Non-viral vector-mediated large-fragment DNA site-directed knock-in system

Abstract

Provided are a method for introducing a target nucleic acid sequence into a primary cell, and a nucleic acid-lipid nanoparticle composition encapsulating a linear DNA. The target nucleic acid sequence is inserted into the genome of the primary cell and / or expressed in the primary cell. The method comprises contacting the primary cell with the nucleic acid-lipid nanoparticle composition encapsulating the linear DNA.

Description

A non-viral vector-mediated large-fragment DNA knock-in system Technical Field

[0001] This application relates to the field of engineered cell modification, and more particularly to a method for introducing DNA molecules into cells, especially the cell nucleus, via liposomes. Background Technology

[0002] Large DNA knock-in has always been a key focus and challenge in gene and cell therapy. Due to the large size and strong negative charge of template DNA molecules, efficient entry into the cell nucleus is difficult, resulting in low knock-in efficiency. Therefore, current methods for large DNA knock-in often employ electroporation or viral delivery. Electroporation uses high voltage to permeate the cell membrane and nuclear membrane, delivering template DNA into the cell nucleus for large DNA knock-in. However, electroporation inevitably causes severe cell damage and death and cannot be widely used in vivo. AAV vectors cannot efficiently integrate the target gene into the genome for stable expression, leading to a gradual decline in efficacy over time. Furthermore, AAV-packaged exogenous DNA has a limited length (typically <4.5kb) and exhibits strong immunogenicity and cytotoxicity, preventing multiple dosing.

[0003] Lipid nanoparticles (LNPs) serve as delivery carriers, efficiently delivering gene-editing tools into cells and mediating the precise insertion of large DNA fragments. Lipid nanoparticles (LNPs) are typically composed of four lipid components: ionizable lipids, cholesterol, auxiliary lipids, and polyethylene glycol (PEG) lipids. These lipid delivery carriers protect nucleic acid payloads from degradation and introduce them into the cytoplasm, thus finding wide application in the delivery of various types of nucleic acid drugs. However, the reported nuclear entry efficiency of large DNA fragments delivered by LNPs is low. Especially for primary human cells, such as primary T cells, the reported LNP delivery efficiency is typically below 5%, failing to meet clinical therapeutic requirements.

[0004] Plasmid DNA can be mass-produced through E. coli fermentation, making it one of the most inexpensive and readily available DNA templates. However, due to the requirements of the fermentation process, circular plasmids contain plasmid replication elements and resistance genes. These exogenous gene sequences can affect the insertion efficiency of the target gene and are prone to immunogenicity. Furthermore, in addition to the conventional circular structure, plasmid DNA easily forms higher-order structures such as supercoiled folds, making batch-to-batch uniformity difficult to control. Besides circular plasmid DNA, there are reported technologies for synthesizing double-ended DNA (with blocked ends) and single-stranded DNA via enzymatic methods, but these are still limited by high production costs and difficulties in scaling up the process, preventing their industrialization.

[0005] Invention Overview

[0006] This application provides a linear DNA template structure suitable as a vector for target gene sequences, which can significantly improve the delivery efficiency and site-specific integration efficiency of the target gene sequence into the genome. Compared with other commonly used DNA template structures, this DNA structure has superior site-specific integration efficiency of the target gene. Furthermore, by optimizing the DNA template sequence, such as by adding nuclear insertion sequences, homologous arms, or linking nuclear localization signal peptides, the efficiency of site-specific integration of the target gene into the genome can be further improved.

[0007] This application also provides a composition for site-directed knock-in of a target gene into the genome of primary cells. The composition comprises the linear DNA structure and gene-editing molecule of this application. The linear DNA structure and gene-editing molecule can be delivered using an LNP. By adjusting the DNA structure, the gene-editing molecule, and the contents of the LNP, this composition achieves a higher efficiency in site-directed integration of the target gene into the genome than other existing similar technologies. It can be used for the construction of engineered cells requiring exogenous gene knock-in expression, such as CAR-T cells.

[0008] Furthermore, this application also provides lipid nanoparticles (LNPs) suitable for DNA delivery, particularly for nuclear DNA delivery. Using this delivery composition can significantly improve the efficiency of nuclear and genome integration of target DNA. For example, when loaded with uncapped linear DNA, the site-directed integration rate of the target gene on the primary cell genome can exceed 13%; when loaded with capped linear DNA, the site-directed integration rate on the primary cell genome can exceed 26%. Moreover, the LNP of this application is particularly advantageous for the site-directed integration of large target gene sequence fragments. Under the same conditions, compared with commonly used C12-200 LNPs and other LNP methods, the LNP provided in this application is more suitable for the delivery of linear DNA, especially capped linear DNA.

[0009] Specifically, the first aspect of this application provides an engineered linear DNA molecule comprising a double-stranded portion formed by hybridization, wherein the 5' end of one or both strands of the double-stranded portion of the linear DNA molecule is connected to the 3' end of its complementary strand by a capping linker. In some embodiments, the capping linker is located on one side of the linear DNA molecule. In some embodiments, the capping linker is located on both sides of the linear DNA molecule. In some embodiments, both ends of the linear DNA molecule contain capping linkers, and the capping linkers at both ends are identical. In some embodiments, both ends of the linear DNA molecule contain capping linkers, and the capping linkers at both ends are different. In some embodiments, both ends of the linear DNA molecule contain capping linkers, and the nucleic acid sequence of the capping linker on one side can be obtained by reversing the nucleotide sequence on the other side. In some embodiments, examples of the linear DNA molecule include, but are not limited to, the three structures shown in FIG18.

[0010] In some embodiments, the capping linker does not contain nucleic acid structures or nucleotides, but is composed of non-nucleic acid structures. In some embodiments, the capping linker is composed of both nucleic acid and non-nucleic acid structures. In some embodiments, the capping linker is composed of one or more nucleotides. In some embodiments, the capping linker is formed by linking one or more nucleotides with other chemical groups. In some embodiments, the capping linker is composed of 1-200 nucleotides, for example, 1-100, 2-60, 3-40, 4-30, or 5-20 nucleotides. In some embodiments, the capping linker includes a double-stranded segment. In some embodiments, the length of the end-cap connector does not exceed 30 nt, for example, not exceeding 29 nt, 28 nt, 27 nt, 26 nt, 25 nt, 24 nt, 23 nt, 22 nt, 21 nt, 20 nt, 19 nt, 18 nt, 17 nt, 16 nt, 15 nt, 14 nt, 13 nt, 12 nt, 11 nt, 10 nt, 9 nt, 8 nt, 7 nt, 6 nt, or 5 nt. In some embodiments, the length of the end-cap connector does not exceed 20 nt, for example, not exceeding 19 nt, 18 nt, 17 nt, 16 nt, 15 nt, 14 nt, 13 nt, 12 nt, 11 nt, 10 nt, 9 nt, 8 nt, 7 nt, 6 nt, or 5 nt. In some embodiments, the length of the end-cap connector does not exceed 10 nt, for example, not exceeding 9 nt, 8 nt, 7 nt, 6 nt, or 5 nt. In some embodiments, the length of the end-cap connector is 4 nt, 3 nt, 2 nt, or 1 nt. In some embodiments, the end-cap connector is composed of T and / or A. In some embodiments, the length of the end-cap connector does not exceed 30 nt, and the end-cap connector is composed of T and / or A. In some embodiments, the length of the end-cap connector does not exceed 20 nt, and the end-cap connector is composed of T and / or A. In some embodiments, the length of the end-cap connector does not exceed 10 nt, and the end-cap connector is composed of T and / or A. In some embodiments, the length of the end-cap connector does not exceed 5 nt, and the end-cap connector is composed of T and / or A. In some embodiments, the end-cap connector is composed of G and / or C. In some embodiments, the length of the end-cap connector does not exceed 30 nt, and the end-cap connector is composed of G and / or C. In some embodiments, the length of the end-cap connector does not exceed 20 nt, and the end-cap connector is composed of G and / or C. In some embodiments, the length of the end-cap connector does not exceed 10 nt, and the end-cap connector is composed of G and / or C. In some implementations, the length of the end cap connector does not exceed 5 nt, and the end cap connector is composed of G and / or C.

[0011] In some embodiments, the capping linker is double-stranded, meaning it has one strand connecting the 5' end of one strand of the double-stranded region of the linear DNA (referred to as the "linking strand") to the 3' end of its complementary strand, and another strand complementary to the long strand (referred to as the "attachment strand"). In some embodiments, each nucleotide in the attachment strand hybridizes complementaryly to the linking strand. In some embodiments, only a portion of the nucleotides in the attachment strand hybridizes complementaryly to the linking strand. In some embodiments, only a portion of the nucleotides in the attachment strand hybridizes complementaryly to the linking strand. In some embodiments, the linking strand and / or the complementary strand is discontinuous, i.e., for example, the complementary strand is discontinuous, with the strands on either side of the break complementary to the linking strand, thereby being connected by the linking strand, and vice versa.

[0012] In some embodiments, the end-cap connector is a single chain that can form a secondary or tertiary structure, for example, by forming one or more hairpin structures or other more complex tertiary structures through reverse repeating sequences. In some embodiments, the end-cap connector does not contain a double-chain region, i.e., it consists of a single chain and the single chain does not form a double-chain region through reverse complementary segments.

[0013] In some embodiments, the 5' end of the linear DNA molecule includes a capping linker that does not contain a double-stranded region, i.e., it consists of a single strand and the single strand does not form a double-stranded region through an inverted complementary segment; the 3' end of the double-stranded DNA molecule does not contain a capping linker. In some embodiments, the 5' end of the linear DNA molecule includes a capping linker that does not contain a double-stranded region, i.e., it consists of a single strand and the single strand does not form a double-stranded region through an inverted complementary segment; the 3' end of the double-stranded DNA molecule also includes a capping linker, which contains a double-stranded region or contains an inverted repeat sequence.

