosed in the previous embodiment further comprises a region defined by amino acid position numbers [158-169], e.g., at least one substitution at A166S; and / or at least one substitution at positions Y527, R518, K525 and / or N463; the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9.

[0111] Typically, the modified highly active PiggyBac transposase contains an amino acid sequence that is at least 85%, at least 90%, and at least 95% identical to the unmodified highly active PiggyBac transposase of SEQ ID NO: 1.

[0112] In one embodiment, the modified high-activity PiggyBac transposase further comprises one or more of the following amino acid substitutions at positions selected from 34, 43, 117, 202, 230, 245, 268, 275, 277, 287, 290, 315, 325, 341, 346, 347, 350, 351, 356, 357, 388, 409, 412, 432, 447, 460, 461, 465, 517, 560, 564, 571, 573, 576, 586, 587, 589, 592, and 594; the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9.

[0113] In one embodiment, the modified highly active PiggyBac transposase has the following amino acid substitutions or combinations of substitutions: V34M, T43I, Y177H, R202K, S230N, R245A, D268N, R275A, R277A, 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, K4 Includes one of 61A, W465A, S517A, T560A, S564P, S571N, S573A, K576A, H586A, I587A, M589V, S592G, F594L, D450N / R372A / K375A, R275A / R277A, K409A / K412A, R460A / K461A, R275A / R277A / N347S / K375A / T560A / S573A / M589V / S592G and R245A / R275A / R277A / R372A / W465A; numbering is based on the wild-type highly active PiggyBac transposase of SEQ ID NO: 9.

[0114] In one embodiment, the modified highly active PiggyBac transposase has the following amino acid substitutions or combinations of amino acid substitutions: - R372A / K375A / D450N, - R372A / K375A / R376A / D450N, ‐K375A / R376A / E377A / E380A / D450N、 ‐R372A / K375A / R376A / E377A / E380A / D450N、 ‐M194V、 ‐M194V / R372A / K375A、 ‐S351A / R372A / K375A / R388A / D450N / W465A / S573A / M589V / S592G / F594L、 ‐R245A / R275A / R277A / R372A / W465A / M589V、 ‐R275A / 325A / R372A / T560A、 ‐N347A / D450N、 ‐N347S / D450N / T560A / S573A / F594L、 ‐R202K / R275A / N347S / R372A / D450N / T560A / F594L、 ‐R275A / N347S / K375A / D450N / S592G、 ‐R275A / N347S / R372A / D450N / T560A / F594L、 ‐R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L ‐R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G、 ‐R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L、 ‐V34M / R275A / G325A / N347S / S351A / R372A / K375A / D450N / T560A / S564P、 ‐G325A / N347S / K375A / D450N / S573A / M589V / S592G、 ‐S230N / R277A / N347S / K375A / D450N、 ‐T43I / R372A / K375A / A411T / D450N、 ‐G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G、 -Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G It includes one of these, and the numbering is based on the wild-type highly active PiggyBac transposase of Sequence ID No. 9.

[0115] In one embodiment, the modified highly active PiggyBac transposase has the following amino acid substitutions or combinations of amino acid substitutions: -K375A / R376A / E377A / E380A / D450N, -R372A / K375A / R376A / E377A / E380A / D450N, -M194V, - M194V / R372A / K375A, -R245A / R275A / R277A / R372A / W465A / M589V, - R275A / 325A / R372A / T560A, -N347A / D450N, -N347S / D450N / T560A / S573A / F594L, -R202K / R275A / N347S / R372A / D450N / T560A / F594L, -R275A / N347S / K375A / D450N / S592G, -R275A / N347S / R372A / D450N / T560A / F594L, -R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L -R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G, -R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L, -G325A / N347S / K375A / D450N / S573A / M589V / S592G, -S230N / R277A / N347S / K375A / D450N, - G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, or Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G It includes one of these, and the numbering is based on the wild-type highly active PiggyBac transposase of Sequence ID No. 9.

[0116] In a preferred embodiment, the modified hyperactive PiggyBac transposase has the following amino acid substitutions or combinations of amino acid substitutions: - R372A / K375A / D450N, -S351A / R372A / K375A / R388A / D450N / W465A / S573A / M589V / S592G / F594L, -R245A / R275A / R277A / R372A / W465A / M589V, -N347A / D450N, -N347S / D450N / T560A / S573A / F594L, -R202K / R275A / N347S / R372A / D450N / T560A / F594L, -R275A / N347S / K375A / D450N / S592G, -R275A / N347S / R372A / D450N / T560A / F594L, -R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L, - R275A / 325A / R372A / T560A, -R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G, -R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L, -G325A / N347S / K375A / D450N / S573A / M589V / S592G, -S230N / R277A / N347S / K375A / D450N, - G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, or -Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G It includes one of these, and the numbering is based on the wild-type highly active PiggyBac transposase of Sequence ID No. 9.

[0117] In one embodiment, the modified highly active PiggyBac transposase has an amino acid sequence selected from among SEQ ID NOs: 1-8, 10-18, 108-113, and 122-130.

[0118] In one embodiment, the modified highly active PiggyBac transposase has an amino acid sequence selected from any of SEQ ID NOs: 1-8 and 10-18.

[0119] In one embodiment, the modified highly active PiggyBac transposase has an amino acid sequence selected from any of SEQ ID NOs: 108 to 113.

[0120] In one embodiment, the modified highly active PiggyBac transposase has an amino acid sequence selected from any of SEQ ID NOs: 122 to 130.

[0121] In one embodiment, the modified high-activity PiggyBac transposase may contain one or more substitutions involved in the conserved catalytic triad, for example at amino acids 268 and / or 346 (e.g., D268N and / or D346N), compared to the wild-type high-activity PiggyBac transposase, and the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9 or the modified high-activity PiggyBac transposase of SEQ ID NO: 11.

[0122] In one embodiment, the modified high-activity PiggyBac transposase may contain one or more substitutions important for excision, for example, at amino acids 287, 287 / 290 and / or 460 / 461 (e.g., K287A, K287A / K290A, and / or R460A / K461A), compared to the wild-type high-activity PiggyBac transposase, and the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9 or the modified high-activity PiggyBac transposase of SEQ ID NO: 12.

[0123] In one embodiment, the modified high-activity PiggyBac transposase may contain one or more substitutions involved in target binding, for example, at amino acids 351, 356, and / or 379 (e.g., S351E, S351P, S351A, and / or K356E), compared to the wild-type high-activity PiggyBac transposase, and the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9 or the modified high-activity PiggyBac transposase of SEQ ID NO: 13.

[0124] In one embodiment, the modified high-activity PiggyBac transposase may include one or more substitutions important for incorporation in amino acids 560, 564, 571, 573, 589, 592, and / or 594 (e.g., T560A, S564P, S571N, S573A, M589V, S592G, and / or F594L) compared to the wild-type high-activity PiggyBac transposase, and may be numbered based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9 or the modified high-activity PiggyBac transposase of SEQ ID NO: 14.

[0125] In one embodiment, the modified high-activity PiggyBac transposase may, compared to hyPB, include one or more substitutions involved in alignment at, for example, amino acids 325, 347, 350, 357 and / or 465 (e.g., G325A, N347A, N347S, T350A and / or W465A), and is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9 or the modified high-activity PiggyBac transposase of SEQ ID NO: 15.

[0126] In one embodiment, the modified high-activity PiggyBac transposase may contain one or more well-conserved substitutions, for example, at amino acids 576 and / or 587 (e.g., K576A and / or I587A), compared to the wild-type high-activity PiggyBac transposase, and may be numbered based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9 or the modified high-activity PiggyBac transposase of SEQ ID NO: 16.

[0127] In one embodiment, the modified high-activity PiggyBac transposase, compared to the wild-type high-activity PiggyBac transposase, for example, at 586 (e.g., H586A), contains Zn 2+ It may contain one or more substitutions involved in binding, and the numbering is based on the wild-type highly active PiggyBac transposase of SEQ ID NO: 9 or the modified highly active PiggyBac transposase of SEQ ID NO: 17.

[0128] In one embodiment, the programmable transposase may include one or more substitutions involved in incorporation, for example, in 315, 341, 372, and / or 375 (e.g., R315A, R341A, R372A, and / or K375A), compared to the wild-type highly active PiggyBac transposase, and is based on the wild-type highly active PiggyBac transposase of SEQ ID NO: 9 or the modified highly active PiggyBac transposase of SEQ ID NO: 18.

[0129] In one embodiment, a modified high-activity PiggyBac transposase is selected for its high specificity for DNA integration into the genome compared with a wild-type high-activity PiggyBac transposase. In one embodiment, each modified high-activity PiggyBac transposase comprises an amino acid sequence having one or more of the modifications disclosed herein, compared with any one of SEQ ID NOs: 9-18 and 108-113, and retains at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequences described in SEQ ID NOs: 1-18 and 108-113.

[0130] As shown in the examples, a newly developed high-activity PiggyBac transposase substitution library has been used to identify modified high-activity PiggyBacs that undergo specific targeting rearrangements. Modified high-activity PiggyBacs with positive targeting rearrangements were identified using such a library.

[0131] In one embodiment, the modified high-activity PiggyBac transposase may include substitutions of one or more amino acids selected from amino acids 245, 275, 275, 277, 325, 347, 351, 372, 375, 388, 450, 465, 560, 564, 573, 589, 592, 594, numbering based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9.

[0132] In one embodiment, the modified high-activity PiggyBac transposase comprises one or more amino acid substitutions selected from R245A, R275A, R275A, R277A, R275A / R277A, G325A, N347A, N347S, S351E, S351P, S351A, R372A, K375A, R388A, D450N, W465A, T560A, S564P, S573A, M589V, S592G, or F594L; the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9.

[0133] In one embodiment, the modified high-activity PiggyBac transposase contains the amino acid substitution D450N, and the numbering is based on the wild-type high-activity PiggyBac transposase of Sequence ID No. 9.

[0134] In one embodiment, the modified high-activity PiggyBac transposase comprises amino acid variant N347A, and the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9. In another embodiment, the modified high-activity PiggyBac transposase comprises amino acid substitution N347S, and the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9.

[0135] In one embodiment, the modified high-activity PiggyBac transposase comprises the double amino acid substitutions D450N and N347A, and the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9. In another embodiment, the modified high-activity PiggyBac transposase comprises the double amino acid substitutions D450N and N347S, and the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9.

[0136] In one embodiment, the modified high-activity PiggyBac transposase comprises amino acid substitutions R372A, K375A, and D450, and the numbering is based on the wild-type high-activity PiggyBac transposase of Sequence ID No. 9.

[0137] In one embodiment, the modified high-activity PiggyBac transposase includes amino acid substitutions R245A and D450, and the numbering is based on the wild-type high-activity PiggyBac transposase of Sequence ID No. 9.

[0138] In one embodiment, the modified high-activity PiggyBac transposase comprises amino acid substitutions R245A, G325A, and S573P, and the numbering is based on the wild-type high-activity PiggyBac transposase of Sequence ID No. 9.

[0139] In one embodiment, the modified high-activity PiggyBac transposase comprises amino acid modifications R245A, G325A, D450, and S573P, and the numbering is based on the wild-type high-activity PiggyBac transposase of Sequence ID No. 9.

[0140] In one embodiment, the modified high-activity PiggyBac transposase includes amino acid substitutions N347S and D450N, and the numbering is based on the wild-type high-activity PiggyBac transposase of Sequence ID No. 9.

[0141] In one embodiment, the modified high-activity PiggyBac transposase includes amino acid substitutions N347A and D450N, and the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9. In one embodiment, this modified high-activity PiggyBac transposase includes the amino acid sequence of SEQ ID NO: 110.

[0142] In one embodiment, the modified high-activity PiggyBac transposase comprises one or more amino acid substitutions selected from L25F, R36A, i42K, G59D, I212K, N245S, K252A and / or Q271L, and the numbering is based on the wild-type high-activity PiggyBac transposase of SEQ ID NO: 9.

[0143] The modified high-activity PiggyBac transposases provided herein can be fused to other elements disclosed herein, such as site-directed DNA-binding proteins capable of binding to and cleaving target nucleic acid sequences, or can be used alone or in combination with such other elements. In one embodiment, the modified high-activity PiggyBac transposase disclosed herein comprises the amino acid sequence of SEQ ID NO: 9, The amino acid at position 34 is either V or M. The amino acid at position 43 is either T or I. The amino acid at position 177 is either Y or H. The amino acid at position 202 is either R or K. The amino acid at position 230 is either S or N. The amino acid at position 245 is A. The amino acid at position 268 is either D or N. The amino acid at position 275 is either R or A. The amino acid at position 277 is either R or A. The amino acid at position 325 is either A or G. The amino acid at position 347 is either N, S, or A. The amino acid at position 351 is E, P, or A. The amino acid at position 372 is either R or A. The amino acid at position 375 is either A or K. The amino acid at position 388 is either R or A. The amino acid at position 409 is either K or A. The amino acid at position 411 is either A or T. The amino acid at position 412 is either K or A. The amino acid at position 450 is either D or N. The amino acid at position 460 is either R or A. The amino acid at position 465 is either W or A. The amino acid at position 517 is either S or A. The amino acid at position 560 is either T or A. The amino acid at position 564 is either P or S. The amino acid at position 571 is either S or N. The amino acid at position 573 is either S or A. The amino acid at position 576 is either K or A. The amino acid at position 586 is either H or A. The amino acid at position 587 is either I or A. The amino acid at position 589 is either M or V. The amino acid at position 592 is G or S, and / or The amino acid at position 594 is either L or F.

[0144] In one embodiment, the transposase is Sleeping Beauty transposase.

[0145] In one embodiment, the Sleeping Beauty transposase comprises or consists of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 73. An exemplary nucleic acid sequence encoding this protein is as described in SEQ ID NO: 72.