[0014] In some embodiments, the 3' end of the linear DNA molecule includes a capping linker that does not contain a double-stranded region, i.e., it consists of a single strand and the single strand does not form a double-stranded region through an inverted complementary segment; the 5' end of the double-stranded DNA molecule does not include a capping linker. In some embodiments, the 3' end of the linear DNA molecule includes a capping linker that does not contain a double-stranded region, i.e., it consists of a single strand and the single strand does not form a double-stranded region through an inverted complementary segment; the 5' end of the double-stranded DNA molecule also includes a capping linker, which contains a double-stranded region or contains an inverted repeat sequence.

[0015] In some embodiments, the linear DNA molecule includes capping linkers at both ends. These capping linkers are double-stranded, meaning they have one chain connecting the 5' end of one strand of the double-stranded region of the linear DNA (referred to as the "linking strand") to the 3' end of its complementary strand, and another strand complementary to the long strand (referred to as the "attachment strand"). In some embodiments, each nucleotide in the attachment strand is complementary to the linking strand. In some embodiments, only a portion of the nucleotides in the attachment strand are complementary to the linking strand. In some embodiments, only a portion of the nucleotides in the attachment strand are complementary to the linking strand. In some embodiments, the linking strand and / or the complementary strand is discontinuous, for example, the complementary strand is discontinuous, with the strands on either side of the break complementary to the linking strand, thus connecting them via the linking strand, and vice versa.

[0016] In some embodiments, the linear DNA molecule includes capping linkers at both ends, and these capping linkers are single-stranded; the single strand can form secondary or tertiary structures, such as one or more hairpin structures or other more complex tertiary structures formed by inverted repeat sequences. In some embodiments, the linear DNA molecule includes capping linkers at both ends, and these capping linkers do not contain double-stranded regions, i.e., they consist of a single strand and the single strand does not form a double-stranded region through inverted complementary segments.

[0017] In some embodiments, the capped connector comprises an inverted repeat sequence. In some embodiments, the capped connector comprises, or is composed of, the loop portion of an adeno-associated virus (AAV) inverted terminal repeat (ITR) structure, or is composed of the loop portion of an AAV ITR. In some embodiments, the loop portion of the AAV ITR is the loop portion of an AAV2 ITR. In this application, the ITR-L-Loop portion and the ITR-R-Loop portion are collectively referred to as the loop portion of the ITR. In some embodiments, the ITR-L-Loop portion is the same as the ITR-R-Loop portion. In some embodiments, the ITR-L-Loop portion is different from the ITR-R-Loop portion. In some embodiments, the ITR-L-Loop portion comprises or is composed of the sequence shown in SEQ ID NO:23. In some embodiments, the ITR-R-Loop portion comprises or is composed of the sequence shown in SEQ ID NO:24.

[0018] In some embodiments, the capping linker does not contain an inverted repeat sequence. In some embodiments, the capping sequence is single-stranded and does not contain double-stranded segments. In some embodiments, the capping sequence is single-stranded and does not contain inverted repeat sequences. In some embodiments, the capping sequence is single-stranded and does not contain double-stranded segments or inverted repeat sequences, and consists of 1-10 nucleotides. In some embodiments, the base sequence of the capping linker is selected from any one or two of the following: 5'-TTTT-3', 5'-TTT-3', 5'-AAA-3', 5'-TTATT-3', 5'-TTTTTT-3', and 5'-CGCGCG-3'.

[0019] In some embodiments, the capping linker includes an ITR-L-Loop portion located on the 5' side of the linear DNA double-stranded portion and / or an ITR-R-Loop portion located on the 3' side of the linear DNA double-stranded portion. In some embodiments, the capping linker on the 5' side of the linear DNA is an ITR-L-Loop portion, and the 3' side of the linear DNA is not capped. In some embodiments, the capping linker on the 3' side of the linear DNA is an ITR-R-Loop portion, and the 5' side of the linear DNA is not capped. In some embodiments, the capping linker on the 5' side of the linear DNA is an ITR-L-Loop portion, and the capping linker on the 3' side of the linear DNA is an ITR-R-Loop portion. In some embodiments, the capping linker on the 5' side of the linear DNA is an ITR-L-Loop portion, and the capping linker on the 3' side of the linear DNA is a single strand without a double-stranded segment. In some embodiments, the capping linker on the 5' side of the linear DNA is an ITR-L-Loop portion, and the capping linker on the 3' side of the linear DNA is a single strand without double-stranded segments. In some embodiments, the capping linker on the 3' side of the linear DNA is an ITR-R-Loop portion, and the capping linker on the 5' side of the linear DNA is a single strand without double-stranded segments. In some embodiments, the capping linker on the 5' side of the linear DNA is an ITR-L-Loop portion, and the capping linker on the 3' side of the linear DNA is 5'-TTTT-3', 5'-TTT-3', 5'-AAA-3', 5'-TTATT-3', 5'-TTTTTT-3', or 5'-CGCGCG-3'. In some embodiments, the capping linker on the 3' side of the linear DNA is an ITR-R-Loop portion, and the capping linker on the 5' side of the linear DNA is 5'-TTTT-3', 5'-TTT-3', 5'-AAA-3', 5'-TTATT-3', 5'-TTTTTT-3', or 5'-CGCGCG-3'. In some embodiments, the capping linker on the 5' side of the linear DNA is an ITR-L-Loop portion, and the length of the capping linker on the 3' side of the linear DNA does not exceed 30nt, 20nt, 10nt, or 5nt. In some embodiments, the capping linker on the 3' side of the linear DNA is an ITR-R-Loop portion, and the length of the capping linker on the 5' side of the linear DNA does not exceed 30nt, 20nt, 10nt, or 5nt.In some embodiments, the capping linker on the 5' side of the linear DNA is an ITR-L-Loop portion; the capping linker on the 3' side of the linear DNA is no longer than 30 nt, 20 nt, 10 nt, or 5 nt and consists of A and / or T. In some embodiments, the capping linker on the 3' side of the linear DNA is an ITR-R-Loop portion; the capping linker on the 5' side of the linear DNA is no longer than 30 nt, 20 nt, 10 nt, or 5 nt and consists of A and / or T. In some embodiments, the capping linker on the 5' side of the linear DNA is an ITR-L-Loop portion; the capping linker on the 3' side of the linear DNA is no longer than 30 nt, 20 nt, 10 nt, or 5 nt and consists of G and / or C. In some embodiments, the capping linker located on the 3' side of the linear DNA is an ITR-R-Loop portion, and the capping linker on the 5' side of the linear DNA is no longer than 30nt, 20nt, 10nt, or 5nt, and consists of G and / or C.

[0020] In some embodiments, the linear DNA contains capping linkers on both sides, and the length of each capping linker does not exceed 30 nt, for example, not exceeding 29 nt, 28 nt, 27 nt, 26 nt, 25 nt, 24 nt, 23 nt, 22 nt, 21 nt, 20 nt, 19 nt, 18 nt, 17 nt, 16 nt, 15 nt, 14 nt, 13 nt, 12 nt, 11 nt, 10 nt, 9 nt, 8 nt, 7 nt, 6 nt, or 5 nt. In some embodiments, the linear DNA contains capping linkers on both sides, and the length of each capping linker does not exceed 20 nt, for example, not exceeding 19 nt, 18 nt, 17 nt, 16 nt, 15 nt, 14 nt, 13 nt, 12 nt, 11 nt, 10 nt, 9 nt, 8 nt, 7 nt, 6 nt, or 5 nt. In some embodiments, the linear DNA contains capping conjugates on both sides, and the length of each capping conjugate does not exceed 10 nt, for example, not exceeding 9 nt, 8 nt, 7 nt, 6 nt, or 5 nt. In some embodiments, the linear DNA contains capping conjugates on both sides, and the length of each capping conjugate is 4 nt, 3 nt, 2 nt, or 1 nt. In some embodiments, the linear DNA contains capping conjugates on both sides, and the capping conjugates are composed of T and / or A. In some embodiments, the linear DNA contains capping conjugates on both sides, and the length of each capping conjugate does not exceed 30 nt, and it is composed of T and / or A. In some embodiments, the linear DNA contains capping conjugates on both sides, and the length of each capping conjugate does not exceed 20 nt, and it is composed of T and / or A. In some embodiments, the linear DNA contains capping conjugates on both sides, and the length of each capping conjugate does not exceed 10 nt, and it is composed of T and / or A. In some embodiments, the linear DNA contains capping conjugates on both sides, and the capping conjugates are no more than 5 nt in length and are composed of T and / or A. In some embodiments, the linear DNA contains capping conjugates on both sides, and the capping conjugates are composed of G and / or C. In some embodiments, the linear DNA contains capping conjugates on both sides, and the capping conjugates are no more than 30 nt in length and are composed of G and / or C. In some embodiments, the linear DNA contains capping conjugates on both sides, and the capping conjugates are no more than 20 nt in length and are composed of G and / or C. In some embodiments, the linear DNA contains capping conjugates on both sides, and the capping conjugates are no more than 10 nt in length and are composed of G and / or C. In some embodiments, the linear DNA contains capping conjugates on both sides, and the capping conjugates are no more than 5 nt in length and are composed of G and / or C.In some embodiments, the linear DNA contains capping linkers on both sides, and the capping linkers are selected from any one or two of the following: 5'-TTTT-3', 5'-TTT-3', 5'-AAA-3', 5'-TTATT-3', 5'-TTTTTT-3', and 5'-CGCGCG-3'. In some embodiments, the linear DNA contains capping linkers on both sides, and the capping linkers are 5'-TTTT-3'.

[0021] In some embodiments, the double-stranded region of the linear DNA molecule begins at the 5' end with a base sequence as shown in SEQ ID NO:51 and ends at the 3' end with a base sequence as shown in SEQ ID NO:52.

[0022] In some embodiments, the linear DNA is further linked to one or more nuclear localization signal peptides (NLS). In some embodiments, the NLS is selected from one, two, or more of monotypic NLS, ditypic NLS, and non-classical proline-tyrosine typic NLS. In some embodiments, the NLS comprises one or more amino acid sequences selected from the following, or its amino acid sequence consists of one or more amino acid sequences selected from the following: SEQ ID NO:40 and SEQ ID NO:48-50. In some embodiments, the NLS comprises a polynucleotide sequence as shown in SEQ ID NO:40, or the amino acid sequence of the NLS is as shown in SEQ ID NO:40. In some embodiments, the one or more NLS are linked to the base portion of one or more nucleotides in the linear DNA. In some embodiments, the one or more NLS are linked to the base portion of one or more T in the linear DNA. In some embodiments, the one or more NLS are linked to the base portion of one or more T in the linear DNA. In some embodiments, one, more, or all of the one or more NLS are linked to one or more nucleotides in the capping linker. In some embodiments, the linear DNA comprises an even number of NLSs, each NLS being connected to both ends of the linear DNA, and the number of NLSs connected to both ends of the linear DNA is the same. In some embodiments, the linear DNA comprises an even number of NLSs and end-capping connectors, each NLS being connected to an end-capping connector at both ends of the linear DNA, and the number of NLSs connected to the end-capping connectors at both ends of the linear DNA is the same. In some embodiments, the linear DNA comprises NLSs and end-capping connectors, the end-capping connectors being 5'-TTTT-3', and the NLSs being connected to the third T from the 5' position of the end-capping connector.

[0023] In some embodiments, the linear DNA molecule contains a target gene and homologous arms. In some embodiments, the linear DNA molecule contains a target gene but does not contain homologous arms. In this application, when the double-stranded portion of the DNA molecule contains homologous arms and a target gene sequence, the homologous arms, the target gene sequence, and the sequence connecting the homologous arms and the target gene together constitute the insert sequence; when the double-stranded portion of the DNA molecule contains a target gene sequence but does not contain homologous arms, the sequence containing the target gene sequence itself is called the insert sequence (or "insertion gene template"); it should be understood that when a nuclear insertion sequence is present between the target gene sequence and the end cap connector, the insert sequence does not contain the nuclear insertion sequence or the sequence between the nuclear insertion sequence and the end cap connector. In some embodiments, the insert sequence is inserted into the genome in the manner shown in Figure 19, i.e., it can be inserted into a gene editing site (or target site) through homologous recombination or non-homologous end repair.

[0024] In some embodiments, the double-stranded portion of the linear DNA molecule comprises, or is composed of, any one or more of, selected from: the target gene sequence, homologous arms, and nuclear insertion sequences; wherein,

[0025] The homologous arms include a homologous arm L located on one side of the 5' end of the target gene sequence and / or a homologous arm R located on one side of the 3' end of the target gene sequence, and

[0026] The nuclear insertion sequence includes a nuclear insertion sequence L located on one side of the 5' end of the target gene sequence and / or a nuclear insertion sequence R located on one side of the 3' end of the target gene sequence.

[0027] In some embodiments, the 5' end of the linear DNA includes the following structure: a capping linker-ITR-L double-stranded region sequence;

[0028] The 3' end of the linear DNA contains the following structure: -ITR-L double-stranded region sequence - capping linker;

[0029] The end-cap connector is not the loop portion of the AAV ITR.

[0030] In some embodiments, the 5' end of the linear DNA includes the following structure: a capping linker-ITR-L double-stranded region sequence;

[0031] The 3' end of the linear DNA contains the following structure: -ITR-L double-stranded region sequence - capping linker;

[0032] The end-cap connector is not the loop portion of the AAV ITR; in some embodiments, the length of the end-cap connector does not exceed 30 nt, for example, not exceeding 29 nt, 28 nt, 27 nt, 26 nt, 25 nt, 24 nt, 23 nt, 22 nt, 21 nt, 20 nt, 19 nt, 18 nt, 17 nt, 16 nt, 15 nt, 14 nt, 13 nt, 12 nt, 11 nt, 10 nt, 9 nt, 8 nt, 7 nt, 6 nt, or 5 nt; in some embodiments, the length of the end-cap connector does not exceed 20 nt, for example, not exceeding 19 nt, 18 nt, 17 nt, 16 nt, 15 nt, 14 nt, 13 nt, 12 nt, 11 nt, 10 nt, 9nt, 8nt, 7nt, 6nt, or 5nt; in some embodiments, the length of the end-sealing connector does not exceed 10nt, for example, not exceeding 9nt, 8nt, 7nt, 6nt, or 5nt; in some embodiments, the length of the end-sealing connector is 4nt, 3nt, 2nt, or 1nt; in some embodiments, the end-sealing connector is composed of T and / or A; in some embodiments, the length of the end-sealing connector does not exceed 30nt, and the end-sealing connector is composed of T and / or A; in some embodiments, the length of the end-sealing connector does not exceed 20nt, and the end-sealing connector is composed of T and / or A; in some embodiments, the length of the end-sealing connector does not exceed 10nt. In some embodiments, the end-sealing connector is composed of T and / or A; in some embodiments, the length of the end-sealing connector is no more than 5nt, and the end-sealing connector is composed of T and / or A; in some embodiments, the end-sealing connector is composed of G and / or C; in some embodiments, the length of the end-sealing connector is no more than 30nt, and the end-sealing connector is composed of G and / or C; in some embodiments, the length of the end-sealing connector is no more than 20nt, and the end-sealing connector is composed of G and / or C; in some embodiments, the length of the end-sealing connector is no more than 10nt, and the end-sealing connector is composed of G and / or C; in some embodiments, the length of the end-sealing connector is no more than 5nt, and The terminator is composed of G and / or C; in some embodiments, the terminator does not contain an inverted repeat sequence; in some embodiments, the terminator sequence is single-stranded and does not contain a double-stranded segment; in some embodiments, the terminator sequence is single-stranded and does not contain a double-stranded segment or an inverted repeat sequence, and is composed of 1-10 nucleotides; in some embodiments, the base sequence of the terminator is selected from any one or two of the following: 5'-TTTT-3', 5'-TTT-3', 5'-AAA-3', 5'-TTATT-3', 5'-TTTTTT-3', and 5'-CGCGCG-3';In some embodiments, the base sequence of the end-capping linker is 5'-TTTT-3'.

[0033] In some embodiments, the 5' end of the linear DNA includes the following structure: full-length ITR-L;

[0034] The 3' end of the linear DNA contains the following structure: full-length ITR-R.

[0035] In some embodiments, the double-stranded portion of the linear DNA molecule is composed of a structure selected from any of the following:

[0036] (1) -Target gene sequence-;

[0037] (2) - Homologous arm L- Target gene sequence - Homologous arm R-;

[0038] (3) - Nuclear sequence L- Target gene sequence - Nuclear sequence R-;

[0039] (4) - Nuclear sequence L-Homologous arm L-Target gene sequence-Homologous arm R-Nuclear sequence R-;

[0040] (5) - Target gene sequence - Nuclear sequence R-;

[0041] (6) - Homologous arm L - Target gene sequence - Homologous arm R - Nuclear sequence R -;

[0042] (7) - Nuclear sequence L- Target gene sequence -; or

[0043] (8) - Nuclear sequence L- Homologous arm L- Target gene sequence - Homologous arm R-;

[0044] The "-" indicates 0, 1 or more nucleotides.

[0045] In some embodiments, exemplary structures of the linear DNA molecule are shown in Figure 16A as doubly closed double-stranded DNA with a nuclear insertion sequence and NLS-modified doubly closed double-stranded DNA with a nuclear insertion sequence. The circular portion in the figure represents a capping linker; it should be understood that this circular portion is not intended to limit the specific structure of the capping linker, but is merely illustrative. In some embodiments, the nuclear insertion sequence comprises an AAV ITR nuclear insertion sequence. In some embodiments, the nuclear insertion sequence comprises an AAV ITR double-stranded region, such as the double-stranded region of AAV ITR-R and / or the double-stranded region of AAV ITR-L. In some embodiments, the gene sequence comprises a nuclear insertion sequence L and / or a nuclear insertion sequence R, wherein the nuclear insertion sequence L is located on one side of the 5' end of the insert sequence, and the nuclear insertion sequence R is located on one side of the 3' end of the insert sequence. In some embodiments, the DNA molecule comprises an insert sequence and capping linkers flanking it, wherein the capping linkers flanking the insert sequence are symmetrical about the halfway point of the insert sequence. In some embodiments, the DNA molecule includes an insert sequence, nuclear insertion sequences flanking it, and capping linkers, wherein the structures flanking the insert sequence are symmetrical about a 1 / 2 mark from the insert sequence.

[0046] In some embodiments, the nuclear insertion sequence on the 5' end side of the insert sequence includes or is composed of an AAV ITR-L double-stranded region; and the nuclear insertion sequence on the 3' end side of the insert sequence includes or is composed of an AAV ITR-R double-stranded region. In some embodiments, the nuclear insertion sequence on the 5' end side of the insert sequence includes or is composed of an AAV ITR-R double-stranded region; and the nuclear insertion sequence on the 3' end side of the insert sequence includes or is composed of an AAV ITR-L double-stranded region. In some embodiments, both the nuclear insertion sequence on the 5' end side of the insert sequence and the nuclear insertion sequence on the 3' end side of the insert sequence include or are both composed of AAV ITR-L double-stranded regions. In some embodiments, the nucleus sequence on the 5' side of the insert sequence and the nucleus sequence on the 3' side of the insert sequence both contain or are composed of AAV ITR-R double-stranded regions. In some embodiments, the nucleus sequence, after being joined with the end-cap connector, forms the full-length ITR sequence. In some embodiments, the nucleus sequence, after being joined with the end-cap connector, forms a stem-loop structure. In some embodiments, the nucleus sequence, after being joined with the end-cap connector, forms a hairpin structure.