[0146] In one embodiment, the transposase is a modified Sleeping Beauty transposase. In one embodiment, the modified Sleeping Beauty transposase includes one or more substitutions compared to the wild-type Sleeping Beauty transposase.

[0147] In one embodiment, one or more substitutions in the Sleeping Beauty transposase are selected from L25F, R36A, I42K, G59D, I212K, N245S, K252A and / or Q271L; the numbering is based on the wild-type Sleeping Beauty transposase of Sequence ID No. 73.

[0148] In one embodiment, the transposase is not a Himar1C9 mutant.

[0149] As will be further detailed below, certain aspects of this disclosure relate to vectors or plasmids (e.g., expression vectors, packaging vectors, etc.) comprising a nucleic acid construct encoding a transposase, in particular, either the highly active PiggyBac transposase or a modified highly active PiggyBac transposase described herein, said vector or plasmid being suitable for expression in host cells, such as mammalian cells, yeast cells, insect cells, plant cells, fungal cells, or algal cells.

[0150] In one embodiment, a first protein (as described above) comprising or consisting of a site-specific DNA-binding protein capable of binding to and cleaving a target nucleic acid sequence, and a second protein (as described above) comprising or consisting of a transposase, are fused together directly or indirectly via a linker to form a fusion protein.

[0151] Any embodiment relating to site-specific DNA-binding proteins on the one hand, and to transposases on the other, also applies to the fusion proteins described herein with necessary modifications.

[0152] Therefore, in one embodiment, the fusion protein is (i) A first protein comprising or consisting of the above-mentioned RNA-induced DNA nuclease, zinc finger protein, or transcription activator-like effector nuclease, and (ii) A second protein containing or consisting of the above-mentioned transposase. It includes or consists of.

[0153] In one embodiment, the fusion protein is (i) A first protein comprising or consisting of the above-mentioned RNA-induced DNA nuclease or zinc finger protein, and (ii) A second protein containing or consisting of the above-mentioned transposase. It includes or consists of.

[0154] In one embodiment, the fusion protein is (i) A first protein comprising or consisting of the above-mentioned RNA-induced DNA nuclease, and (ii) A second protein containing or consisting of the above-mentioned transposase. It includes or consists of.

[0155] In one embodiment, the fusion protein is (i) A first protein comprising or consisting of the above-mentioned CAS9 protein or its variant, and (ii) the above-mentioned highly active PiggyBac transposase or a modified highly active PiggyBac transposase; in particular, a second protein comprising or consisting of the above-mentioned modified highly active PiggyBac transposase. It includes or consists of.

[0156] In one embodiment, the first protein and the second protein can be oriented in any order in the fusion protein.

[0157] In one embodiment, the fusion protein comprises or consists of a first protein fused directly or indirectly via a linker at the C-terminus of a second protein. In other words, the fusion protein comprises or consists of (i) a second protein (i.e., a transposase); (ii) optionally a linker; and (iii) a first protein (i.e., a site-directed DNA-binding protein, preferably an RNA-induced DNA nuclease; more preferably a Cas9 protein or a variant thereof) from the N-terminus to the C-terminus.

[0158] In one embodiment, the fusion protein comprises or consists of a first protein fused directly or indirectly via a linker at the N-terminus of a second protein. In other words, the fusion protein comprises or consists of (i) a first protein (i.e., a site-directed DNA-binding protein, preferably an RNA-induced DNA nuclease; more preferably a Cas9 protein or a variant thereof) from the N-terminus to the C-terminus; (ii) optionally a linker; and (iii) a second protein (i.e., a transposase).

[0159] In one embodiment, the fusion protein includes a linker.

[0160] A suitable example of a linker is a peptidic linker between a first protein and a second protein (in any order).

[0161] In one embodiment, the peptidic linker is (GGS) n , Sequence ID 114 (GGGGS) n , (G) n , Sequence ID 115 (EAAAK) n , XTEN linker, and (XP) n A selection is made from a group containing or consisting of motifs and any combination thereof, where n is an independent integer between 1 and 50.

[0162] In one embodiment, the linker is encoded by a nucleic acid sequence that is 12 to 24 amino acids long or 36 to 72 nucleotides long.

[0163] In one embodiment, the linker is an XTEN linker or (GGS) n It's a linker.

[0164] In one embodiment, the linker is selected from the linkers shown in Table 1.

[0165] Table 1: Linker [Table 1]

[0166] In one embodiment, the linker includes an amino acid sequence selected from the group comprising or comprising SEQ ID NOs: 41, SEQ ID NOs: 43, SEQ ID NOs: 45, SEQ ID NOs: 47, SEQ ID NOs: 49, SEQ ID NOs: 51, SEQ ID NOs: 53, SEQ ID NOs: 55, or any combination thereof, each encoded by the exemplary nucleic acid sequences of SEQ ID NOs: 40, SEQ ID NOs: 42, SEQ ID NOs: 44, SEQ ID NOs: 46, SEQ ID NOs: 48, SEQ ID NOs: 50, SEQ ID NOs: 52, and SEQ ID NOs: 54, respectively.

[0167] In one embodiment, the linker comprises or consists of the amino acid sequence of SEQ ID NO: 41, which is encoded by the exemplary nucleic acid sequence of SEQ ID NO: 40.

[0168] Fusion proteins obtained from the expression of any of the nucleic acid constructs provided in this disclosure are also provided herein.

[0169] In one embodiment, the fusion protein is a triple fusion protein.

[0170] Such triple fusion proteins, -One first protein (i.e., one site-specific DNA-binding protein) and two second proteins (i.e., two transposases); or - Two first proteins (i.e., two site-specific DNA-binding proteins) and one second protein (i.e., one transposase) It may include or consist of.

[0171] In one embodiment, the triple fusion comprises or consists of one first protein (i.e., one site-specific DNA-binding protein) and two second proteins (i.e., two transposases), and the triple fusion is located from the N-terminus to the C-terminus. -(i) site-specific DNA-binding protein, (ii) first transposase; (iii) second transposase; or -(i) first transposase; (ii) site-specific DNA-binding protein; (iii) second transposase; or -(i) first transposase; (ii) second transposase; (iii) site-specific DNA-binding protein Includes.

[0172] In one embodiment, the first and second transposases are identical. In another embodiment, the first and second transposases are different. For example, the first transposase may be a highly active PiggyBac transposase, and the second transposase may be a modified highly active PiggyBac transposase selected from any of the modified highly active PiggyBac transposases described herein. Alternatively, both the first and second transposases may be modified highly active PiggyBac transposases, each possessing different substitutions or combinations of substitutions described herein.

[0173] In one embodiment, the first and second transposases are capable of forming a functional dimer.

[0174] In one embodiment, the triple fusion comprises or consists of two first proteins (i.e., two site-specific DNA-binding proteins) and one second protein (i.e., one transposase), and the triple fusion is located from the N-terminus to the C-terminus. -(i) a first site-specific DNA-binding protein, (ii) a second site-specific DNA-binding protein; (iii) a transposase; or -(i) a first site-specific DNA-binding protein; (ii) a transposase; (iii) a second site-specific DNA-binding protein; or -(i) transposase; (ii) first site-specific DNA-binding protein; (iii) second site-specific DNA-binding protein Includes.

[0175] In one embodiment, the first and second site-specific DNA-binding proteins are identical. In another embodiment, the first and second site-specific DNA-binding proteins are different. For example, the first site-specific DNA-binding protein may be a Cas9 protein, and the second site-specific DNA-binding protein may be a Cas9 protein variant selected from any of the Cas9 protein variants described herein. Alternatively, both the first and second site-specific DNA-binding proteins may be CAS9 protein variants, but each is a different variant.

[0176] In one embodiment, the triple fusion protein may include a linker between two or three of its proteins.

[0177] (i) a second protein comprising or consisting of the above transposase; or a nucleic acid construct encoding the second protein, and (ii) an RNA-binding protein capable of binding to at least one specific RNA sequence; or a nucleic acid construct encoding the RNA-binding protein Fusion proteins including the above are also disclosed herein.

[0178] In one embodiment, the fusion protein includes the linker described above.

[0179] In one embodiment, the second protein comprises or consists of a transposase, wherein the transposase is the highly active PiggyBac of SEQ ID NO: 9. In one embodiment, the second protein comprises or consists of a transposase, wherein the transposase is a modified highly active PiggyBac having one or more amino acid mutations compared to the highly active PiggyBac of SEQ ID NO: 9. In particular, the modified highly active PiggyBac may be any of those disclosed herein.

[0180] In one embodiment, the transposase / RNA-binding protein fusion can be further fused to a first protein containing or comprising the above-mentioned site-specific DNA-binding protein.

[0181] In some embodiments, the RNA-binding protein is the MS2 bacteriophage coat protein (MCP) or a fragment thereof.

[0182] In some embodiments, the MCP has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with sequence number 132 (for example, encoded by the nucleic acid sequence of sequence number 131).

[0183] In some embodiments, the RNA-binding protein is capable of binding to at least one specific RNA sequence, the RNA sequence comprising a tetraloop. The term "tetraloop" is used interchangeably with the terms "stemloop" and "hairpin loop."

[0184] In some embodiments, at least one tetraloop is an MS2 RNA tetraloop binding sequence.

[0185] In some embodiments, the tetraloop is contained within the guide RNA (gRNA). In certain embodiments, the gRNA is in a complex with the Cas9 protein described above.

[0186] In some embodiments, the gRNA includes at least one MS2 RNA tetraloop binding sequence. In some embodiments, the gRNA includes two or more MS2 RNA tetraloop binding sequences.

[0187] In some embodiments, a gRNA containing at least one MS2 RNA tetraloop linking sequence has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 134 (e.g., encoded by the DNA sequence of SEQ ID NO: 133).

[0188] In some embodiments, the MCP in the fusion protein non-covalently binds to at least one MS2 RNA tetraloop binding sequence contained within the gRNA itself, which is non-covalently bound to the Cas9 protein; in particular, the binding of the fusion protein to the Cas9 / gRNA complex directs the cleavage activity of the modified, highly active PiggyBac transposase to a site specifically recognized by the Cas9 / gRNA complex.

[0189] As will be further detailed below, certain aspects of this disclosure also include vectors or plasmids (e.g., expression vectors, packaging vectors, etc.) comprising nucleic acid constructs encoding the fusion proteins described herein; said vectors or plasmids are preferably suitable for expression in host cells, such as mammalian cells, yeast cells, insect cells, plant cells, fungal cells, or algal cells.

[0190] According to the present invention, the composition may include a first protein and / or a second protein (or a fusion protein including both) as either the above-mentioned protein or as a nucleic acid construct encoding these proteins.

[0191] Therefore, in one embodiment, the composition of the present invention is a) A nucleic acid construct encoding the first protein, comprising or consisting of a site-specific DNA-binding protein capable of binding to and cleaving the target nucleic acid sequence; (b) a nucleic acid construct encoding the second protein described above, comprising or consisting of a transposase; and c) A nucleic acid construct comprising or consisting of laminin-α2 protein, a transgene encoding a functional variant or fragment thereof. It includes or consists of.

[0192] In another embodiment, the composition of the present invention is a) A nucleic acid construct encoding the above-mentioned fusion protein comprising (i) a first protein comprising or comprising a site-specific DNA-binding protein capable of binding to and cleaving a target nucleic acid sequence, and (ii) a second protein comprising or comprising a transposase; and b) A nucleic acid construct comprising or consisting of a transgene encoding the laminin-α2 protein, a functional variant or fragment thereof. It includes or consists of.

[0193] In one embodiment, the nucleic acid construct encoding the fusion protein further includes a nucleic acid sequence encoding a linker between the first protein and the second protein; or, in the case of a triple fusion protein, between the two proteins or between the three proteins.

[0194] According to this disclosure, the first protein and the second protein, or a fusion protein comprising or consisting of the first protein and the second protein, enable and / or facilitate site-specific insertion of a transgene in a genome, particularly a transgene encoding the laminin-α2 protein, a functional variant or fragment thereof.

[0195] Some embodiments include, - A nucleic acid construct that codes for the first protein; or - A nucleic acid construct that codes for a second protein; or - A nucleic acid construct encoding a first protein and a nucleic acid construct encoding a second protein; or - Nucleic acid constructs encoding fusion proteins or triple fusion proteins This applies to plasmids or vectors (e.g., expression vectors) containing any of the following.

[0196] In some embodiments, the plasmid is a packaging plasmid. In some embodiments, the plasmid further comprises a polynucleotide encoding a capsid protein, e.g., gag and pol. In some embodiments, the plasmid is combined with a second plasmid (envelope plasmid) containing a polynucleotide encoding a viral envelope protein; and a third plasmid containing a nucleic acid construct containing the LAMA2 transgene. When this combination is introduced into a producing cell line (e.g., eukaryotic cells, prokaryotic cells and / or cell lines), viral particles are produced containing the nucleic acid construct encoding the LAMA2 transgene and a nucleic acid construct encoding either the first protein, the second protein, both the first and second proteins, or a fusion protein.

[0197] In some embodiments, the plasmid is combined with a second plasmid (a packaging plasmid lacking functional integrase) containing polynucleotides encoding capsid proteins, e.g., gag and pol; a third plasmid (an envelope plasmid) containing polynucleotides encoding viral envelope proteins; and a fourth plasmid containing a nucleic acid construct including the LAMA2 transgene. When this combination is introduced into a producing cell line (e.g., eukaryotic and prokaryotic cells and / or cell lines), viral particles are produced that contain the nucleic acid construct including the LAMA2 transgene and a nucleic acid construct encoding either a first protein, a second protein, both first and second proteins, or a fusion protein.

[0198] In one embodiment, the first protein, the second protein, both the first and second proteins or a fusion protein, and / or the LAMA2 transgene are delivered to the cell using lentiviral particles.

[0199] In one embodiment, the nucleic acid construct comprises a first polynucleotide sequence encoding a first protein comprising or consisting of a site-directed DNA-binding protein engineered to bind to a target nucleic acid sequence; a second polynucleotide sequence encoding a second protein comprising or consisting of a transposase enabling insertion of a LAMA2 transgene into the genome; and optionally, a third polynucleotide sequence comprising a nucleic acid sequence encoding a linker between the first and second polynucleotides. In some embodiments, the first protein is the zinc finger protein or Cas9 protein or a variant thereof; and / or the second protein is the high-activity PiggyBac transposase or a modified high-activity PiggyBac transposase.

[0200] Examples of linkers suitable for generating fusion proteins are listed above.

[0201] In some embodiments, a linker is not required because the first protein is expressed from a different plasmid than the second protein.

[0202] In one embodiment, instead of using a linker, the first and / or second polynucleotide sequences each comprise nucleic acids encoding the first and second proteins, and further include an additional nucleotide at at least one of their ends to enable the linker to function.

[0203] In one embodiment, the nucleic acid construct is in the form of DNA or RNA.

[0204] Vectors containing any of the nucleic acid constructs provided in this disclosure are also provided herein. In particular, the vectors are suitable for expression in mammalian cells, yeast cells, insect cells, plant cells, fungal cells, or algal cells. Host cells containing any of the nucleic acid constructs or vectors provided in this disclosure are also provided herein.

[0205] According to the present invention, the composition further comprises a nucleic acid construct comprising a transgene encoding a laminin-α2 protein, a functional variant or fragment thereof, also referred to herein as the "LAMA2 transgene."

[0206] In one embodiment, the LAMA2 transgene may be any nucleic acid sequence encoding laminin-α2 protein, particularly wild-type mammalian laminin-α2 protein, preferably human laminin-α2 protein, a functional variant or fragment thereof.

[0207] In one embodiment, the LAMA2 transgene may include any of the following: the LAMA2 wild-type gene (or a functional variant or fragment thereof), LAMA2 cDNA (or a functional variant or fragment thereof), or LAMA2 minigene (or a functional variant or fragment thereof).

[0208] In one embodiment, the LAMA2 transgene includes the full-length LAMA2 gene.

[0209] The laminin-α2 chain protein is encoded by the LAMA2 gene (gene ID: 3908, updated November 24, 2020), transcribed, and translated into a 390 kDa protein. Laminin-α2 is a component of the cruciate heterotrimer known as laminin-211 (or merosine), which consists of three subunits (alpha, beta, and gamma) linked to each other by disulfide bonds.

[0210] The coding sequences of numerous different mammalian laminin-α2 proteins, including but not limited to those derived from humans, pigs, chimpanzees, dogs, cattle, mice, rabbits, or rats, are publicly known in the art and can be readily found in sequence databases. Alternatively, the coding sequence can be readily determined by those skilled in the art based on the polypeptide sequence.

[0211] In particular, the LAMA2 transgene can encode either the human laminin subunit α2 isoform a precursor (see NCBI: NP_000417, submitted October 22, 2020) or the laminin-subunit α2 isoform b precursor (see NCBI: NP_001073291.2, submitted October 24, 2020).

[0212] In one embodiment, the LAMA2 transgene preferably encodes the human laminin subunit α2 isoform a precursor of Sequence ID No. 74.

[0213] After translation, the laminin-α2 chain is typically cleaved into an N-terminal and a C-terminal fragment that are associated with each other by non-covalent bonds.

[0214] In one embodiment, the LAMA2 transgene is preferably laminin-subunit α2 isoform X1 (NCBI reference: XP_005267038.1, SEQ ID NO: 116), laminin-subunit α2 isoform X2 (NCBI reference: XP_011534122.1, SEQ ID NO: 117), laminin-subunit α2 isoform X3 (NCBI reference: XP_005267039.1, SEQ ID NO: 118), laminin-subunit α2 isoform X1 It is possible to encode different human mature isoforms of the laminin-α2 protein, including or selected from the group consisting of laminin-subunit α2 isoform X4 (NCBI reference: XP_016866340.1, SEQ ID NO: 119), laminin-subunit α2 isoform X5 (NCBI reference: XP_01686634.1, SEQ ID NO: 120), and laminin-subunit α2 isoform X6 (NCBI reference: XP_005267038.1, SEQ ID NO: 121).

[0215] As used herein, the terms “fragment” or “functional fragment” refer to a laminin-α2 protein or the nucleic acid encoding it, and mean a protein, polypeptide, or nucleic acid derived from the laminin-α2 protein that still retains the activity of the full-length laminin-α2 protein but whose sequence is not 100% identical to that of the full-length protein, polypeptide, or nucleic acid. A functional fragment may have more, fewer, or the same number of residues as the corresponding native molecule and / or may contain one or more amino acid or nucleotide substitutions. Preferably, a functional fragment refers to a mature form of the laminin-α2 protein that does not contain a signal peptide at the N-terminus of the protein. Those skilled in the art can easily determine which part of the laminin-α2 protein a signal peptide corresponds to, for example, based on information published on the Uniprot database.

[0216] In one embodiment, the LAMA2 transgene includes the sequence of SEQ ID NO: 75. In one embodiment, the LAMA2 transgene includes a human LAMA2 minigene containing the synthetic intron of SEQ ID NO: 82. In one embodiment, the synthetic intron is located between the nucleic acid bases at positions 3925 and 3926 of the coding sequence of SEQ ID NO: 75.

[0217] In one embodiment, the LAMA2 transgene may be any nucleic acid sequence encoding a functional laminin-α2 protein variant that retains the activity of the full-length laminin-α2 protein. In particular, the laminin-α2 protein variant retains the ability to bind to the laminin-211β- and γ-subunits via disulfide bonds to form functional cruciate laminin-211 or laminin-221. Laminins form an independent network and are associated with the type IV collagen network via enterin, fibronectin, and perlecan. They also bind to the cell membrane via integrin receptors and other plasma membrane molecules such as dystroglycan glycoprotein complexes and lutheran blood group glycoproteins. Through these interactions, laminins make a critical contribution to cell adhesion and differentiation, cell shape and migration, maintenance of tissue phenotype, and promotion of tissue survival.

[0218] Preferably, as used herein, the terms “variant” or “functional variant” refer to a polypeptide having an amino acid or nucleotide sequence that has at least 70, 75, 80, 85, 90, 95, or 99% sequence identity with the wild-type laminin-α2 protein sequence, with respect to the laminin-α2 protein.

[0219] More preferably, the terms “variant” or “functional variant” refer to a polypeptide having an amino acid sequence different from the wild-type laminin-α2 protein sequence by 30, 25, 20, 15, 10 or fewer than 5 substitutions, insertions and / or deletions. In one embodiment, the variant differs from the wild-type laminin-α2 protein sequence by one or more conservative substitutions, preferably by 15, 10 or fewer than 5 conservative substitutions. Examples of conservative substitutions are substitutions within the groups of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (methionine, leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and minor amino acids (glycine, alanine, serine, and threonine).

[0220] Therefore, in one embodiment, the LAMA2 transgene includes a sequence having at least 70, 75, 80, 85, 90, 95, or 99% sequence identity with sequence number 75.

[0221] In one embodiment, the LAMA2 transgene may include an optimized sequence encoding the laminin-α2 protein, a variant thereof, or a fragment thereof.

[0222] In the context of nucleic acid sequences, the term "optimization" refers to codon optimization, which typically involves changing codons that exhibit a bias towards humans to synonymous codons that do not exhibit a bias towards humans (i.e., codons that encode the same amino acid residue). Therefore, changes in codons do not result in any amino acid changes in the encoded protein.

[0223] According to the present invention, the LAMA2 transgene is included in the nucleic acid construct.

[0224] In one embodiment, the nucleic acid construct includes a LAMA2 transgene ligated to one or more regulatory sequences that direct the expression of the transgene in a cell.

[0225] The promoter contains a transcriptional regulatory sequence that mediates the expression of the LAMA2 transgene upon introduction into host cells. The promoter may be any polynucleotide that exhibits transcriptional activity in cells, including mutant promoters, cleavage promoters, and hybrid promoters. The promoter may be a constitutive or inducible promoter, preferably a constitutive promoter, and more preferably a potent constitutive promoter.

[0226] Examples of suitable promoters include, but are not limited to, the CMV promoter of SEQ ID NO: 76, the CAG promoter of SEQ ID NO: 77, the EF1-α promoter of SEQ ID NO: 78, the SV-40 promoter of SEQ ID NO: 79, and the hybrid albumin enhancer-α1-antitrypsin (EalbAAT) liver-specific promoter of SEQ ID NO: 80.

[0227] In alternative embodiments, the LAMA2 transgene may also be inserted into the cell's genome such that its expression is promoted by an endogenous promoter at or near the integration site. In particular, a nucleic acid construct containing the LAMA2 transgene may include splice acceptors to ensure gene expression when the transgene is integrated into an intron of an actively expressed gene, such as endogenous LAMA2 or an albumin gene, in order to control endogenous expression.

[0228] Splice acceptor sites provide a signal that targets the sequence following the expressed splice acceptor site (Padgett et al., 1988. Annu Rev Biochem. 55:1119-1150). Splice acceptor sites are nucleotide sequences commonly involved in RNA splicing to remove intronic RNA sequences. Splice acceptor sites typically participate in the excision of introns, binding to an RNA-protein complex called a spliceosome, being cleaved, and then binding to an already cleaved splice donor site. Splice acceptor sequences are well known in the art and can be readily obtained from genes located between exons and introns that mediate splicing. Alternatively, splice acceptor sites may be synthesized chemically or enzymatically. Splice acceptor (SA) sites typically end with a highly conserved AG dinucleotide. The remaining nucleotides in the sequence are mainly cytidine and / or thymidine. In one embodiment, the splice acceptor has the sequence of Sequence ID No. 81.

[0229] Regulatory sequences may also include appropriate transcription start, termination, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); and / or sequences that enhance protein stability. For example, numerous innate, constitutive, inducible, and / or tissue-specific expression regulatory sequences are known in the art and can be used to promote the expression of LAMA2 transgenes. Typically, LAMA2 transgenes are ligated to act on transcription promoters and transcription terminators.

[0230] Poly(A) signaling typically consists of a) a consensus sequence AAUAAA, which has been shown to be necessary for both 3' end cleavage and polyadenylation of pre-messenger RNA (pre-mRNA) and to facilitate downstream transcription termination, and b) upstream and downstream appendages of the AAUAAA sequence that control the utilization efficiency of AAUAAA as a poly(A) signal. There is considerable diversity in these motifs in mammalian genes.

[0231] In one embodiment, the polyadenylation signal sequence in the nucleic acid construct is a polyadenylation signal sequence of a mammalian gene or a viral gene. Suitable polyadenylation signals include, but are not limited to, the SV40 early polyadenylation signal, the SV40 late polyadenylation signal, the HSV thymidine kinase polyadenylation signal, the protamine gene polyadenylation signal, the adenovirus 5EIb polyadenylation signal, the growth hormone polyadenylation signal, the PBGD polyadenylation signal, and in silico-designed polyadenylation signals (synthesized). In one embodiment, the polyadenylation signal sequence is poly(A) having sequences including or selected from the group consisting of sequence numbers 83, 84, and 85.

[0232] In one embodiment, the nucleic acid construct containing the LAMA2 transgene also includes an insulator element to increase the transgene expression level, avoid silencing, and reduce expression variability.

[0233] Insulators are a complex class of cis-acting regulatory sequences that prevent heterochromatin diffusion and gene silencing (barrier activity), and possess enhancer-blocking activity. Insulators are typically 300 bp to 2000 bp in length. There are many examples of insulators, including CTCF insulators, gypsy insulators, and insulators containing β-globin loci.

[0234] In one embodiment, the insulator element sequence is adjacent to both ends of the nucleic acid construct.

[0235] In one embodiment, the nucleic acid construct containing the LAMA2 transgene includes insulator elements of sequence numbers 86 and 87.

[0236] In one embodiment, the transposase disclosed herein recognizes an inverted terminal repeat (ITR) element possessing a transposase binding site and catalyzes the excision and subsequent re-incorporation of the element between the ITRs.

[0237] Therefore, in certain embodiments, the nucleic acid construct containing the LAMA2 transgene has inverted terminal sequences (ITRs) that possess transposase binding sites, preferably the 5'-ITR and 3'-ITR of sequence numbers 88 and 89, adjacent to each other.

[0238] In one embodiment, an increase in the size between ITRs positively regulates the introduction of the LAMA2 transgene into genomic DNA.

[0239] In one embodiment, the size between ITRs is at least 105 bp. In one embodiment, the size between ITRs is at least 200 bp. In one embodiment, the size between ITRs is at least 300 bp.

[0240] As used herein, the term “at least 300 bp” means 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 kb, 2 kb or more.

[0241] In one embodiment, the nucleic acid construct containing the LAMA2 transgene is included in the expression vector.

[0242] Examples of suitable vectors include, but are not limited to, recombinant integrative or non-integrative viral vectors, and vectors derived from recombinant bacteriophage DNA, plasmid DNA or cosmid DNA. In one embodiment, the vector is a plasmid vector, a minicircle vector or a doggybone DNA donor vector. Preferably, the vector is a recombinant integrative or non-integrative viral vector. Examples of recombinant viral vectors include, but are not limited to, vectors derived from herpes virus, retrovirus, lentivirus, vaccinia virus, adenovirus, adeno-associated virus or bovine papillomavirus.

[0243] In one embodiment, the viral vector is a lentivirus or retrovirus vector. Thus, the present disclosure also relates to viral particles comprising a nucleic acid construct comprising a LAMA2 transgene, or an expression vector comprising said nucleic acid construct, as described above. In particular, the nucleic acid construct or expression vector can be packaged into a viral capsid to generate "viral particles", also named "viral vector particles".