[0047] In some embodiments, the linear DNA, from its 5' to 3' end, comprises: a capping linker L, a linker sequence L, a nuclear insertion sequence L, an insertion sequence, a nuclear insertion sequence R, a linker sequence R, and a capping linker R, wherein the nuclear insertion sequence R and the nuclear insertion sequence L may be the same or different, and the capping linker L and the capping linker R may be the same or different. The linker sequence R and the linker sequence L each consist of 0, 1, or more base pairs and may be the same or different. In some embodiments, the linear DNA, from its 5' to 3' end, comprises: a capping linker L, a linker sequence L, a nuclear insertion sequence L, an insertion sequence, a nuclear insertion sequence R, a linker sequence R, and a capping linker R, wherein the nuclear insertion sequence R and the nuclear insertion sequence L may be the same or different, and the capping linker L and the capping linker R may be the same or different. The base sequence of the linker sequence R is shown in SEQ ID NO:52, and the base sequence of the linker sequence L is shown in SEQ ID NO:51. In some embodiments, the linear DNA, from the 5' to the 3' end, comprises: a capping linker L, a linker sequence L, a nuclear insertion sequence L, an insertion sequence, a nuclear insertion sequence R, a linker sequence R, and a capping linker R, wherein the base sequence of the linker sequence R is as shown in SEQ ID NO:52, the base sequence of the linker sequence L is as shown in SEQ ID NO:51, the base sequence of the nuclear insertion sequence L is as shown in SEQ ID NO:25, and the base sequence of the nuclear insertion sequence R is as shown in SEQ ID NO:26.

[0048] In some embodiments, the double-stranded portion comprises a target gene sequence and a 2A peptide coding sequence located on one side of the 5' and / or 3' end of the target gene sequence. In some embodiments, the double-stranded portion comprises the target gene sequence and a promoter sequence located on one side of the 5' end of the target gene sequence. In some embodiments, the double-stranded portion comprises the target gene sequence and a tailing signal sequence located on one side of the 3' end of the target gene sequence.

[0049] In some embodiments, the double-stranded portion of the linear DNA molecule is composed of a structure selected from any of the following:

[0050] (1)-2A peptide coding sequence-target gene sequence-;

[0051] (2)-Homologous arm L-2A peptide coding sequence-Target gene sequence-Homologous arm R-;

[0052] (3) - Nuclear insertion sequence L-2A peptide coding sequence - Target gene sequence - Nuclear insertion sequence R-;

[0053] (4) - Nuclear insertion sequence L-Homologous arm L-2A peptide coding sequence - Target gene sequence - Homologous arm R-Nuclear insertion sequence R-;

[0054] (5) - Promoter - Target gene sequence - Tail signal -;

[0055] (6)-Homologous arm L-promoter-target gene sequence-tailing signal-homologous arm R-;

[0056] (7) - Nuclear insertion sequence L-promoter-target gene sequence-tailing signal-nuclear insertion sequence R-;

[0057] (8) - Nuclear insertion sequence L-Homologous arm L-Promoter-Target gene sequence-Tailing signal-Homologous arm R-Nuclear insertion sequence R-;

[0058] (9)-2A peptide coding sequence-target gene sequence-2A peptide coding sequence-;

[0059] (10)-Homologous arm L-2A peptide coding sequence-Target gene sequence-2A peptide coding sequence-Homologous arm R-;

[0060] (11)-Nuclear sequence L-2A peptide coding sequence-Target gene sequence-2A peptide coding sequence-Nuclear sequence R-;

[0061] (12)-Nuclear insertion sequence L-Homologous arm L-2A peptide coding sequence-Target gene sequence-2A peptide coding sequence-Homologous arm R-Nuclear insertion sequence R-;

[0062] (13) - Target gene sequence - 2A peptide coding sequence -;

[0063] (14)-Homologous arm L-Target gene sequence-2A peptide coding sequence-Homologous arm R-;

[0064] (15)-Nuclear sequence L-Target gene sequence-2A peptide coding sequence-Nuclear sequence R-;

[0065] (16)-Nuclear insertion sequence L-Homologous arm L-Target gene sequence-2A peptide coding sequence-Homologous arm R-Nuclear insertion sequence R-;

[0066] (17)-2A peptide coding sequence-target gene sequence-nuclear sequence R-;

[0067] (18)-Homologous arm L-2A peptide coding sequence-Target gene sequence-Homologous arm R-Nuclear sequence R-;

[0068] (19)-2A peptide coding sequence-target gene sequence-2A peptide coding sequence-nuclear sequence R-;

[0069] (20)-Homologous arm L-2A peptide coding sequence-Target gene sequence-2A peptide coding sequence-Homologous arm R-Nuclear sequence R-;

[0070] (21) - Target gene sequence - 2A peptide coding sequence - Nuclear insertion sequence R-;

[0071] (22)-Homologous arm L-Target gene sequence-2A peptide coding sequence-Homologous arm R-Nuclear sequence R-;

[0072] (23) - Promoter - Target gene sequence - Tail signal - Nuclear sequence R-;

[0073] (24) - Homologous arm L - Promoter - Target gene sequence - Tailing signal - Homologous arm R - Nuclear insertion sequence R -

[0074] (25)-Nuclear insertion sequence L-2A peptide coding sequence-Target gene sequence-;

[0075] (26)-Nuclear sequence L-Homologous arm L-2A peptide coding sequence-Target gene sequence-Homologous arm R-;

[0076] (27)-Nuclear sequence L-2A peptide coding sequence-Target gene sequence-2A peptide coding sequence-;

[0077] (28)-Nuclear insertion sequence L-Homologous arm L-2A peptide coding sequence-Target gene sequence-2A peptide coding sequence-Homologous arm R-;

[0078] (29) - Nuclear sequence L- Target gene sequence - 2A peptide coding sequence -;

[0079] (30)-Nuclear sequence L-Homologous arm L-Target gene sequence-2A peptide coding sequence-Homologous arm R-;

[0080] (31) - Nuclear insertion sequence L-promoter-target gene sequence-tailing signal-; and

[0081] (32) - Nuclear insertion sequence L- Homologous arm L- Promoter - Target gene sequence - Tail addition signal - Homologous arm R-,

[0082] The "-" indicates 0, 1 or more nucleotides.

[0083] In some implementations, the nuclear sequence is selected from one or more of the following: FadR nuclear sequence, ETS1 nuclear sequence, NFAT nuclear sequence, STAT2 nuclear sequence, LEF1 nuclear sequence, and AAV ITR nuclear sequence.

[0084] In some embodiments, at least one of the above-described nuclear insertion sequences contains an AAV ITR nuclear insertion sequence. In some embodiments, the linear DNA molecule does not contain the BB' and CC' regions of an AAV ITR, and at least one of the nuclear insertion sequences contains an AAV ITR nuclear insertion sequence. In some embodiments, the AAV ITR nuclear insertion sequence contains the complete D region of the AAV ITR. In some embodiments, the AAV ITR nuclear insertion sequence contains the complete terminal break site (TRS) sequence of the AAV ITR. In some embodiments, the AAV ITR nuclear insertion sequence contains the core TRS sequence of the AAV ITR. In some embodiments, the AAV ITR nuclear insertion sequence contains the complete Rep binding element (RBE) sequence of the AAV ITR. In some embodiments, the AAV ITR nuclear insertion sequence contains the complete D region and the complete TRS sequence of the AAV ITR, but does not contain the complete RBE. In some embodiments, the AAV ITR nuclear insertion sequence contains the complete D region and the complete TRS sequence of the AAV ITR, but does not contain any portion of the RBE sequence. In some embodiments, the AAV ITR core sequence includes the complete D region of the AAV ITR and the complete TRS sequence, and does not include any other AAV ITR sequences. In some embodiments, the AAV ITR core sequence includes the complete D region of the AAV ITR and the TRS core region sequence. In some embodiments, the AAV ITR core sequence includes the complete D region of the AAV ITR and the TRS core region sequence, and does not include any other parts of the AAV ITR. In some embodiments, the AAV ITR core sequence includes the complete D region of the AAV ITR and the TRS core region sequence, and does not include the complete RBE sequence. In some embodiments, the AAV ITR core sequence includes the complete D region of the AAV ITR and the TRS core region sequence, and does not include any part of the RBE sequence. In some embodiments, the AAV ITR core sequence includes the complete D region of the AAV ITR and the complete RBE sequence. In some embodiments, the AAV ITR core sequence includes the complete D region of the AAV ITR and the complete RBE sequence, but does not include the TRS core region sequence of the AAV ITR. In some embodiments, the AAV ITR nuclear insertion sequence includes the complete D region of the AAV ITR and the complete RBE sequence, but does not include any part of the AAV ITR TRS sequence.In some embodiments, the AAV ITR core insertion sequence comprises the complete D region of the AAV ITR and the complete RBE sequence, but does not contain any other part of the AAV ITR. In some embodiments, the AAV ITR core insertion sequence comprises the complete TRS sequence of the AAV ITR and the complete RBE sequence. In some embodiments, the AAV ITR core insertion sequence comprises the complete TRS sequence of the AAV ITR and the complete RBE sequence, but does not contain any sequence from the D region of the AAV ITR. In some embodiments, the AAV ITR core insertion sequence comprises the complete TRS sequence of the AAV ITR and the complete RBE sequence, but does not contain the complete D region sequence of the AAV ITR. In some embodiments, the AAV ITR core insertion sequence comprises the complete TRS sequence of the AAV ITR and the complete RBE sequence, but does not contain any other part of the AAV ITR. In some embodiments, the AAV ITR core insertion sequence comprises the TRS core region sequence of the AAV ITR and the complete RBE sequence. In some embodiments, the AAV ITR nuclear insertion sequence includes the TRS core region sequence of the AAV ITR and the complete RBE sequence, but does not include the complete AAV ITR D region sequence. In some embodiments, the AAV ITR nuclear insertion sequence includes the TRS core region sequence of the AAV ITR and the complete RBE sequence, but does not include any sequence from the AAV ITR D region. In some embodiments, the AAV ITR nuclear insertion sequence includes the TRS core region sequence of the AAV ITR and the complete RBE sequence, but does not include sequences from any other part of the AAV ITR.

[0085] In some embodiments, at least one of the nuclear insertion sequences contains an AAV ITR nuclear insertion sequence, the linear DNA molecule does not contain the BB' and CC' regions of the AAV ITR, and:

[0086] (1) The AAV ITR nuclear sequence does not contain a complete TRS sequence or does not contain a TRS core region sequence;

[0087] (2) The AAV ITR nuclear insertion sequence does not contain a complete RBE sequence;

[0088] (3) The AAV ITR nuclear insertion sequence does not contain a complete TRS sequence and does not contain a complete RBE sequence; or

[0089] (4) The AAV ITR nuclear sequence does not contain the TRS core region sequence and does not contain the complete RBE sequence.