[0244] In one embodiment, the nucleic acid construct or expression vector is packaged into a capsid derived from lentivirus or AAV to generate "lentiviral particles" or "AAV particles".

[0245] According to the invention, the first protein comprises or consists of a site-specific DNA-binding protein capable of binding to and cleaving a target nucleic acid sequence.

[0246] As used herein, "target sequence" or "target nucleic acid sequence" or "target site" is a sequence that defines a portion of a nucleic acid in a genome to which a binding molecule binds, provided that sufficient conditions for binding exist.

[0247] The first protein can be engineered to bind to any selected sequence within the genome of a cell, referred to as the "target nucleic acid sequence". For example, the sequence 5'-GAATTC-3' is the target site of the EcoRI restriction endonuclease.

[0248] In one embodiment, the target nucleic acid sequence is within a safe harbor locus in the genome of the cell. In one embodiment, the target nucleic acid sequence is within the endogenous LAMA2 gene in the genome of the cell. In particular, the first and / or second protein can be engineered to bind to and / or integrate into a transgene encoding the laminin-α2 protein within a safe harbor locus.

[0249] A "safe harbor locus" refers to a region of the genome of a cell that can appropriately express an integrated substance without disrupting endogenous gene structure or function. Safe harbor loci include, but are not limited to, AAVSl (intron 1 of PPP1R12C), HPRT, HI 1, hRosa26, albumin, and the F-A region. A safe harbor locus can be an exon or intron of a ubiquitously expressed gene and / or a gene that is tissue-specifically expressed (e.g., muscle). The safe harbor locus can be selected from the group consisting of exon 1, intron 1, or exon 2 of PPP1R12C, exon 1, intron 1, or exon 2 of HPRT, and exon 1, intron 1, or exon 2 of hRosa26, intron 1 of the albumin gene. A safe harbor locus can also include a region of the genome that lacks an endogenous gene and has open chromatin that allows for the expression of an integrated transgene without disrupting genome structure or function.

[0250] In one embodiment, the first and / or second protein is engineered to bind to the LAMA2 transgene and integrate it into the endogenous LAMA2 gene, particularly intron 1 of the endogenous LAMA2 gene, within the genome of the cell.

[0251] In one embodiment, the first protein is optionally combined with a guide RNA and binds to a target nucleic acid sequence selected from the group consisting of one of sequence numbers 90 to 97.

[0252] In one embodiment, the composition of the present invention further comprises guide RNA.

[0253] As used herein, “guide RNA,” “gRNA,” or “single guide RNA” refers to a nucleic acid that facilitates the specific targeting or homing of a gRNA / Cas complex to a target nucleic acid.

[0254] In particular, gRNA refers to RNA molecules including transactivated crRNA (tracrRNA) and crRNA. Preferably, the guide RNA corresponds to crRNA and tracrRNA that can be used separately or fused together. When the target sequence forms a pair with the complementary sequence, the Cas protein is recruited to bind to and cleave the DNA at the target sequence.

[0255] In one embodiment, the guide RNA is manipulated to include a sequence complementary to a portion of the target sequence, preferably selected from the group consisting of any one of sequence numbers 90 to 97.

[0256] As used herein, the term “complementary sequence” refers to a sequence of polynucleotides (e.g., a portion of crRNA or tracRNA) that can hybridize to another portion of a polynucleotide under standard low-stringent conditions. Preferably, the sequences are complementary to each other according to the complementarity between two nucleic acid strands, which depends on inter-strand Watson-Crick base pairing, i.e., intrinsic base pairing between adenine and thymine (AT) nucleotides and between guanine and cytosine (GC) nucleotides.

[0257] The disclosure also relates to a method for incorporating a LAMA2 transgene into a target nucleic acid sequence within the genome of a cell. The method includes introducing the composition into a cell such that a first protein and a second protein (either separately or as part of the fusion protein described above) cleave the target nucleic acid sequence and incorporate the LAMA2 transgene within the target nucleic acid sequence.

[0258] The method involves introducing into cells a nucleic acid construct comprising a first protein and a second protein (either separately or as part of the above-mentioned fusion protein) or one or more nucleic acid constructs encoding them; guide RAN; and a transgene encoding a laminin-α2 protein, a functional variant or fragment thereof.

[0259] These elements can be synthesized intracellularly as a result of introducing nucleic acid constructs or expression vectors encoding the elements into the cell, as described above. Alternatively, the elements may be generated extracellularly and then introduced into the cell.

[0260] In one embodiment, a nucleic acid construct, expression vector, or viral particle containing the above-mentioned LAMA2 transgene is introduced into the cell.

[0261] In one embodiment, one or more polynucleotides encoding the first protein and the second protein (either separately or as part of the fusion protein described above) may be transfected in mRNA form, which are then directly introduced into cells, for example, by electroporation or lipid nanoparticles.

[0262] In one embodiment, the guide RNA may also be generated extracellularly and then introduced into the cell, for example, by electroporation or lipid nanoparticles.

[0263] In one embodiment, the first protein and the second protein (either separately or as part of the fusion protein described above) can be introduced into cells either alone or pre-complexed with guide RNA.

[0264] In one embodiment, the guide RNA and / or the first and second proteins (either separately or as part of the fusion protein described above) are encoded by a nucleic acid construct or expression vector.

[0265] Products (proteins and nucleic acids) according to the present invention may be delivered intracellularly or into intracellular compartments by physical methods such as liposome delivery means, polymer carriers, chemical carriers, lipoplexes, polyplexes, dendrimers, nanoparticles, emulsions, natural endocytosis or phagocytic pathways, and electroporation, among others.

[0266] The nucleic acid construct or expression vector can be introduced into cells by any method known in the art, and non-limiting examples include stable transformation methods in which the nucleic acid construct or expression vector is integrated into the cell genome, transient transformation methods in which the nucleic acid construct or expression vector is not integrated into the cell genome, and virus-mediated methods. For example, transient transformation methods include, for example, microinjection, electroporation, or particle collision.

[0267] The nucleic acid molecules, nucleic acid constructs, or expression vectors according to the present invention can be introduced into cells using any known technique, including but not limited to calcium phosphate transfection, DEAE-dextran transfection, electroporation, microinjection, bioristic, viral infection, or liposome-mediated transfection.

[0268] In one embodiment, RNA encoding a first protein and a second protein (either separately or as part of the above fusion protein), preferably guide RNA or mRNA, can be generated in vitro, for example, by in vitro transcription. The RNA can then be introduced into cells by electroporation (for example, as described in Almasbak et al., 2011. Cytotherapy. 13(5):629-640; Rabinovich et al., 2009. Hum Gene Ther. 20(1):51-61; and Beatty et al., 2013. Cancer Immunol Res. 2(2):112-20).

[0269] Alternatively, the RNA may be introduced by other means such as liposomes or cationic molecules.

[0270] In one embodiment, the nucleic acid construct(s) or vector(s) introduced into the cell can be expressed episomally or integrated into the genome of the cell.

[0271] In one embodiment, polynucleotides such as guide RNA and mRNA encoding a nucleic acid construct containing or comprising a first protein and a second protein (either separately or as part of the above fusion protein) and / or the LAMA2 transgene can be delivered to cells or patients by nanoparticles, preferably lipid nanoparticles (LNPs). LNPs can be generated using any lipid or combination of lipids known in the art. Examples of lipids used to generate LNPs include DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Examples of cationic lipids include 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids include DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids include PEG-DMG, PEG-CerC14, and PEG-CerC20.

[0272] In one embodiment, the method is either in vitro or ex vivo. The in vitro method is performed on cell cultures.

[0273] In another embodiment, the disclosure relates to engineered isolated cells that can be obtained or obtained by the above-described method. The engineered cells include at least the transgene encoding the laminin-α2 protein described above.

[0274] In one embodiment, the manipulated isolated cells contain at least one transgene encoding laminin-α2, which includes one or more regulatory sequences that direct the expression of the transgene in the cell, as described above.

[0275] In some embodiments, the manipulated isolated cells contain at least one transgene encoding laminin-α2, including the adjacent ITRs described above.

[0276] In one embodiment, the size between ITRs is at least 105 bP, at least 200 bp, at least 300 bp or more.

[0277] The manipulated cells described herein may be used for ex vivo gene therapy. In such embodiments, a first protein and a second protein (either separately or as part of the above fusion protein), a guide RNA and a LAMA2 transgene, or the above nucleic acid construct(s), expression vector(s), or viral particle(s) are introduced into the cells. The isolated cells can then be transplanted into a patient or subject. This implantation step can be achieved using any implantation method known in the art. For example, the genetically modified cells may be injected directly into the patient's blood, or into a desired muscle, or otherwise administered to the patient. The transplanted cells may be autologous, allogeneic, or heterologous. In particular, the cells may be myocytes isolated from the skeletal muscle, smooth muscle, or cardiac muscle of a donor or patient, or mesenchymal stem cells derived from bone marrow or peripheral blood. For clinical use, cell isolation is usually performed under Good Manufacturing Practice (GMP) conditions.

[0278] Suitable cells include, but are not limited to, eukaryotic cells and prokaryotic cells and / or eukaryotic cell lines and prokaryotic cell lines. Preferably, the cells are eukaryotic cells such as mammalian cells, which include, but are not limited to, humans, non-human primates such as great apes; chimpanzees; monkeys, and domestic animals such as orangutans, dogs and cats, as well as domestic animals such as horses, cattle, pigs, sheep and goats, or other mammalian species such as mice, rats, guinea pigs, rabbits and hamsters. A person skilled in the art will select more appropriate cells depending on the patient or subject to be transplanted.

[0279] The manipulated cells may also be muscle cells. As used herein, the term “muscle” refers to cardiac muscle (i.e., heart) and skeletal muscle. As used herein, the term “muscle cells” refers to muscle cells, myotubes, myoblasts, and / or satellite cells.

[0280] The manipulated cells may be self-renewing and pluripotent cells such as stem cells or induced pluripotent stem cells. Suitable stem cells include, for example, embryonic stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells, neural stem cells, and mesenchymal stem cells. Stem cells are preferably mesenchymal stem cells. Mesenchymal stem cells (MSCs) can differentiate into at least one of osteoblasts, chondrocytes, adipocytes, or muscle cells and can be isolated from any type of tissue. Generally, MSCs are isolated from bone marrow, adipose tissue, umbilical cord, or peripheral blood. Methods for obtaining them are well known to those skilled in the art. Induced pluripotent stem cells (also known as iPS cells or iPSC cells) are a type of pluripotent stem cell that can be produced directly from adult cells. Yamanaka et al. induced iPS cells by introducing the Oct3 / 4, Sox2, Klf4, and c-Myc genes into mouse and human fibroblasts and expressing these genes in the cells (WO2007 / 069666). Thomson et al. subsequently generated human iPS cells using Nanog and Lin28 instead of Klf4 and c-Myc (WO2008 / 118820). In certain embodiments, the cells are myoblasts. Myoblasts may be iPSC-derived, including stem cells, e.g., iPSC cells derived from patients with muscle disorders such as muscular dystrophy.

[0281] The compositions according to this disclosure are preferably used in the form of a pharmaceutical composition comprising a therapeutically effective amount of the product according to the present invention, such as a nucleic acid construct encoding the LAMA2 transgene, a nucleic acid construct encoding a first protein and a second protein (either separately or as part of the above-mentioned fusion protein), a polynucleotide or nucleic acid construct including guide RNA, or an expression vector or viral particle encoding the product.

[0282] In the context of this disclosure, therapeutically effective dose means a dose sufficient to reverse, alleviate or inhibit the progression of the disorder or condition to which such term applies, or to reverse, alleviate or inhibit the progression of one or more symptoms of the disorder or condition to which such term applies.

[0283] The effective dose is determined and adjusted according to factors such as the composition used, the route of administration, the physical characteristics of the individual being considered (such as sex, age, and weight), concomitant medications, and other factors recognized by those skilled in the medical field.

[0284] In various embodiments of this disclosure, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or vehicle.

[0285] A "pharmaceutically acceptable carrier" refers to a vehicle that, when administered to a mammal, particularly a human, does not cause any adverse allergic or other troublesome response. A pharmaceutically acceptable carrier or excipient refers to any kind of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid.

[0286] Preferably, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for an injectable formulation. These may be a dry composition, particularly a lyophilized composition, that enables the formation of an injectable solution upon addition of isotonic, sterile saline (such as monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or a mixture of such salts), or optionally sterile water or saline.

[0287] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or suspensions. The solution or suspension may contain cell-compatible additives. The solution or suspension may contain additives compatible with non-viral vectors, viral vectors, and nanoparticles, and which do not interfere with the entry of components into target cells. In all cases, the form must be sterile and fluid enough to allow for easy syringe operation. It must be stable under manufacturing and storage conditions and preserved against contamination by microorganisms such as bacteria and fungi. Examples of suitable solutions are buffers such as phosphate-buffered saline (PBS) or Ringer's lactate.

[0288] The compositions relating to this disclosure are used for therapeutic purposes, particularly for the treatment of LAMA2-associated muscular dystrophy.

[0289] LAMA2-associated muscular dystrophy is a disorder that causes weakness and wasting (atrophy) of the muscles used for movement (skeletal muscles). The severity of this condition varies from severe early-onset to mild late-onset.

[0290] Early-onset LAMA2-associated muscular dystrophy is evident at birth or within the first few months of life. It is considered part of a class of muscle disorders called congenital muscular dystrophy, and is sometimes referred to as congenital muscular dystrophy type 1A. Affected infants may have severe muscle weakness, lack of muscle tone (hypotonia), limited spontaneous movement, and joint deformities (contractures). Weakness of the facial and throat muscles can lead to difficulty feeding, as well as an inability to grow at the expected rate and gain weight. Respiratory failure due to weakness of the chest muscles can cause a weak cry and breathing problems, and may frequently lead to lung infections that are potentially life-threatening.

[0291] As affected children grow, they often develop abnormal, gradually worsening lateral curvature of the spine (scoliosis) and inward curvature of the spine (lordosis). Children with early-onset LAMA2-associated muscular dystrophy often fail to develop the ability to walk. Speech difficulties may be due to weakness of the facial muscles and enlargement of the tongue. Seizures occur in about one-third of individuals with early-onset LAMA2-associated muscular dystrophy, and cardiac complications rarely occur in this form of the disorder.

[0292] The symptoms of late-onset LAMA2-associated muscular dystrophy become apparent later in childhood or adulthood and are similar to those of a group of muscle disorders classified as limb-girdle muscular dystrophy. In late-onset LAMA2-associated muscular dystrophy, the muscles most affected are the muscles closest to the body (proximal muscles), particularly those of the shoulder, upper arm, pelvic region, and thigh. Children with late-onset LAMA2-associated muscular dystrophy experience delayed development of motor skills, such as walking, but generally reach the ability to walk without assistance. Over time, they may develop spinal stiffness, joint contractures, scoliosis, and respiratory problems. However, most affected individuals retain the ability to walk and climb stairs.

[0293] The Disclosure also provides a method for treating LAMA2-associated muscular dystrophy according to the Disclosure, comprising administering a therapeutically effective amount of the above-mentioned composition, manipulated cells, or pharmaceutical composition to a patient.

[0294] "Therapeutic effective dose" refers to the amount and duration of medication that is effective in achieving the desired therapeutic outcome and preventing, delaying, or reversing at least one sign or symptom of LAMA2-related muscular dystrophy, such as skeletal muscle weakness and atrophy. The therapeutic effective dose of the products of this disclosure, or pharmaceutical compositions containing them, may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the product or pharmaceutical composition to induce the desired response in the individual. The administration regimen may be adjusted to provide the optimal therapeutic response. The therapeutic effective dose is also typically the amount in which the therapeutically beneficial effects of the product or pharmaceutical composition outweigh any toxic or adverse effects.

[0295] As used herein, the terms “patient” or “individual” mean a mammal. Preferably, the patient or individual as used herein is a human.

[0296] In the context of this disclosure, as used herein, the terms “to treat” or “treatment” mean to reverse, mitigate or inhibit the progression of LAMA2-associated muscular dystrophy or condition to which such terms apply, or to reverse, mitigate or inhibit the progression of one or more symptoms of the disorder or condition to which such terms apply, in particular to a reduction in skeletal muscle weakness and atrophy and / or improvement in skeletal muscle function.

[0297] The pharmaceutical compositions of this disclosure are generally administered to patients in doses and for a duration effective in inducing a therapeutic effect, in accordance with known procedures.

[0298] Administration may be systemic or local. Systemic administration may preferably be parenteral administration such as subcutaneous (SC) or intramuscular (IM), intravascular administration such as intravenous (IV) or intra-arterial; intraperitoneal (IP) administration; intradermal (ID) administration; interstitial administration or others. Administration may be by injection or perfusion, for example. In some preferred embodiments, administration is parenteral, preferably intravascular administration such as intravenous (IV) or intra-arterial. Implementations of this disclosure employ the prior art, which is within the scope of the art of the art unless otherwise specified. Such art is fully described in the literature.

[0299] Kits for carrying out the methods disclosed herein are also provided herein. The kits may include nucleic acid constructs encoding the first protein and the second protein (either separately or as part of the above-described fusion protein), and nucleic acid constructs comprising the LAMA2 transgene described herein. In some embodiments, the kits may include lentiviral particles comprising nucleic acid constructs comprising or comprising the LAMA2 transgene described herein and the nucleic acid constructs comprising the LAMA2 transgene described herein.

[0300] The kit may further include instructions for using the kit's components to carry out the method. Instructions for carrying out the method are generally recorded on a suitable recording medium. For example, the instructions can be printed on a material such as paper or plastic. Thus, the instructions may be present in the kit as an accompanying document, on the label of the kit or its component containers (i.e., associated with packaging or subpackaging), etc. In other embodiments, the instructions exist as an electronic storage data file on a suitable computer-readable storage medium, such as a CD-ROM or diskette. In yet another embodiment, the actual instructions are not present in the kit, but a means for obtaining the instructions from a remote source, for example via the internet, is provided. An example of this embodiment is a kit that includes a web address from which the instructions can be viewed and / or downloaded. Like the instructions, this means for obtaining the instructions is recorded on a suitable material.

[0301] This disclosure is usually, (i) a first protein as defined herein, or a nucleic acid encoding the first protein, (ii) a second protein as defined herein, or a nucleic acid encoding the second protein, and (iii) A nucleic acid construct containing or comprising a LAMA2 transgene The present invention relates to a composition or kit comprising a first composition containing the following.

[0302] This disclosure also states, (i) A fusion protein as defined herein, or a nucleic acid encoding the fusion protein (ii) A nucleic acid construct containing or comprising a LAMA2 transgene The present invention also relates to a composition or kit comprising a first composition containing the first composition containing the first composition.

[0303] In one embodiment, the composition or kit comprises a minicircle, plasmid, or viral vector, particularly a non-integrated viral vector, for example, or an exogenous nucleic acid within a non-integrated lentiviral vector. In one embodiment, the composition or kit disclosed herein is contained within nanoparticles.

[0304] In one embodiment, the nucleic acid construct encoding the first and / or second protein (either separately or as part of the fusion protein described above) is in the form of RNA, DNA, or protein, and the polynucleotide sequence encoding the LAMA2 transgene is in the form of RNA or DNA, depending on the delivery method. In particular, the polynucleotide sequence encoding the exogenous nucleic acid is in the form of RNA.

[0305] In one embodiment, the composition or kit is virus-free, and the packaging vector is nanoparticles, such as polymer nanoparticles or lipid nanoparticles. The packaging vector may also be a carrier bound to the elements of the composition. In some embodiments, the composition comprises a viral vector, particularly lentiviral particles.

[0306] In one embodiment, the composition or kit comprises, contained in or conjugated to a package vector, (a) a nucleic acid construct in the form of RNA encoding either a first protein and a second protein as part of the above-mentioned fusion protein (e.g., Cas9 and transposase), (b) optionally, a guide RNA (e.g., as separate linear single-stranded RNA molecules), and (c) a polynucleotide comprising or consisting of a LAMA2 transgene for insertion into DNA (e.g., in the vector).

[0307] In one embodiment, the composition comprises (a) a first protein and a second protein described herein (e.g., Cas9 and transposase) in the form of proteins (either separately or as part of the above-described fusion protein), (b) optionally, a guide RNA (e.g., as separate linear single-stranded RNA molecules) on which the fusion protein and the guide RNA form a ribonucleic acid protein complex (RNP), and (c) a polynucleotide comprising or consisting of a LAMA2 transgene for insertion (e.g., into the vector) in the form of DNA.

[0308] In one embodiment, the composition comprises (a) a first protein and a second protein, either separately in the form of DNA or as part of the above-mentioned fusion protein (e.g., Cas9 and transposase), (b) optionally, a guide RNA (e.g., as separate linear RNA molecules or as DNA in the vector), and (c) a polynucleotide comprising or consisting of a LAMA2 transgene for insertion in the form of DNA (e.g., in the vector).

[0309] In one embodiment, the composition comprises (a) either a first protein or a second protein in the form of a separate protein or as part of the above-mentioned fusion protein (e.g., Cas9 and integrase), (b) optionally, a guide RNA (e.g., as a separate RNA molecule that forms a complex with the fusion protein), and (c) a polynucleotide comprising or consisting of a LAMA2 transgene for insertion, contained in or bound to a package vector. In certain embodiments, the packaging vector is a lentiviral particle. In some embodiments, the first protein and the second protein, either separately or as part of the above-mentioned fusion protein, are bound to the lentiviral capsid by gag-pol or VPR (viral protein R). In some embodiments, the polynucleotide comprising or consisting of a LAMA2 transgene is in the form of RNA as the payload for integrase.

[0310] In one embodiment, if the first protein is a ZFP, a guide RNA is not required.

[0311] This disclosure is usually, (i) A first protein and a second protein, either separately or as part of the above-mentioned fusion protein, comprising the amino acid sequences of the first guide RNA nickase Cas9, the SpCas9 nickase of sequence number 65, and a modified highly active PiggyBac, or a nucleic acid that encodes the first protein and the second protein, and (ii) First guide RNA nucleic acid A first composition comprising, (iii) A first protein and a second protein, either separately or as part of the above-mentioned fusion protein, comprising the amino acid sequences of the second guide RNA nickase Cas9, the SaCas9 nickase of sequence number 66, and a modified highly active PiggyBac, or a nucleic acid encoding the first protein and the second protein, (iv) Second guide RNA nucleic acid A second composition containing, Furthermore, nucleic acid constructs including LAMA2 transgenes A kit that includes, The kit relates to a first protein and a second protein, each (in each of the first and second compositions), which are capable of heterodimerization, can perform double cuts determined by first and second guide RNAs at adjacent sites in a genomic DNA region, and can optionally insert the nucleic acid between adjacent sites.

[0312] Several additional embodiments are disclosed below. E1: a) Preferably a fusion protein comprising a first protein consisting of a site-directed DNA-binding protein capable of binding to a target nucleic acid sequence contained in intron 1 of albumin; Lama2 or Rosa26 locus; and a second protein consisting of a transposase; or a nucleic acid construct, preferably mRNA encoding the fusion protein; b) a nucleic acid construct comprising a laminin-α2 protein, or a transgene encoding a functional variant or fragment thereof.

[0313] E2: The transgene encodes the laminin-α2 protein of sequence number 74.

[0314] The nucleic acid construct of E3:b) includes a promoter selected from the group consisting of the CMV promoter of SEQ ID NO: 76, the CAG promoter of SEQ ID NO: 77, the EF1-alpha promoter of SEQ ID NO: 78, the SV-40 promoter of SEQ ID NO: 79, and the EalbAAT promoter of SEQ ID NO: 80, or the splice acceptor of SEQ ID NO: 81.

[0315] The nucleic acid construct of E4:b) preferably includes a poly(A) signal sequence selected from the group consisting of SEQ ID NOs. 83 to 85.

[0316] The nucleic acid construct of E5:b) may also include an insulator element preferably selected from the group consisting of SEQ ID NOs: 86-87.

[0317] The nucleic acid construct of E6:b) is adjacent to inverted terminal sequences (ITRs), preferably the 5'-ITR and 3'-ITR of sequence numbers 88 and 89.

[0318] The nucleic acid construct of E7:b) may be contained in a vector selected from the group consisting of plasmid vectors, minicircle vectors, doggybone DNA donor vectors, lentiviral vectors, and retroviral vectors.

[0319] E8: The site-specific DNA-binding protein is an RNA-induced nuclease containing a Cas protein, and the composition further comprises a guide RNA containing a sequence complementary to a target nucleic acid sequence for incorporating the LAMA2 transgene into a specific site in the cell genome, preferably the Cas protein being the Cas9 protein of Streptococcus pyogenes.

[0320] E9: The guide RNA contains a complementary sequence of a sequence selected from the group consisting of sequence numbers 90-97.

[0321] E10: The transposase is a modified high-activity PiggyBac transposase or Sleepy Beuty transposase, preferably a modified high-activity PiggyBac transposase containing one or more amino acid mutations that increase cleavage activity compared to unmodified high-activity PiggyBac, and one or more amino acid mutations that decrease DNA binding activity compared to unmodified high-activity PiggyBac.

[0322] E11: The highly active PiggyBac transposase is a modified highly active PiggyBac transposase containing a mutation in at least one amino acid selected from the group consisting of M194, R245, G325, R372, K375, R376, E377, E380, D450, and S573, preferably a mutation in amino acids D450, R372, and K375, where the position numbers correspond to the amino acid numbers of the unmodified highly active PiggyBac of Sequence ID No. 9.

[0323] E12: The transposase is preferably fused to the N-terminus of the site-specific DNA-binding protein by a linker, preferably a peptide linker containing GGS, XTEN, or FOKI, more preferably XTEN of SEQ ID NO: 53.

[0324] E13: The composition is packaged within nanoparticles.

[0325] E14: An in vitro method for incorporating a LAMA2 transgene into a target nucleic acid sequence within the genome of a cell, comprising introducing the above composition into a cell, wherein the manipulated cell obtained by the method contains the LAMA2 transgene incorporated into its genome.

[0326] E15: A pharmaceutical composition comprising the above-mentioned composition or manipulated cells in combination with one or more pharmaceutically acceptable excipients of any choice.

[0327] E16: Compositions, manipulated cells, or pharmaceutical compositions for use in the treatment of merosine-deficient congenital muscular dystrophy type 1 (MDC1A), particularly in subjects where such treatment is needed.