[0090] In some embodiments, at least one of the nuclear insertion sequences comprises an AAV ITR nuclear insertion sequence, the linear DNA molecule does not contain the BB' and CC' regions of the AAV ITR, and the AAV ITR nuclear insertion sequence is a truncated version of the AAV ITR, and:

[0091] (1) The D region is located in the truncated body of the AAV ITR;

[0092] (2) At least one of the D regions and at least one of the TRS sequences are located in the same AAV ITR truncated body; and / or

[0093] (3) At least one of the D regions, at least one of the TRS sequences, and at least one of the RBE sequences are located in the same AAV ITR truncated form. It should be understood that when the AAV ITR nuclear insertion sequence is a truncated form of the AAV ITR, it forms a double-stranded region with its complementary strand, and any one of the strands can be a truncated form of the AAV ITR.

[0094] In some embodiments, the AAV ITR is the ITR of AAV2. In some embodiments, the linear DNA molecule contains one, two, or more AAV ITR nuclear insertion sequences. In some embodiments, each AAV ITR nuclear insertion sequence independently comprises or is selected from any one or more of the following base sequences: SEQ ID NO:25 to 26, SEQ ID NO:53 to 56, and SEQ ID NO:74-77.

[0095] In some embodiments, the nuclear insertion sequence comprises one or more sequences selected from the following base sequences: such as those shown in SEQ ID NO:3-7, 25-28, 53-56 and SEQ ID NO:74-77.

[0096] In some embodiments, the nuclear insertion sequence L in the linear DNA molecule from 5' to 3' comprises an AAV ITR-L nuclear insertion sequence and another nuclear insertion sequence besides the AAV ITR nuclear insertion sequence, and the nuclear insertion sequence R from 5' to 3' comprises another nuclear insertion sequence besides the AAV ITR nuclear insertion sequence and an ITR-R nuclear insertion sequence. In some embodiments, the nuclear insertion sequence L in the linear DNA molecule from 5' to 3' consists of an AAV ITR-L nuclear insertion sequence and another nuclear insertion sequence besides the AAV ITR nuclear insertion sequence, and the nuclear insertion sequence R from 5' to 3' consists of another nuclear insertion sequence besides the AAV ITR nuclear insertion sequence and an ITR-R nuclear insertion sequence. In some embodiments, the nuclear insertion sequence L in the linear DNA molecule comprises or is an AAV ITR-L nuclear insertion sequence, and the nuclear insertion sequence R comprises or is an AAV ITR nuclear insertion sequence.

[0097] In some embodiments, the nuclear insertion sequence L in the linear DNA molecule, from 5' to 3', comprises an AAV ITR-L double-stranded region sequence and another nuclear insertion sequence besides the AAV ITR nuclear insertion sequence, and the nuclear insertion sequence R, from 5' to 3', comprises another nuclear insertion sequence besides the AAV ITR nuclear insertion sequence and an ITR-R double-stranded region sequence. In some embodiments, the nuclear insertion sequence L in the linear DNA molecule, from 5' to 3', consists of an AAV ITR-L double-stranded region sequence and another nuclear insertion sequence besides the AAV ITR nuclear insertion sequence, and the nuclear insertion sequence R, from 5' to 3', consists of another nuclear insertion sequence besides the AAV ITR nuclear insertion sequence and an ITR-R double-stranded region sequence. In some embodiments, the nuclear insertion sequence L in the linear DNA molecule comprises or is an AAV ITR-L double-stranded region sequence, and the nuclear insertion sequence R comprises or is an AAV ITR double-stranded region sequence.

[0098] In some embodiments, the double-stranded portion of the linear DNA molecule is composed of a structure selected from any of the following:

[0099] (1)-AAV ITR-L nuclear insertion sequence-NFAT nuclear insertion sequence-2A peptide coding sequence-target gene sequence-NFAT nuclear insertion sequence-AAV ITR-R nuclear insertion sequence-;

[0100] (2)-AAV ITR-L nuclear insertion sequence-NFAT nuclear insertion sequence-homologous arm L-2A peptide coding sequence-target gene sequence-homologous arm R-NFAT nuclear insertion sequence-AAV ITR-R nuclear insertion sequence-;

[0101] (3) -AAV ITR-L nuclear insertion sequence -NFAT nuclear insertion sequence -2A peptide coding sequence - target gene sequence -2A peptide coding sequence -NFAT nuclear insertion sequence -AAV ITR-R nuclear insertion sequence -;

[0102] (4) -AAV ITR-L nuclear insertion sequence-NFAT nuclear insertion sequence-homologous arm L-2A peptide coding sequence-target gene sequence-2A peptide coding sequence-homologous arm R-NFAT nuclear insertion sequence-AAV ITR-R nuclear insertion sequence-;

[0103] (5)-AAV ITR-L nuclear insertion sequence-NFAT nuclear insertion sequence-target gene sequence-2A peptide coding sequence-NFAT nuclear insertion sequence-AAV ITR-R nuclear insertion sequence-;

[0104] (6)-AAV ITR-L nuclear insertion sequence-NFAT nuclear insertion sequence-homologous arm L-target gene sequence-2A peptide coding sequence-homologous arm R-NFAT nuclear insertion sequence-AAV ITR-R nuclear insertion sequence-;

[0105] (7) - Homologous arm L-promoter-peptide coding sequence-target gene sequence-tailing signal-homologous arm R-;

[0106] (8) -AAV ITR-L nuclear entry sequence-NFAT nuclear entry sequence-promoter-target gene sequence-tailing signal-NFAT nuclear entry sequence-AAV ITR-R nuclear entry sequence-;

[0107] (9) -AAV ITR-L nuclear insertion sequence -NFAT nuclear insertion sequence - homologous arm L - promoter - target gene sequence - tailing signal - homologous arm R -NFAT nuclear insertion sequence -AAV ITR-R nuclear insertion sequence -

[0108] (10)-2A peptide coding sequence-target gene sequence-NFAT nuclear insertion sequence-AAV ITR-R nuclear insertion sequence-;

[0109] (11)-Homologous arm L-2A peptide coding sequence-Target gene sequence-Homologous arm R-NFAT nuclear insertion sequence-AAV ITR-R nuclear insertion sequence-;

[0110] (12)-2A peptide coding sequence-target gene sequence-2A peptide coding sequence-NFAT nuclear insertion sequence-AAV ITR-R nuclear insertion sequence-;

[0111] (13)-Homologous arm L-2A peptide coding sequence-Target gene sequence-2A peptide coding sequence-Homologous arm R-NFAT nuclear insertion sequence-AAV ITR-R nuclear insertion sequence-;

[0112] (14) - Target gene sequence - 2A peptide coding sequence - NFAT nuclear insertion sequence - AAV ITR-R nuclear insertion sequence -;

[0113] (15)-Homologous arm L-Target gene sequence-2A peptide coding sequence-Homologous arm R-NFAT nuclear insertion sequence-AAV ITR-R nuclear insertion sequence-;

[0114] (16) - Promoter - Target gene sequence - Tail signal - NFAT nuclear insertion sequence - AAV ITR-R nuclear insertion sequence -;

[0115] (17) - Homologous arm L-promoter - Target gene sequence - Tailing signal - Homologous arm R-NFAT nuclear insertion sequence - AAV ITR-R nuclear insertion sequence -

[0116] (18)-AAV ITR-L nuclear insertion sequence-NFAT nuclear insertion sequence-2A peptide coding sequence-target gene sequence-;

[0117] (19)-AAV ITR-L nuclear insertion sequence-NFAT nuclear insertion sequence-homologous arm L-2A peptide coding sequence-target gene sequence-homologous arm R-;

[0118] (20)-AAV ITR-L nuclear insertion sequence-NFAT nuclear insertion sequence-2A peptide coding sequence-target gene sequence-2A peptide coding sequence-;

[0119] (21)-AAV ITR-L nuclear insertion sequence-NFAT nuclear insertion sequence-homologous arm L-2A peptide coding sequence-target gene sequence-2A peptide coding sequence-homologous arm R-;

[0120] (22)-AAV ITR-L nuclear insertion sequence-NFAT nuclear insertion sequence-target gene sequence-2A peptide coding sequence-;

[0121] (23)-AAV ITR-L nuclear insertion sequence-NFAT nuclear insertion sequence-homologous arm L-target gene sequence-2A peptide coding sequence-homologous arm R-;

[0122] (24)-AAV ITR-L nuclear insertion sequence-NFAT nuclear insertion sequence-promoter-target gene sequence-tailing signal-;

[0123] (25)-AAV ITR-L nuclear sequence-NFAT nuclear sequence-homologous arm L-promoter-target gene sequence-tailing signal-homologous arm R-.