[0328] The present invention will now be illustrated with reference to the attached drawings, along with the following examples, which are not limiting. [Brief explanation of the drawing]

[0329] [Figure 1] Figure 1: Diagram of different Lama2 payloads generated for therapeutic gene replacement in the Lama2 animal model of MCD1A. [Figure 2] Figure 2: Viral copy number of Hek293 T cells infected with GFP or Lama2-loaded lentivirus. Titer was calculated based on qPCR estimation of copy number. The increase in cargo size due to Lama2 reduces the efficiency of viral generation by two orders of magnitude. [Figure 3]Figure 3: FACS analysis of circulating leukocytes (A) and infiltrating muscle cells (B) isolated from mice 4 weeks post-transplantation. Comparison of empty lentiviral transduction and GFP-loaded virus is shown. [Figure 4] Figure 4: Experimental design and workflow for ex vivo lentiviral-mediated Lama2 gene transfer into bone marrow-derived cells to treat the MCD1A model. [Figure 5] Figure 5: Relative muscle strength of treated and untreated MCD1A models compared to wild-type animals at 6 and 12 weeks after transplantation of bone marrow-derived cells expressing Lama2 (A). Increase in muscle strength after treatment in the same animals (B). In both cases, the results were analyzed using grip strength assays. [Figure 6] Figure 6: Kaplan-Meier survival curves over time for treated and untreated MCD1A models in wild-type animals. [Figure 7] Figure 7: Experimental design and workflow for in vivo virus-free, transposase-mediated Lama2 gene transduction into hepatocytes to treat the MCD1A model. [Figure 8] Figure 8: Transgene copy number (A;C) and transgene expression (B;D) measured by qPCR and RT-qPCR, respectively, four weeks after treatment in hepatocytes. Data for the RFP gene reporter stably incorporated by highly active PiggyBac (A;B) and data for the Lama2 gene stably incorporated by the programmable transposase Cas9 fused to the manipulated PiggyBac (C;D) are shown. [Figure 9] Figure 9: Elisa study on the presence of laminin in circulating serum at 1 and 4 weeks post-treatment of MDC1A models with and without simultaneous transfection of the insertion mechanism for stable expression. [Figure 10]Figure 10: (A) The Rosa26 safe harbor in the mouse genome was targeted using the RFP transposon alone (episome) or with hyPB or FICAT R372A_K375A_D450N mRNA, delivered in vivo with the JetPEI reagent. The relative copy number of the RFP transgene in the liver was measured by semi-quantitative qPCR and normalized to the relative double copy of the Tfrc gene (diploid genome). (B) Liver integration of the minicircle luciferase transposon. The minicircle luciferase transposon, sgRNA targeting the Rosa26 locus, and FiCAT (Cas9-hyPB R372A_K375A_D450N) mRNA were delivered by hydrodynamic injection, and the luciferase signal was monitored. (C) Junction PCR between the transposon 3'ITR and the Rosa26 locus in liver genomic DNA. Mice were hydrodynamically injected with FiCAT R372A_K375A_D450N plasmid DNA or mRNA, gRNA targeting the Rosa26 locus, and a minicircle transposon GFP payload, and sacrificed 5 weeks post-injection. PCR was performed to amplify genome + strand integrations. n=2-3 animals / condition, numbers correspond to different individuals. 66% of mice treated with FiCAT mRNA or pDNA showed targeted insertions. The size of the bands detected by FICAT corresponds to the expected size of the amplified insertions. Larger bands were detected in episomal samples, which are considered background. [Figure 11]Figure 11: (a) C2C12 cells were transduced with either the RFP transposon alone (episome) or in combination with FiCAT R372A_K375A_D450N and gRNA (spacer 271.1) targeting the Lama2 gene. RFP-positive cells were monitored for 2 weeks post-transduction. The mean ± SD of n=2 technical replicates was plotted. Representative image of n=3. (b) Karyoplot showing insertions detected in the c2c12 genome. (c) Junction PCR between the 3' ITR and the Lama2 locus in +-chain (1,3) and --chain (2,4) payload insertions, comparing enriched populations treated with FiCAT (3,4) and episome (1,2) (lower panel). Representative image of n=3. (d) Coverage at the on-target junction (Lama2 site). [Figure 12] Figure 12: Targeted insertion of a 1 / 2 GFP transposon at the AAVS1 site of a reporter cell line compared to insertion of a 300 bp ITR size. ITR sizes of 105 bp, 200 bp, and 300 bp were tested for insertion of a 1 / 2 GFP containing Cas9 fused to either a highly active PiggyBaC(hyPB)R372A_K375A_D450N(Cas9-PBx3) or a hyPB R372A_K375A_D450N(Cas9-PBx3-PBx3) dimer. [Figure 13] Figure 13. Programmable transposases could be manipulated with different Cas variants, including CasX, CjCas9 Cpf1, and SaCas9, and some of these yielded similar results for programmable insertion at the target site, as did SpCas9. Each of the tested Cas variants was targeted to a specific target region in a split GFP reporter cell line containing three independent gRNAs. [Figure 14]Figure 14. Programmable insertion activity of FiCAT R372A_K375A_D450N using four different nuclease proteins. SpCas9 was used as a control for programmable insertion by gRNA-TRAC-1 alone (left). Each nuclease was used with three independent gRNAs (1-3) for targeted insertion into a 1 / 2 GFP reporter cell line. [Figure 15] Figure 15. (a) Editing activity by CasX (left) and Cpf1 (center). (b) Editing activity by SaCas9 (left) and CjCas9 (center). Mean %+ / -SD of indel-containing read information for representative images of two technical repeats of N=3 biological replications. SpCas9 targeting the TRAC-1 site was used as a reference (right). [Figure 16] Figure 16. The programmable transposase can be manipulated as a dimerized polypeptide of two hyPB domains and Cas9 nuclease, resulting in better programmable insertion compared to Cas9-hyPB. A split GFP reporter cell line was used for programmable insertion of the split GFP transposon into the target site. A variant of hyPB R372A_K375A_D450N was used for monomer or dimer fusion to Cas9. Conditions: 1. Negative control using hyPB alone as the insertion mechanism; 2. Positive control of Cas9-hyPB R372A-K375A-D450N in a pcDNA expression vector; 3. Positive control of Cas9-hyPB R372A-K375A-D450N in a lentiviral expression vector; 4. Cas9 nuclease fused to two units of hyPB R372A-K375A-D450N at the C-terminus; 5. Cas9 nuclease fused to two units of hyPB R372A-K375A-D450N, one at the C-terminus and the other at the N-terminus. [Figure 17]Figure 17. After several cycles of selecting cells that had undergone programmable transposition, it became possible to select the best combination of mutants from the library. We identified several mutants that, when fused to Cas9, exhibited superior enrichment and programmable insertion ability compared to Cas9-hyPB R372A_K375A_D450N. [Figure 18] Figure 18. On-target efficiency improved with each selection cycle. Bulk variants selected from each cycle were co-transfected into reporter cell lines with AAVS1-targeting gRNA and 1 / 2 GFP transposons. Plasmid amounts were corrected for PB copy number and normalized for cloning efficiency. [Figure 19] Figure 19. (a) On-target efficiency of the top selected candidates. Six individual candidates were selected based on the highest on-target activity among 96 random clones selected from the last cycle. The individual on-target activities were compared with Cas9-hyPB R372A-K375A-D450N. (b) Logos indicating the dominant PB residues in the top on-target activity variants. [Figure 20] Figure 20. Double-strand breaks by Cas9 and a single gRNA (gRNA-TCR1 or AAVS1-3), or by nickase Cas9 and two gRNAs targeting nearby sites (gRNA-TCR1 and AAVS1-3), and a programmable DNA-binding domain (ZnF) fused with a modified hyPB (mutant R372A-K375A-D405N), result in targeted insertion. Simultaneous localization of the double-strand break and PB at the insertion site is necessary for efficient on-target insertion. This can be achieved by nuclease Cas9 or by double breaks by nickase Cas9. [Figure 21] Figure 21.1 / 2 Programmable insertion activity of the dimer hyPB R372A-K375A-D450N fused with either SpCas9 or SaCas9 for targeted insertion in GFP reporter cell lines. [Figure 22]Figure 22. On-target efficiency improves with increasing number of selected cycles. (a) Bulk variants selected from each cycle were co-transfected into reporter cell lines with AAVS1-targeting gRNA and 1 / 2 GFP transposons. Plasmid amounts were corrected for PB copy number and normalized for cloning efficiency. (B) Lentiviruses expressing bulk variants from each cycle were generated and used to infect reporter cell lines. [Figure 23] Figure 23. Specific target incorporation of Cas9_PB libraries co-transfected with gRNA tcr1 and 1 / 2 GFP MC transposons after 4 and 5 enrichment cycles, compared to single mutant FiCAT (hyPB R372A-K375A-D450N) isolated from bulk variants. [Figure 24] Figure 24.1 / 2 Relative comparison of programmable insertion activity for targeted insertion in GFP reporter cell lines. (a) Comparison of hyPB R372A-K375A-D450N (left) fused with SpCas9 protein and hyPB R372A-K375A-D450N (right) with SpCas9 added separately and fused with MCP protein. (B) Comparison of three hyPB mutants (R372A-K375A-D450N; R202K-R275A-N347S-R372A-D450N-T560A-F594L; and R275A-N347S-R372A-D450N-T560A-F594L) with SpCas9 added separately and fused with MCP protein. [Figure 25] Figure 25.1 / 2 Relative comparison of programmable insertion activity for targeted insertion in GFP reporter cell lines. (a) Comparison of co-expression of hyPB R372A-K375A-D450N and SpCas9 protein (left) and a fusion protein with SpCas9 containing hyPB R372A-K375A-D450N (right). (B) Relative comparison of three hyPB mutants co-expressed with SpCas9 (R372A-K375A-D450N; R202K-R275A-N347S-R372A-D450N-T560A-F594L; and R275A-N347S-R372A-D450N-T560A-F594L). [Figure 26] Figure 26. Relative comparison of programmable insertion activity for targeted insertion in 1 / 2 GFP reporter cell lines when a first fusion protein containing SpCas9 and hyPB R372A-K375A-D450N is co-expressed with a second fusion protein containing MCP protein and hyPB variants (R372A-K375A-D450N; R202K-R275A-N347S-R372A-D450N-T560A-F549L; and R275A-N347S-R372A-D450N-T560A-F594L). [Figure 27] Figure 27. Relative comparison of programmable insertion activity for targeted insertion in a 1 / 2 GFP reporter cell line when a fusion protein containing SpCas and hyPB R372A-K375A-D450N is co-expressed with three hyPB variants: R372A-K375A-D450N; R202K-R275A-N347S-R372A-D450N-T560A-F549L; and R275A-N347S-R372A-D450N-T560A-F594L. [Figure 28] Figure 28. Comparison of programmable insertion activity for targeted insertion in 1 / 2 GFP reporter cell lines between SpCas9 fused with the hyPB R272A-K275A-D450N dimer (left) and SpCas9 fused with the first hyPB R272A-K275A-D450N and the second hyPB mutant (right). [Examples]

[0330] 1. LAMA2 Payload Construction A LAMA2 coding sequence containing a leader sequence for excretion, relating to either the house mouse (Mus musculus) (SEQ ID NO: 94) or Homo sapiens (SEQ ID NO: 75), can be used. The LAMA2 coding sequence may include sequences that recode a portion of the sequence for payload-specific detection and tracking after gene transfer, in order to distinguish it from endogenous genes present in the organism being gene-edited by the inventors.

[0331] The LAMA2 gene is cloned downstream of a constitutive promoter (SEQ ID NOs. 76-79), a tissue-specific promoter (SEQ ID NOs. 80), or a splice acceptor (SEQ ID NOs. 81). The version with the splice acceptor can be incorporated into the first intron of an actively expressed gene to control endogenous expression.

[0332] This coding sequence may also have a synthetic intron (SEQ ID NO: 82) for a minigene version of the payload. The expression cassette may include polyA signals (SEQ ID NOs: 83-85) and insulator elements (SEQ ID NOs: 86-87).

[0333] The payload is, 1. Plasmid vectors containing bacterial replication origins and antibiotic resistance. 2. A minicircle vector containing, in addition to plasmid vector components, 32 SceI restriction enzyme target sites and two AttB and AttP recombination sites adjacent to the Lama2 expression cassette. 3. A doggy-born DNA donor vector that, in addition to plasmid vector components, also includes a TelN target site adjacent to the Lama2 expression cassette for linearization and protection of the cargo ends of the donor DNA. 4. A lentiviral / retroviral manipulation element that can be manipulated to reduce in size to allow for additional space for therapeutic cargo, in addition to a lentiviral or gamma-retroviral payload vector having a terminal repeat sequence adjacent to the Lama2 expression cassette. It can take the form of...

[0334] In all of these cases, the Lama2 expression cassette can be flanked by an inverted terminal sequence (ITR) for transposase recognition (Piggybac and Sleeping Beauty).

[0335] Figure 1 shows different cargoes containing the Lama2 transgene.

[0336] The inventors further investigated whether the size between ITRs in the payload affects the binding ability of the fusion protein and, consequently, its transcription into the genome. For targeted insertion with payload efficiency, three sizes between ITRs were tested: FiCAT (a highly active PiggyBac transposase mutated with Cas9+ residue R372A_K375A_D450N), a FiCAT dimer (a dimer of the highly active PiggyBac transposase mutated with Cas9+ residue R372A_K375A_D450N), and approximately 300 bp, 200 bp, and 105 bp for a minicircle (MC) payload of a 1 / 2 GFP transposon containing gRNA targeting the AAVS1 site of a reporter cell line (Figure 12). The optimal sizes between ITRs were 300 bp and 200 bp, while the 105 bp size resulted in lower targeted insertion efficiency.

[0337] In addition to lentivirus and retrovirus-based delivery, different Lama2 payloads can be delivered to target cells using polyplexes and lipid nanoparticles, or transduced as naked nucleic acids by electroporation or microinjection.

[0338] 2. Ex vivo transduction of Lama2 payload Lentiviral particles containing the mouse Lama2 coding sequence were generated under the control of the CMV promoter using a standard protocol for second-generation lentiviral generation. Considering the large payload size, the particles were enriched, and viral titers were calculated by qPCR estimation of the viral copy number after infection with Hek293T cells (Figure 2).

[0339] Although the generation efficiency was twice as low as that of the reporter gene GFP, this titer is sufficient for ex vivo transduction of cells and can be used for cell therapy in the Lama2 mouse model. Two different approaches were taken. Isolated muscle stem cells were transduced and transplanted into the muscle of the recipient model. Alternatively, hematopoietic progenitor cells derived from bone marrow cells could be transduced with a lentivirus containing a Lama2 expression cassette, and then these progenitor cells could be transplanted into a conditioned model by intravenous injection.