[0124] (26)-AAV ITR-L double-stranded region sequence-NFAT nuclear insertion sequence-2A peptide coding sequence-target gene sequence-NFAT nuclear insertion sequence-AAV ITR-R double-stranded region sequence-;

[0125] (27)-AAV ITR-L double-stranded region sequence-NFAT nuclear insertion sequence-homologous arm L-2A peptide coding sequence-target gene sequence-homologous arm R-NFAT nuclear insertion sequence-AAV ITR-R double-stranded region sequence-;

[0126] (28)-AAV ITR-L double-stranded region sequence-NFAT nuclear insertion sequence-2A peptide coding sequence-target gene sequence-2A peptide coding sequence-NFAT nuclear insertion sequence-AAV ITR-R double-stranded region sequence-;

[0127] (29)-AAV ITR-L double-stranded region sequence-NFAT nuclear insertion sequence-homologous arm L-2A peptide coding sequence-target gene sequence-2A peptide coding sequence-homologous arm R-NFAT nuclear insertion sequence-AAV ITR-R double-stranded region sequence-;

[0128] (30)-AAV ITR-L double-stranded region sequence-NFAT nuclear insertion sequence-target gene sequence-2A peptide coding sequence-NFAT nuclear insertion sequence-AAV ITR-R double-stranded region sequence-;

[0129] (31)-AAV ITR-L double-stranded region sequence-NFAT nuclear insertion sequence-homologous arm L-target gene sequence-2A peptide coding sequence-homologous arm R-NFAT nuclear insertion sequence-AAV ITR-R double-stranded region sequence-;

[0130] (32)-Homologous arm L-Promoter-Peptide coding sequence-Target gene sequence-Tailing signal-Homologous arm R-;

[0131] (33)-AAV ITR-L double-stranded region sequence-NFAT nuclear entry sequence-promoter-target gene sequence-tailing signal-NFAT nuclear entry sequence-AAV ITR-R double-stranded region sequence-;

[0132] (34) -AAV ITR-L double-stranded region sequence -NFAT nuclear insertion sequence - homologous arm L - promoter - target gene sequence - tailing signal - homologous arm R -NFAT nuclear insertion sequence -AAV ITR-R double-stranded region sequence -

[0133] (35)-2A peptide coding sequence-target gene sequence-NFAT nuclear insertion sequence-AAV ITR-R double-stranded region sequence-;

[0134] (36)-Homologous arm L-2A peptide coding sequence-Target gene sequence-Homologous arm R-NFAT nuclear insertion sequence-AAV ITR-R double-stranded region sequence-;

[0135] (37)-2A peptide coding sequence-target gene sequence-2A peptide coding sequence-NFAT nuclear insertion sequence-AAV ITR-R double-stranded region sequence-;

[0136] (38)-Homologous arm L-2A peptide coding sequence-Target gene sequence-2A peptide coding sequence-Homologous arm R-NFAT nuclear insertion sequence-AAV ITR-R double-stranded region sequence-;

[0137] (39) - Target gene sequence - 2A peptide coding sequence - NFAT nuclear insertion sequence - AAV ITR-R double-stranded region sequence -;

[0138] (40)-Homologous arm L-Target gene sequence-2A peptide coding sequence-Homologous arm R-NFAT nuclear insertion sequence-AAV ITR-R double-stranded region sequence-;

[0139] (41) - Promoter - Target gene sequence - Tail signal - NFAT nuclear insertion sequence - AAV ITR-R double-stranded region sequence -;

[0140] (42) - Homologous arm L-promoter - Target gene sequence - Tailing signal - Homologous arm R-NFAT nuclear insertion sequence - AAV ITR-R double-stranded region sequence -

[0141] (43)-AAV ITR-L double-stranded region sequence-NFAT nuclear insertion sequence-2A peptide coding sequence-target gene sequence-;

[0142] (44)-AAV ITR-L double-stranded region sequence-NFAT nuclear insertion sequence-homologous arm L-2A peptide coding sequence-target gene sequence-homologous arm R-;

[0143] (45)-AAV ITR-L double-stranded region sequence-NFAT nuclear insertion sequence-2A peptide coding sequence-target gene sequence-2A peptide coding sequence-;

[0144] (46)-AAV ITR-L double-stranded region sequence-NFAT nuclear insertion sequence-homologous arm L-2A peptide coding sequence-target gene sequence-2A peptide coding sequence-homologous arm R-;

[0145] (47)-AAV ITR-L double-stranded region sequence-NFAT nuclear insertion sequence-target gene sequence-2A peptide coding sequence-;

[0146] (48)-AAV ITR-L double-stranded region sequence-NFAT nuclear insertion sequence-homologous arm L-target gene sequence-2A peptide coding sequence-homologous arm R-;

[0147] (49)-AAV ITR-L double-stranded region sequence-NFAT nuclear insertion sequence-promoter-target gene sequence-tailing signal-;

[0148] (50)-AAV ITR-L double-stranded region sequence-NFAT nuclear insertion sequence-homologous arm L-promoter-target gene sequence-tailing signal-homologous arm R-.

[0149] (51)-AAV ITR-L nuclear insertion sequence-2A peptide coding sequence-target gene sequence-NFAT nuclear insertion sequence-AAV ITR-R nuclear insertion sequence-;

[0150] (52)-AAV ITR-L nuclear insertion sequence-Homologous arm L-2A peptide coding sequence-Target gene sequence-Homologous arm R-AAV ITR-R nuclear insertion sequence-;

[0151] (53)-AAV ITR-L nuclear insertion sequence-2A peptide coding sequence-target gene sequence-2A peptide coding sequence-NFAT nuclear insertion sequence-AAV ITR-R nuclear insertion sequence-;

[0152] (54)-AAV ITR-L nuclear insertion sequence-Homologous arm L-2A peptide coding sequence-Target gene sequence-2A peptide coding sequence-Homologous arm R-AAV ITR-R nuclear insertion sequence-;

[0153] (55)-AAV ITR-L nuclear insertion sequence-target gene sequence-2A peptide coding sequence-NFAT nuclear insertion sequence-AAV ITR-R nuclear insertion sequence-;

[0154] (56)-AAV ITR-L nuclear insertion sequence-homologous arm L-target gene sequence-2A peptide coding sequence-homologous arm R-AAV ITR-R nuclear insertion sequence-;

[0155] (57)-Homologous arm L-Promoter-Peptide coding sequence-Target gene sequence-Tailing signal-Homologous arm R-;

[0156] (58)-AAV ITR-L nuclear insertion sequence-promoter-target gene sequence-tailing signal-AAV ITR-R nuclear insertion sequence-;

[0157] (59)-AAV ITR-L nuclear insertion sequence-Homologous arm L-Promoter-Target gene sequence-Tailing signal-Homologous arm R-AAV ITR-R nuclear insertion sequence-

[0158] (60)-2A peptide coding sequence-target gene sequence-AAV ITR-R nuclear insertion sequence-;

[0159] (61)-Homologous arm L-2A peptide coding sequence-Target gene sequence-Homologous arm R-AAV ITR-R nuclear insertion sequence-;

[0160] (62)-2A peptide coding sequence-target gene sequence-2A peptide coding sequence-AAV ITR-R nuclear insertion sequence-;

[0161] (63)-Homologous arm L-2A peptide coding sequence-Target gene sequence-2A peptide coding sequence-Homologous arm R-AAV ITR-R nuclear insertion sequence-;

[0162] (64) - Target gene sequence - 2A peptide coding sequence - AAV ITR-R nuclear insertion sequence -;

[0163] (65)-Homologous arm L-Target gene sequence-2A peptide coding sequence-Homologous arm R-AAV ITR-R nuclear insertion sequence-;

[0164] (66) - Promoter - Target gene sequence - Tail signal - AAV ITR-R nuclear insertion sequence -;

[0165] (67)-Homologous arm L-promoter-target gene sequence-tailing signal-Homologous arm R-AAV ITR-R nuclear insertion sequence-

[0166] (68)-AAV ITR-L nuclear insertion sequence-2A peptide coding sequence-target gene sequence-;

[0167] (69)-AAV ITR-L nuclear sequence-homologous arm L-2A peptide coding sequence-target gene sequence-homologous arm R-;

[0168] (70)-AAV ITR-L nuclear insertion sequence-2A peptide coding sequence-target gene sequence-2A peptide coding sequence-;

[0169] (71)-AAV ITR-L nuclear insertion sequence-homologous arm L-2A peptide coding sequence-target gene sequence-2A peptide coding sequence-homologous arm R-;

[0170] (72)-AAV ITR-L nuclear insertion sequence-target gene sequence-2A peptide coding sequence-;

[0171] (73)-AAV ITR-L nuclear sequence-homologous arm L-target gene sequence-2A peptide coding sequence-homologous arm R-;

[0172] (74)-AAV ITR-L nuclear insertion sequence-promoter-target gene sequence-tailing signal-; and

[0173] (75)-AAV ITR-L nuclear sequence-homologous arm L-promoter-target gene sequence-tailing signal-homologous arm R-.

[0174] (76)-AAV ITR-L double-stranded region sequence-2A peptide coding sequence-target gene sequence-NFAT nuclear insertion sequence-AAV ITR-R double-stranded region sequence-;

[0175] (77)-AAV ITR-L double-stranded region sequence-Homologous arm L-2A peptide coding sequence-Target gene sequence-Homologous arm R-AAV ITR-R double-stranded region sequence-;

[0176] (78)-AAV ITR-L double-stranded region sequence-2A peptide coding sequence-target gene sequence-2A peptide coding sequence-NFAT nuclear insertion sequence-AAV ITR-R double-stranded region sequence-;

[0177] (79)-AAV ITR-L double-stranded region sequence-Homologous arm L-2A peptide coding sequence-Target gene sequence-2A peptide coding sequence-Homologous arm R-AAV ITR-R double-stranded region sequence-;

[0178] (80)-AAV ITR-L double-stranded region sequence-target gene sequence-2A peptide coding sequence-NFAT nuclear insertion sequence-AAV ITR-R double-stranded region sequence-;

[0179] (81)-AAV ITR-L double-stranded region sequence-Homologous arm L-Target gene sequence-2A peptide coding sequence-Homologous arm R-AAV ITR-R double-stranded region sequence-;

[0180] (82)-Homologous arm L-Promoter-Peptide coding sequence-Target gene sequence-Tailing signal-Homologous arm R-;

[0181] (83)-AAV ITR-L double-stranded region sequence-promoter-target gene sequence-tailing signal-AAV ITR-R double-stranded region sequence-;

[0182] (84)-AAV ITR-L double-stranded region sequence-Homologous arm L-Promoter-Target gene sequence-Tailing signal-Homologous arm R-AAV ITR-R double-stranded region sequence-

[0183] (85)-2A peptide coding sequence-target gene sequence-AAV ITR-R double-stranded region sequence-;

[0184] (86)-Homologous arm L-2A peptide coding sequence-Target gene sequence-Homologous arm R-AAV ITR-R double-stranded region sequence-;

[0185] (87)-2A peptide coding sequence-target gene sequence-2A peptide coding sequence-AAV ITR-R double-stranded region sequence-;

[0186] (88)-Homologous arm L-2A peptide coding sequence-Target gene sequence-2A peptide coding sequence-Homologous arm R-AAV ITR-R double-stranded region sequence-;

[0187] (89) - Target gene sequence - 2A peptide coding sequence - AAV ITR-R double-stranded region sequence -;