[0340] As a proof-of-concept for ex vivo treatment, hematopoietic progenitor cells derived from bone marrow were isolated, transduced with a GFP-expressing lentivirus, and then transplanted into conditioned mice. Engraftment was then examined at 4 weeks post-treatment by measuring GFP expression in circulating blood leukocytes, and positive results were found (Figure 3A). In addition, the infiltration of circulating cells into inflamed muscle was also examined, and positive results were found (Figure 3B).

[0341] Next, considering the clear infiltration and payload expression of ex vivo-transduced hematopoietic progenitor cells, this experiment was repeated using Lama2-expressing lentiviral particles (Figure 4). Muscle strength was measured by a grip strength assay in mice that had recovered muscle strength 12 weeks post-treatment (Figures 5A and 5B). In addition, survival of the treated Lama2 animal model was observed (Figure 6), demonstrating the therapeutic value of ex vivo cell transduction and transplantation for treating MDC1A patients.

[0342] In a separate series of experiments, the inventors performed LAMA2-targeted insertion of an RFP reporter into the C2C12 mouse myoblast cell line, achieving an efficiency of approximately 20% (Figure 11). On-target and off-target efficiencies were measured using junction PCR and (STAT)-PCR. This improvement in efficiency was attributed to the use of a fusion protein called FiCAT, which contains Cas9 and a highly active PiggyBac transposase mutated at residue R372A_K375A_D450N.

[0343] 3. In vivo transduction of Lama2 payload Considering the liver's ability to function as a bioreactor for protein synthesis in vivo, we explored the possibility of transduction of a Lama2 expression vector into in vivo hepatocytes of an animal model using a transposase-based, virus-free gene delivery approach.

[0344] An enhanced, highly active PiggyBac transposase, formulated in vivo using JetPei (PolyPlus), or an engineered highly active PiggyBac fusion transposase containing a programmable nuclease protein (SpCas9) as either a plasmid vector or mRNA molecule, and a transposon Lama2 plasmid vector (Figure 7) were intravenously injected into an animal model. Tissue was collected 3–4 weeks later for liver payload copy number (RFP data as proof of concept) (Figure 8A, C), expression analysis in the liver (RFP, Figure 8C; Lama2, Figure 8D), and circulating blood for Lama2 (Figure 9). 100 μL of blood was collected 1 week after injection, and whole blood was collected at the endpoint.

[0345] The transgene copy number in hepatocytes four weeks after treatment showed positive results, and compared to the RFP reporter gene, similar transduction efficiency was confirmed with payloads of different sizes (Figure 8A, B). The case where the transposase was not delivered simultaneously was referred to as episomal delivery. Transgene expression related to the RFP reporter and Lama2 gene was also confirmed in hepatocytes (Figure 8B, D), and Lama2 expression was detected in blood at 1 and 4 weeks after delivery (Figure 9).

[0346] 4. Tools improved for in vivo gene delivery With the aim of improving programmable transposases for in vivo gene delivery, we used both plasmid DNA (pDNA) and mRNA in FiCAT (Cas9-hyPB R372A_K375A_D450N) and delivered them to mouse liver targeting the Rosa26 genome-safe harbor (for proof of concept, a transposon encoding RFP or luciferase) in either plasmid or MC form. A higher transgene copy number was observed compared to the endogenous gene TFRC (Figure 10A), and transgene expression was maintained over time (Figure 10B). Newly formed on-target insertions were measured using PCR of the junction between the 3'ITR and the genomic locus (Figure 10C).

[0347] 4.1. Results of Cas variants fused with hyPB To further characterize the ability of hyPB to perform programmable transpositions, the SpCas9 mudile of the programmable transposase under test was replaced with other Cas proteins with nuclease activity from different organisms (i.e., SaCas9 of SEQ ID NO: 72, Cpf1 of SEQ ID NO: 74, CasX of SEQ ID NO: 75, and CjCas9 of SEQ ID NO: 29). For Hershey cell line transfection, specific gRNAs targeting the upstream region of a split GFP reporter were designed and cloned. Targeted transpositions were measured by GFP expression (Figure 13).

[0348] These results were confirmed in a separate experimental line: good programmable insertion activity was obtained for CjCas9 and LbCpf1, but CasX did not achieve programmable insertion in our assay. In particular, SaCas9 had the highest level of programmable insertion among the Cas proteins tested, at a similar level to SpCas9 fused with modified hyPB (Figure 14). Indels were determined for the different Cas proteins used, and three different gRNAs were designed for each protein by Ilumina NGS and are shown for normalization purposes (Figure 15).

[0349] These positive results demonstrate that hyPB, manipulated for programmable rearrangement, is useful for sequence-specific nuclease modules.

[0350] 4.2. Results of adding Cas9 fused with a PB mutant dimer Considering the properties of PB, which acts as a dimer during rearrangement, the inventors attempted to create fusion proteins of Cas9 and the hyPB R372A-K375A-D450N mutant. The inventors compared the on-target activity of these fusions with that of the Cas9-PB mutant alone. The inventors observed better performance of the Cas9-PB-PB fusion, but the PB-Cas9-PB fusion did not outperform the Cas9-PB monomer fusion (Figure 16). For dimer fusion with Cas9, the inventors used a re-encoded version of the hyPB R372A-K375A-D450N mutant to facilitate cloning and expression.

[0351] Interestingly, when Cas9 fused with the dimer hyPB R372A-K375A-D450N is SaCas9 rather than SpCas9, the activity is further increased (Figure 21). The performance improvement of SaCas9 with the dimer hyPB compared to SpCas9 with the dimer hyPB is consistent with the results obtained with monomer hyPB (Figures 13 and 14).

[0352] 4.3. Results of ZNF-PB mutant rescue The inventors desired to further explore the role of double-strand break (DSB) activity induced near the (4-nucleotide) target sites of two gRNAs that promote single-strand breaks by the SpCas9 nickase variant (D10A) in order to promote target integration while reducing target activity by non-inducible DSBs at off-target sites. The inventors used a zinc finger-PB fusion for the targeted localization of the transposon by fusing the D450N variant with the R372A_K375A_D450N variant, and complemented this with two on-site single-strand breaks by an independent nickase Cas9, or a single DSB by a Cas9 nuclease.

[0353] When Znf-PB fusions were combined with the introduction of a double-strand break (DSB) near the Znf binding site and a single or dual gRNA-inducible Cas9 (either a nuclease or nickas), they showed no rescued targeted insertion activity, or only very low activity (Figure 20).

[0354] 4.4. Results of Cas9-hyPB mutant variants To further explore mutant combinations that could perform programmable transposition with better efficiency, we performed several cycles of cell selection to reconstitute GFP by programmable insertion of a split GFP reporter system. Interestingly, several combinations were observed that outperformed Cas9-hyPB R372A-K375A-D450N (Figure 17). Of particular note is the hyPB variant fused to Cas9 mutated with AA:A351-A372-A375-A388-N450-A465-A573-V589-G592-L594 (also identified as SEQ ID NO: 2) in hyPB, which is several times more abundant in the positive cell population compared to R372A-K375A-D450N (SEQ ID NO: 1); A245-A275-A277-A372-A465-V589 (SEQ ID NO: 3) and A275-A325-A372-A560 (SEQ ID NO: 4) are also abundant, albeit to a lesser degree.

[0355] In a separate experimental line, Twist Bioscience generated a PiggyBacDNA library, cloned it into a lentiviral vector by fusion with Cas9, and transformed it into stb4 competent cells to ensure a 100-fold variant complexity. The plasmid was purified with maxiprep and co-transfected into Hek293T cells with a lentiviral packaging plasmid. The lentivirus was used to infect a 1 / 2GFP reporter cell line. The infected cells were transfected with gRNAs targeting the 1 / 2GFP transposon and the AAVS1 sequence. GFP-positive cells were selected by flow cytometry and their genomic DNA was extracted. PB was amplified from the extracted gDNA and re-cloned into a lentiviral vector to restart a new cycle. The most high-performing programmable transposase variant was selected and individually transfected with AAVS1 gRNA and MC1 / 2GFP.

[0356] First, 96 variants were randomly selected, and the highest-performing variants were screened separately (Figure 18). Summarizing the best PB amino acid variants for high on-target insertion, the importance of mutations D450N, R372A, and K375A is confirmed, but other important residues contributing to improved target efficiency are highlighted (Figure 19B). The six PB variants with the highest on-target efficiency were selected (Figure 19A). Individual on-target activity was determined using FiCAT(Cas9-hyPB). Compared to R372A-K375A-D450N), the following variants were significantly improved (two-sided t-test): N347A-D450N; N347S-D450N-T560A-S573A-F594L; R202K-R275A-N347S-R372A-D450N-T560A-F594L; R275A-N347S-K375A-D450N-S592G; R275A-N347S-R372A-D450N-T560A-F594L; and R275A-R277A-N347S-R372A-D450N-T560A-S564P-F594L.

[0357] This experiment was repeated and confirmed (Figure 22A). The inventors also generated lentiviruses expressing the bulk variant of each cycle and infection reporter cell lines whose titers were corrected with the PB variant CN, demonstrating that on-target efficiency similarly improved with each cycle (Figure 22B). After enriching the cas9_PB library for 4 and 5 cycles, single mutants were isolated from the bulk variants. The mutants were separately tested by transfecting the on-target reporter cell lines with the FiCAT mutant, gRNA tcr1, and 1 / 2GFP MC transposon. The best FiCAT mutant is shown compared to FiCAT R372A_K375A_D450N (Figure 23). Individual on-target activities are shown for FiCAT(Cas9-hyPB). Compared to R372A_K375A_D450N), the following variants exist: R202K-R275A-N347S-R372A-D450N-T560A-F594L; R245A-N347S-R372A-D450N-T560A-S564P-S573A-S592G; R275A-N34 7S-R372A-D450N-T560A-F594L;N347A-D450N;R277A-G325A-N347A-R375A-D45 0N-T560A-S564p-S573A-S592G-F594L;N347S-D450N-T560A-S573A-F594L;V34 M-R275A-G325A-N347S-S351A-R372A-K375A-D450N-T560A-S564P;G325A-N347 S-K375A-D450N-S573A-M589V-S592G;S230N-R277A-N347S-K375A-D450N;T43I Significant improvements have been made to the following models: -R372A-K375A-A411T-D450N;G325A-N347S-S351A-K375A-D450N-S573A-M589V-S592G;Y177H-R275A-G325A-K375A-D450N-T560A-S564P-S592G.

[0358] The superiority of the mutants R202K-R275A-N347S-R372A-D450N-T560A-F594L, R275A-R277A-N347S-R372A-D450N-T560A-S564P-F594L, and R275A-N347S-R372A-D450N-T560A-F594L compared to mutant R372A-K375A-D450N was further demonstrated in triple fusion proteins containing SpCas9 and two hyPBs (Figure 28).

[0359] 4.5. Results of non-covalent bonding between Cas9 and hyPB In addition to covalent bonding of Cas9 and hyPB R372A-R375A-D450N via a linker, the inventors used the MS2-MCP system to link Cas9 to a fusion protein consisting of the MCP protein and hyPB R372A-R375A-D450N via a modified gRNA containing a tetraloop of the MS2 sequence that binds to the MCP protein.

[0360] The combination of the MCP-hyPB R372A-R375A-D450N fusion protein and Cas9 showed improved programmable insertion activity compared to the Cas9-hyPB R372A-R375A-D450N fusion protein (Figure 24A). In addition, MCP proteins were fused with other hyPB variants and combined with SpCas9 to perform programmable rearrangements. Both variants used (R202K-R275A-N347S-R372A-D450N-T560A-F594L and R275A-N347S-R372A-D450N-T560A-F594L) outperformed R372A-R375A-D450N (Figure 24B).

[0361] 4.6. Results of Cas9 and hyPB decoupling for programmable dislocations The inventors also tested the performance of SpCas9 with hyPB R372A-R375A-D450N without using either a linker or the MS2-MCP system. The inventors co-expressed SpCas9 and hyPB R372A-R375A-D450N in the same cells and recorded increased programmable insertion activity compared to the Cas9-hyPB R372A-R375A-D450N fusion protein (Figure 25A). To achieve the activity of programmable translocation, the number of tests of hyPB mutant variants that do not fuse with Cas9 but are co-expressed and act together was expanded (Figure 25B).

[0362] 4.7. Results of co-expression of Cas9-hyPB and MCP-hyPB fusion proteins To obtain a dimeric version of the fusion with one of the non-covalently bound monomers, the hyPB R372A-R375A-D450N mutant fused with the MCP protein and the hyPB mutant fused with SpCas9 were co-transfected simultaneously. Some hyPB mutants fused with SpCas9 were compared for specific target integration (Figure 26).

[0363] 4.8. Results of co-expression of Cas9-hyPB variants and hyPB variants In a similar manner, the inventors independently co-transfected the SpCas9 hyPB R372A-R375A-D450N fusion protein and the hyPB mutant to obtain a dimeric version of the fusion with one of the unbound monomers (Figure 27).

[0364] Materials and methods Lentivirus generation, concentration, and titer To generate the virus, cells were co-transfected with pSICO(GFP) or pLV-Lama2 (Lama2) and pmd2.g (VSVG = envelope) and pax2 (including packaging proteins containing IN), and sometimes co-transfected with a plasmid containing only wt-integrase to rescue the non-infectious integrase. First, 6×10 5100 HEK392T cells (passage 8) were seeded into the wells of a 6-well tissue plate and incubated overnight. Five hours before the start of virus production, the cell medium in each well was replaced with 1.7 mL of medium containing 1:1000 CD (chloroquine diphosphate; preservation solution = 25 mM). Plasmids were infected with a molar ratio of 1.6:1.32:0.72:3.32 (pSICO:PAX2:VSVG:wtIN-rescue). PEI (polyethyleneimine; preservation solution = 1 mg / mL) was used as the transfection reagent, with 3 μL of PEI used for every 1 μg of total DNA used for transfection. DNA was diluted in 83 μL of Opti-MEM (Thermo Scientific; #31985062), and PEI was diluted in another 83 μL. After mixing both solutions, they were incubated at room temperature for 15-20 minutes. Each transfection mix was added dropwise to cells in CD medium. Cells were incubated overnight. The culture medium was replaced by the following day, and 2.5 mL of fresh medium was added. The next day, the cell supernatant was centrifuged at 1000 rpm for 5 minutes and passed through a 45 μm filter. It was ultracentrifuged at 19,500 rpm at 4°C for 90 minutes and resuspended overnight in a 1:100 ratio to the original volume of medium. The supernatant was stored at -80°C.