[0188] (90)-Homologous arm L-Target gene sequence-2A peptide coding sequence-Homologous arm R-AAV ITR-R double-stranded region sequence-;

[0189] (91) - Promoter - Target gene sequence - Tail addition signal - AAV ITR-R double-stranded region sequence -;

[0190] (92)-Homologous arm L-promoter-target gene sequence-tailing signal-Homologous arm R-AAV ITR-R double-stranded region sequence-

[0191] (93)-AAV ITR-L double-stranded region sequence-2A peptide coding sequence-target gene sequence-;

[0192] (94)-AAV ITR-L double-stranded region sequence-homologous arm L-2A peptide coding sequence-target gene sequence-homologous arm R-;

[0193] (95)-AAV ITR-L double-stranded region sequence-2A peptide coding sequence-target gene sequence-2A peptide coding sequence-;

[0194] (96)-AAV ITR-L double-stranded region sequence-homologous arm L-2A peptide coding sequence-target gene sequence-2A peptide coding sequence-homologous arm R-;

[0195] (97)-AAV ITR-L double-stranded region sequence-target gene sequence-2A peptide coding sequence-;

[0196] (98)-AAV ITR-L double-stranded region sequence-homologous arm L-target gene sequence-2A peptide coding sequence-homologous arm R-;

[0197] (99)-AAV ITR-L double-stranded region sequence-promoter-target gene sequence-tailing signal-; and

[0198] (100)-AAV ITR-L double-stranded region sequence-homologous arm L-promoter-target gene sequence-tailing signal-homologous arm R-;

[0199] The "-" indicates 0, 1 or more nucleotides.

[0200] In some implementations, the "-" above represents 0 nucleotides, meaning that the elements connected by "-" are directly linked by phosphate ester bonds or other derivative bonds with similar functions (such as thiophosphate bonds).

[0201] In some implementations, the above "AAV" refers to AAV2.

[0202] In some implementations, the aforementioned AAV ITR-L double-stranded region sequence is the double-stranded region sequence of AAV2 ITR-L, and the aforementioned AAV ITR-R double-stranded region sequence is the double-stranded region sequence of AAV2 ITR-R.

[0203] In some embodiments, the AAV ITR-L double-stranded region sequence comprises or is the sequence shown in SEQ ID NO:27, and the AAV ITR-R double-stranded region sequence comprises or is the sequence shown in SEQ ID NO:28. In some embodiments, the AAV ITR-L double-stranded region sequence comprises or is the sequence shown in SEQ ID NO:28, and the AAV ITR-R double-stranded region sequence comprises or is the sequence shown in SEQ ID NO:27. In some embodiments, the AAV ITR-L double-stranded region sequence and the AAV ITR-R double-stranded region sequence comprise or are the sequences shown in SEQ ID NO:27. In some embodiments, the AAV ITR-L double-stranded region sequence and the AAV ITR-R double-stranded region sequence comprise or are the sequences shown in SEQ ID NO:28.

[0204] In some embodiments, the sequences of AAV ITR-L and AAV ITR-R are the same or different. In some embodiments, the sequences of AAV ITR-L and / or AAV ITR-R are as shown in SEQ ID NO:29. In some embodiments, the sequences of AAV ITR-L and / or AAV ITR-R are as shown in SEQ ID NO:30. In some embodiments, the sequence of AAV ITR-L is as shown in SEQ ID NO:29, and the sequence of AAV ITR-R is as shown in SEQ ID NO:30. In some embodiments, the sequence of AAV ITR-L is as shown in SEQ ID NO:30, and the sequence of AAV ITR-R is as shown in SEQ ID NO:29.

[0205] In some embodiments, the length of each homologous arm on either side of the linear DNA molecule is independently about 100 to 2000 bp, for example, about 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1600 bp, or 1800 bp. In some embodiments, the length of each homologous arm on either side of the linear DNA molecule is independently about 100 to 800 bp. In some embodiments, the total length of the homologous arms in the linear DNA molecule is about 100 to 2000 bp, for example, about 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1600 bp, or 1800 bp. In some embodiments, the total length of homologous arms in the linear DNA molecule is about 100 to 2000 bp, for example, about 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1600 bp, or 1800 bp. In some embodiments, the total length of homologous arms in the linear DNA molecule is about 200 to 1600 bp. As used herein, the total length of homologous arms is the sum of the lengths of homologous arms on both sides of the linear DNA molecule; that is, when the linear DNA molecule has homologous arms on only one side, the length of the homologous arm on that side is the total length; when the linear DNA molecule has homologous arms on both sides, the sum of the lengths of the homologous arms on both sides is the total length. In some embodiments, the ratio of the total length of homologous arms to the length of the target gene sequence is about 40% to 60%, for example, about 50%. In some embodiments, the target gene sequence in the linear DNA molecule is approximately 500-5000 bp in length, for example, 1000-3000 bp. In some embodiments, the target gene is a chimeric antigen receptor (CAR) coding gene. In some embodiments, the CAR target gene coding sequence is approximately 1500 nt in length.

[0206] In some embodiments, the homologous arms in the linear DNA molecule can undergo homologous recombination with the target gene sequence. The target gene is the sequence of the gene at the insertion site in the cell's genome after the linear DNA molecule is introduced into the cell. The sequence can be a coding region sequence or a non-coding region sequence, or it can contain both coding and non-coding regions. Those skilled in the art should understand that homologous recombination requires the target gene and the homologous arm to have a certain degree of sequence identity, for example, approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, or approximately 100% sequence identity.

[0207] Those skilled in the art should know that the target gene sequence can be located anywhere in the genome. As long as there is a need to insert the target gene sequence, those skilled in the art can design a suitable homologous arm so that the target gene sequence can be inserted into the target gene sequence.

[0208] In some implementations, the target gene is selected from any of the following:

[0209] Human GAPDH gene, human IL2RA gene, and human TRAC gene.

[0210] A second aspect of this application also provides a gene editing composition for site-specific insertion of a target gene sequence, comprising the linear DNA molecule of the first aspect described above, and a gene editing molecule. The types of gene editing molecules are known in the art, and the gene editing molecules of this application encompass all protein and / or nucleic acid molecules that can facilitate the insertion of a fragment (e.g., a target gene sequence) from the linear DNA molecule into a target gene site or target gene sequence. In some embodiments, the gene editing molecule unwinds the target gene sequence and / or creates gaps (e.g., single-strand breaks and / or double-strand breaks) in the target gene sequence. In some embodiments, the double-strand breaks produce sticky ends or blunt ends. In some embodiments, the gene editing molecule comprises one or more enzymes selected from the group consisting of transposases, nucleases, and helicases. In some embodiments, the gene editing molecule comprises mRNA selected from the group consisting of transposases, nucleases, and helicases. In some embodiments, the gene editing molecule comprises a DNA sequence selected from the group consisting of one or more enzymes: transposases, nucleases, and helicases; the DNA sequence can be expressed as the enzyme in eukaryotic cells. In some embodiments, the gene-editing molecule comprises a nucleic acid programmable DNA-binding protein (napDNAbp), which can recognize the target gene sequence through a nucleic acid molecule that hybridizes complementary to the target gene sequence and perform enzymatic cleavage or other enzymatic reactions (e.g., deamination and transamination) in or near the target gene sequence. In some embodiments, the gene-editing molecule comprises one or more enzymes selected from or encoding the enzymes: CRISPR-associated proteins (Cas) (e.g., CRISPR-associated protein 9 (Cas9), Cpf1, IscB, TnpB, etc.), IIS-type restriction enzymes, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), Ago (Argonaute) proteins, macronucleases, and homing endonucleases (e.g., megaTAL).

[0211] When the gene-editing molecule contains the napDNAbp or a nucleic acid encoding the napDNAbp, the gene-editing composition further contains the nucleic acid molecule that hybridizes complementaryly to the target gene sequence. The nucleic acid molecule is, in some embodiments, a DNA molecule, in some embodiments, an RNA molecule, and in some embodiments, a hybrid of DNA and RNA. In some embodiments, the napDNAbp is a CRISPR-associated protein (Cas) or a Cas variant. In some embodiments, the Cas is Cas9 or a variant thereof. In some embodiments, the gene-editing molecule contains the Cas enzyme and its guide RNA (gRNA). In some embodiments, the gene-editing molecule contains a Cas9 variant or a nucleic acid molecule encoding the Cas9 variant, the Cas9 variant containing, or having a mutation relative to the Cas9 reference sequence, the following mutations: K526A, R691A, Q695A, and H698A;

[0212] The Cas9 reference sequence is shown in SEQ ID NO:41. In some embodiments, the amino acid sequence of the Cas9 variant described above comprises an amino acid sequence as shown in SEQ ID NO:42, or the mRNA sequence of the Cas comprises a nucleic acid sequence as shown in SEQ ID NO:37.

[0213] In some embodiments, the gene-editing molecule comprises Cas or Cas mRNA and a guide RNA (gRNA). In some embodiments, the gene-editing molecule comprises Cas or Cas mRNA and a guide RNA (gRNA). In some embodiments, the gene-editing molecule comprises type II Cas or type II Cas mRNA and a guide RNA (gRNA). In some embodiments, the gRNA is a single guide RNA (sgRNA). In some embodiments, the gene-editing molecule comprises type II Cas or type II Cas mRNA and sgRNA. In some embodiments, the gene-editing molecule contains Cas9. The mass ratio of mRNA to sgRNA is approximately 1:30-30:1, for example, approximately 1:8-8:1, or for example, approximately 1:29, 1:28, 1:27, 1:26, 1:25, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1: 6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, or 29:1. In some embodiments, the ratio of the total mass of Cas9 mRNA and sgRNA contained in the gene-editing molecule to the total mass of the linear DNA is about 3:1 to 1:3, for example, about 2:3, 4:3, or 6:7. In some embodiments, the gene editing composition comprises Cas9 mRNA, sgRNA and linear DNA molecules in a mass ratio of approximately 1:1:2, 1:1:3, 2:2:3, 3:3:7, 3:3:5, 4:4:9 or 4:4:7.