[0365] To determine viral titer, HEK293T cells were infected with the generated virus, and the number of GFP-positive cells was counted as GFP was encoded in the viral packaging sequence. Therefore, 75,000 HEK393T cells were seeded per well in a 6-well plate. Cells were infected with a mixture of 1 mL of medium containing 1:1000 polybrene and 500 μL of previously generated viral supernatant (1:3). The medium was changed by the next day. The following day, the medium was aspirated, and cells were detached using 200 μL of trypsin. 800 μL of normal medium was added to stop the reaction, and the cells were analyzed by cytometry. In the case of non-fluorescent Lama2 payloads, gDNA was extracted from the cells, and qPCR was performed to determine the copy number of the payload relative to the genome.

[0366] In vivo Lama2 delivery The animal experiment procedures were approved by the Animal Ethics Committee of the Barcelona Biomedical Research Park. C57BL / 6J or Lama2 models (dj2y / dj2y), 8-10 weeks old, were used in this study. Animals were purchased from Jackson Laboratories, and both males and females were used without distinction.

[0367] Donor mice were sacrificed at 8 weeks of age, and bone marrow cells were collected from the hind limb bones. Lineage-negative depletion was performed using the Mouse Lineage Cell Depletion Kit (Miltenyi Biotec) according to the manufacturer's instructions. The cells were transduced overnight with lentivirus in stem cell culture medium containing appropriate stimulants (StemCell Technologies). These cells were then collected and transplanted into recipient mice by post-orbital injection (after radiation pretreatment).

[0368] Blood and tibialis muscle samples were collected from mice treated four weeks post-transplant. Blood samples were processed using lysis buffer (Thermo Fisher Scientific) according to the manufacturer's instructions before FACS analysis. Tibialis muscle samples were finely chopped and further treated with liberase / dispase digestion to isolate infiltrating cells before FACS analysis.

[0369] Grip strength assays were performed at 6 and 12 weeks post-treatment using the Bioseb device in accordance with the manufacturer's instructions.

[0370] hyPB mRNA was prepared using the RiboMAX Large-Scale RNA Generation System-T7 (Promega) according to the manufacturer's instructions. Rosa26 gRNA(25) was purchased from IDT. hyPB mRNA, Rosa26-targeting gRNA, and PB512-B or Lama2 transposon were injected posteriorly into the orbit in a ratio of 1:2.5:2.5. A total of 55 μg of nucleic acid was complexed with in vivo-JetPEI (Polyplus transfection) at an NP ratio of 7. Animals were euthanized 10 days post-injection, and their livers were isolated and homogenized. Genomic DNA and RNA were extracted from the liver samples. Transposon-relative copy number and relative expression of the transgene relative to the Tfrc endogenous gene were obtained by qPCR, RT-qPCR, or Elisa assay, respectively.

[0371] Luciferase expression was imaged at different time points after administration of FiCAT-gRNA-transposon or transposon control using the IVIS spectral imaging system (Caliper Life Sciences). Images were taken 5 minutes after intraperitoneal injection of D-luciferin potassium salt (Gold Biotechnology) according to the manufacturer's instructions.

[0372] Collection of gDNA and RNA from the liver Genomic DNA extraction was performed according to the DNeasy® blood & tissue kit protocol (QIAGEN®). Liver tissue was homogenized with PBS (phosphate-buffered saline). 20 μL of proteinase K (included in the kit) was added with 200 μL of buffer AL. After vortexing, the sample was incubated at 56°C for 10 minutes. 200 μL of ethanol (96-100%) was added, and after brief vortexing, the mixture was transferred to a Dneasy mini-spin column in a 3 mL collection tube and centrifuged at 8000 rpm for 1 minute. The spin column was moved to a new 2 mL collection tube, and 500 μL of buffer AW1 was added. The tube was centrifuged at 8000 rpm for 1 minute. This washing step was repeated with buffer AW2 (centrifugation for 3 minutes). Next, the spun sample was transferred to a new 1.5 mL microcentrifuge tube, 200 μL of buffer AE was added to the center of the spin column membrane, and the tube was allowed to stand for 1 minute to elute the DNA. Subsequently, the sample was centrifuged at 8000 rpm for 1 minute. The concentration was measured using NanoDrop One® (ThermoFisher Scientific).

[0373] qPCR and RT-qPCR Genomic DNA samples were analyzed by qPCR at 25 ng / μL and 10 ng / μL dilutions. Each 5 μL dilution was mixed with 4.4 μL of PowerUP SYBR Green Master Mix (Fisher Scientific) and 0.3 μL of forward and reverse oligonucleotides. Oligonucleotides targeting the endogenous genes Lama2, RFP, and TfrC were used (SEQ ID NOs: 100-105).

[0374] Reverse transcription from mRNA to cDNA was performed using a high-capacity RNA-to-cDNA kit (Thermo Fisher Scientific). 1 μg of mRNA sample was mixed with 10 μL of buffer and 1 μL of enzyme mix in a 20 μL reaction mixture. The reaction mixture was incubated at 37°C for 2 hours and inactivated at 80°C for 10 minutes. The RT product was diluted 10-fold, and 5 μL of the sample was mixed with 4.4 μL of PowerUP SYBR Green Master Mix (Fisher Scientific) and 0.3 μL of oligonucleotides fw and rv for qPCR. Oligonucleotides targeting the endogenous genes Lama2, RFP, and GAPDH were used (SEQ ID NOs: 102-107).

[0375] Enzyme-linked immunosorbent assay (ELISA) Whole blood was centrifuged at 1200 × g for 10 minutes at 4°C using a benchtop centrifuge. Plasma was separated and stored at 80°C until ELISA assay. A human laminin-subunit alpha-2 (LAMA-2) ELISA kit (Cusabio) was used. 20 μL of plasma was diluted with 80 μL of sample buffer. The diluted sample was placed in a plate and incubated for 2 hours. The wells were aspirated but not washed, and 100 μL of biotin antibody was added. The plate was incubated for 1 hour and washed three times with wash buffer. 100 μL of HRP-avidin was added to each well and incubated at 37°C for 1 hour. Five careful washing steps were performed. 90 μL of TMB substrate was added, and the plate was incubated at 37°C for 20 minutes. 50 μL of stop solution was added, the plate was lightly tapped, and the optical density was measured at 450 nm within 5 minutes using a plate reader.

[0376] Fluorescence-activated cytometry (FACS) After isolating circulating leukocytes and infiltrating cells from muscle tissue, emGFP expression was measured (BD LSR Fortessa; BD Biosciences. Blue 488nm laser with 530 / 30 filter and yellow-green 561nm laser with 610 / 20 filter).

Claims

1. a) a first protein comprising or consisting of a site-specific DNA binding protein capable of binding to and cleaving a target nucleic acid sequence; or a nucleic acid construct encoding said first protein; b) a second protein comprising or consisting of a transposase; or a nucleic acid construct encoding said second protein; and c) a nucleic acid construct comprising a transgene encoding a laminin-α2 protein, or a functional variant or fragment thereof; A composition comprising:

2. The composition described in claim 1, wherein the introduced gene encodes a laminin-α2 protein having the sequence of sequence number 74.

3. The composition of claim 1 , wherein the first protein and the second protein of a) and b) are fused.

4. The composition described in claim 1, wherein the first protein and the second protein of a) and b) are fused via a linker.

5. The composition of claim 1, wherein the nucleic acid construct of c) comprises a promoter selected from the group consisting of a CMV promoter of SEQ ID NO: 76, a CAG promoter of SEQ ID NO: 77, an EF1-alpha promoter of SEQ ID NO: 78, an SV-40 promoter of SEQ ID NO: 79, and an EalbAAT promoter of SEQ ID NO:

80.

6. The composition of claim 1, wherein the nucleic acid construct of c) comprises a splice acceptor of SEQ ID NO:

81.

7. The composition of claim 1, wherein the nucleic acid construct of c) comprises a poly(A) signal sequence and / or an insulator element.

8. The composition described in claim 7, wherein the poly(A) signal sequence is selected from the group consisting of SEQ ID NOs: 83 to 85 and / or the insulator element is selected from the group consisting of SEQ ID NOs: 86 to 87.

9. The composition of claim 1, wherein the nucleic acid construct of c) is flanked by inverted terminal repeats (ITRs).

10. The composition of claim 9, wherein the ITRs are the 5'-ITR and 3'-ITR of SEQ ID NOs: 88 and 89.

11. The composition of claim 1, wherein the nucleic acid construct of c) is contained in a vector selected from the group consisting of a plasmid vector, a minicircle vector, a doggybone DNA donor vector, a lentiviral vector and a retroviral vector.

12. 2. The composition of claim 1, wherein the site-specific DNA binding protein is an RNA-guided nuclease comprising a Cas protein, and the composition further comprises a guide RNA comprising a sequence complementary to a target nucleic acid sequence for integrating the LAMA2 transgene into a specific site in the genome of a cell.

13. The composition described in claim 12, wherein the Cas protein is a S. pyogenes Cas9 protein.

14. The composition of claim 12, wherein the guide RNA comprises any one of SEQ ID NOs: 90-97.

15. 2. The composition of claim 1, wherein the transposase is a modified hyperactive Piggybac transposase or a Sleepy Beauty transposase.

16. The composition described in any one of claims 1 to 9, wherein the transposase is a modified hyperactivity PiggyBac transposase comprising one or more amino acid mutations that increase excision activity compared to unmodified hyperactivity Piggybac, and one or more amino acid mutations that decrease DNA binding activity compared to unmodified hyperactivity Piggybac.

17. 17. The composition of claim 16, wherein the hyperactive PiggyBac transposase is a modified hyperactive PiggyBac transposase comprising at least one mutation of an amino acid selected from the group consisting of V34, T43, Y177, M194, R202, S230, R245, R275, R277, G325, S351, N347, R372, K375, R376, E377, E380, A411, D450, T560, S564, S573, M589, S592, and F594, wherein the position numbers correspond to the amino acid numbers of the unmodified hyperactive Piggybac of SEQ ID NO:

9.

18. The composition of claim 16, wherein the hyperactive PiggyBac transposase is a modified hyperactive PiggyBac transposase comprising at least one mutation of an amino acid selected from the group consisting of Y177, R202, S230, R245, R275, R277, G325, N347, S351, E377, D450, R372, and K375, E377, T560, S564, S573, M589, S592, F594, and the position numbers correspond to the amino acid numbers of the unmodified hyperactive Piggybac of SEQ ID NO:

9.

19. 17. The composition of claim 16, wherein the hyperactive PiggyBac transposase is a modified hyperactive PiggyBac transposase comprising at least one mutation of an amino acid selected from the group consisting of M194, R245, R275, R277, G325, R372, K375, R376, E377, E380, D450 and S573, and the position numbers correspond to the amino acid numbers of the unmodified hyperactive Piggybac of SEQ ID NO:

9.

20. The composition described in claim 16, wherein the hyper-activity PiggyBac transposase is a modified hyper-activity PiggyBac transposase comprising at least one mutation of an amino acid selected from the group consisting of D450, R372 and R375, and the position numbers correspond to the amino acid numbers of the unmodified hyper-activity Piggybac of SEQ ID NO:

9.

21. The composition of claim 1 , wherein the transposase is N-terminally fused to the site-specific DNA binding protein by a linker.

22. The composition of claim 1, wherein the transposase is fused at its N-terminus to the site-specific DNA binding protein by a peptidic linker comprising GGS, XTEN, or FOKI.

23. The composition of claim 1 , wherein the composition is packaged within a nanoparticle.

24. 24. An in vitro method for integrating a LAMA2 transgene into a target nucleic acid sequence in the genome of a cell, comprising introducing into the cell a composition according to any one of claims 1 to 23.

25. 25. An engineered cell obtainable by the method of claim 24, wherein the engineered cell comprises a nucleic acid integrated into its genome, the nucleic acid comprising a transgene encoding a laminin-α2 protein flanked by engineered sequences for integrase and / or transposase mediated gene insertion.

26. 26. The engineered cell of claim 25, wherein the engineered sequences flanking the transgene comprise or consist of SEQ ID NOs: 88 and 89.

27. ​​The engineered cell described in claim 25, wherein the size between the ITRs is at least 300 bp.

28. The engineered cell of claim 25, wherein the transgene encodes a full-length laminin-α2 protein.

29. 29. A composition according to any one of claims 1 to 23, or an engineered cell according to any one of claims 25 to 28, for use in therapy in a subject in need thereof, in particular for use in the treatment of merosin-deficient congenital muscular dystrophy type 1A (MDC1A).

30. Use of a composition according to any one of claims 1 to 23, or an engineered cell according to any one of claims 25 to 28, for the preparation of a medicament for treating a disease.

31. A composition according to any one of claims 1 to 23, or an engineered cell according to any one of claims 25 to 28, or: a) a first protein comprising or consisting of a site-specific DNA binding protein capable of binding to and cleaving a target nucleic acid sequence, or a nucleic acid construct encoding said first protein; b) a second protein comprising or consisting of a transposase, or a nucleic acid construct encoding said second protein; and c) a nucleic acid construct comprising a transgene encoding a laminin-α2 protein, preferably the laminin-α2 protein of SEQ ID NO: 74, or a functional variant or fragment thereof. Including the kit.