[0214] In some embodiments, the molar ratio of the linear DNA molecule to the transposase, nuclease, or helicase or its mRNA in the gene editing composition is approximately 3:1 to 1:3, for example, approximately 2:1 to 1:2, approximately 1:1, etc. In some embodiments, the molar ratio of the linear DNA molecule to the Cas enzyme or its mRNA in the gene editing composition is approximately 3:1 to 1:3, for example, approximately 2:1 to 1:2, approximately 1:1, etc. In some embodiments, the gene editing composition comprises a linear DNA molecule, a Cas enzyme (or its mRNA), and sgRNA; wherein the molar ratio of the linear DNA molecule to the Cas enzyme (or its mRNA) is approximately 3:1 to 1:3, for example, approximately 2:1 to 1:2, approximately 1:1, etc.

[0215] A third aspect of this application also provides a nucleic acid-lipid nanoparticle composition comprising lipid nanoparticles (LNPs) and nucleic acid comprising linear DNA molecules.

[0216] In this application, LNPs can be divided into at least two categories:

[0217] (1) A first type of LNP that carries a linear DNA molecule, wherein the first type of LNP can be any LNP that facilitates DNA delivery into the nucleus; wherein, in some embodiments, the first type of LNP may further carry gene editing molecules in addition to carrying a linear DNA molecule.

[0218] (2) A second type of LNP that carries gene-editing molecules but does not carry linear DNA molecules; the second type of LNP is any LNP known to facilitate the delivery of gene-editing molecules.

[0219] The nucleic acid-lipid nanoparticle composition of this application contains at least a first type of LNP. In some embodiments, the nucleic acid-lipid nanoparticle composition of this application contains only the first type of LNP. In some embodiments, the nucleic acid-lipid nanoparticle composition of this application contains only the first type of LNP, wherein the first type of LNP encapsulates the aforementioned linear DNA molecule of the first aspect and does not encapsulate gene editing molecules. In some embodiments, the nucleic acid-lipid nanoparticle composition of this application contains only the first type of LNP, wherein the first type of LNP encapsulates the aforementioned linear DNA molecule and gene editing molecules, that is, the first type of LNP encapsulates the gene editing composition of the second aspect.

[0220] In some embodiments, the nucleic acid-lipid nanoparticle composition of this application comprises a first type of LNP and a second type of LNP. In some embodiments, the nucleic acid-lipid nanoparticle composition of this application comprises a first type of LNP and a second type of LNP, wherein the first type of LNP encapsulates the linear DNA molecule described in the first aspect and does not encapsulate the gene editing molecule described in the second aspect. In some embodiments, the nucleic acid-lipid nanoparticle composition of this application comprises a first type of LNP and a second type of LNP, wherein the first type of LNP encapsulates the gene editing composition described in the second aspect.

[0221] In some embodiments, the nucleic acid-lipid nanoparticle composition comprises the first type of LNP and a gene-editing molecule, wherein the gene-editing molecule is not encapsulated in the first type of LNP. In some embodiments, the linear DNA molecule and the gene-editing molecule constitute the gene-editing composition of the second aspect described above. In some embodiments, the first type of LNP partially or entirely encapsulates the linear DNA molecule, and the first type of LNP partially or entirely encapsulates the gene-editing molecule; in some embodiments, the first type of LNP partially or entirely encapsulates Cas mRNA and / or the gRNA. In some embodiments, the first type of LNP partially or entirely encapsulates the linear DNA molecule, and the first type of LNP does not encapsulate the gene-editing molecule.

[0222] In some embodiments, the nucleic acid-lipid nanoparticle composition comprises a first type LNP and a second type LNP, wherein the first type LNP does not contain the gene editing molecule. In some embodiments, the second type LNP contains some or all of Cas mRNA and / or gRNA.

[0223] In some embodiments, the lipid moiety of the LNP comprises cationic lipids, accessory lipids, structural lipids, and amphiphilic lipids. In some embodiments, the lipid moiety of the LNP is composed of cationic lipids, accessory lipids, structural lipids, and amphiphilic lipids. In some embodiments, the lipid moiety of the LNP comprises cationic lipids, phospholipids, cholesterol or derivatives thereof, and amphiphilic lipids. In some embodiments, the lipid moiety of the LNP is composed of cationic lipids, phospholipids, cholesterol or derivatives thereof, and amphiphilic lipids.

[0224] In some embodiments, the lipid component of the LNP is:

[0225] (a) Cationic lipids, which account for about 30 mol% to 55 mol% of the total lipids in the LNP;

[0226] (b) Accessory lipids, which constitute about 5 mol% to 50 mol% of the total lipids in the LNP, for example, 10% to 45%;

[0227] (c) Structural lipids, wherein the structural lipids constitute 3 mol% to 60 mol% of the total lipids in the LNP, for example, about 20 mol% to 50 mol%; and

[0228] (d) Amphiphilic lipids, comprising approximately 0.5 mol% to 3 mol% of the total lipids in the LNP, for example, approximately 1 mol% to 3 mol%.

[0229] In some embodiments, the cationic lipid in the LNP is selected from at least one of SM102, ALC0315, DLin-MC3-DMA, C12-200 or its isomers, C14-4, 306Oi10, Lipid 5, LP-01, DOTMA, DODMA, DLin-KC2-DMA, DOTAP, and DC-Chol.

[0230] In some embodiments, the accessory lipid in the LNP is a phospholipid. In some embodiments, the phospholipid is selected from at least one of 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine (DOPE), distearylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dioleoyllecithin (DOPC), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), 1,2-bis(diphenylphosphine)ethane (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearylphosphatidylethanolamine (DSPE), and SOPE, or a lipid modified with anionic or cationic modifying groups.

[0231] In some embodiments, the structural lipids in the LNP may be selected from, but are not limited to, the group consisting of: cholesterol and its derivatives, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, rapeseed sterol, tomatidine, tomatine, ursolic acid, α-tocopherol, and mixtures thereof. In some embodiments, the structural lipid in the LNP composition is cholesterol.

[0232] In some embodiments, the amphiphilic lipid in the LNP is selected from at least one of DMG-PEG, PEG-c-DMG, PEG-C14, PEG-c-DMA, PEG-DSPE, ALC0159, PEG-PE, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, Tween-20, Tween-80, PEG-DPG, PEG-s-DMG, DAA, PEG-c-DOMG, and GalNAc-PEG-DSG. In some embodiments, the nucleic acid-lipid nanoparticle composition contains DMG-PEG. In some embodiments, the nucleic acid-lipid nanoparticle composition contains DMG-PEG2000.

[0233] In some embodiments, the linear DNA molecule is a linear double-stranded DNA molecule according to any of the first aspects described above.

[0234] In some embodiments, the cationic lipid in the first type of LNP is C12-200 or its isomer. In some embodiments, the accessory lipid in the LNP is DOPE. In some embodiments, in the first type of LNP, the cationic lipid is C12-200, the phospholipid is DOPE, the structural lipid is cholesterol, and the amphiphilic lipid is DMG-PEG. In some embodiments, in the first type of LNP, the cationic lipid is C12-200, the phospholipid is DOPE, the structural lipid is cholesterol, and the amphiphilic lipid is DMG-PEG 2000.

[0235] In some embodiments, the amphiphilic lipids comprise approximately 31 mol%-50 mol%, 32 mol%-49 mol%, 33 mol%-48 mol%, 34 mol%-47 mol%, 35 mol%-46 mol%, 36 mol%-45 mol%, 30 mol%-40 mol%, 31 mol%-39 mol%, 32 mol%-38 mol%, 33 mol%-37 mol%, 34 mol%-36 mol%, or 34.5 mol%-35.5 mol% of the total lipids in the first type of LNP.

[0236] In some embodiments, the auxiliary lipids constitute approximately 6 mol%-49 mol%, 7 mol%-47 mol%, 8 mol%-44 mol%, 9 mol%-43 mol%, 11 mol%-41 mol%, 12 mol%-38 mol%, 15 mol%-35 mol%, 16 mol%-34 mol%, 17 mol%-33 mol%, 18 mol%-32 mol%, 19 mol%-31 mol%, or 19.5 mol%-30.5 mol% of the total lipids in the first type of LNP.

[0237] In some embodiments, the structural lipids constitute approximately 4 mol%-59 mol%, 5 mol%-58 mol%, 6 mol%-57 mol%, 7 mol%-56 mol%, 10 mol%-55 mol%, 12 mol%-54 mol%, 14 mol%-53 mol%, 16 mol%-52 mol%, 18 mol%-51 mol%, 19 mol%-50 mol%, 40 mol%-49 mol%, 41 mol%-48 mol%, 42 mol%-47 mol%, 39 mol%-46 mol%, and 41 mol%-45 mol% of the total lipids in the first type of LNP. 42mol%-44mol%, 42.5mol%-43.5mol%, 41.25mol%-43.25mol%, 42.6mol%-49.9mol%, 42.7mol%-49.8mol%, 42.8mol%-49.7mol%, 42.9mol%-49. 6mol%, 43.1mol%-49.6mol%, 43.2mol%-49.5mol%, 43.21mol%-49.4mol%, 43.22mol%-49.3mol%, 43.23mol%-49.2mol%, or 43.24mol%-49.4mol%.

[0238] In some embodiments, the amphiphilic lipids account for approximately 0.5 mol%-2.95 mol%, 0.55 mol%-2.90 mol%, 0.60 mol%-2.85 mol%, 0.65 mol%-2.80 mol%, 0.70 mol%-2.75 mol%, 0.75 mol%-2.70 mol%, 0.80 mol%-2.65 mol%, 0.85 mol%-2.60 mol%, 0.90 mol%-2.55 mol%, 0.95 mol%-2.45 mol%, 1.0 mol%-2.40 mol% of the total lipids in the first type of LNP, and 1.05 mol%-2.40 mol%. mol%-2.35mol%, 1.10mol%-2.30mol%, 1.15mol%-2.25mol%, 1.20mol%-2.20mol%, 1.25mol%-2.15mol%, 1.30mol%-2.10mol%, 1.35mol%-2. 05mol%, 1.40mol%-2.00mol%, 1.45mol%-1.95mol%, 1.50mol%-1.90mol%, 1.55mol%-1.85mol%, 1.60mol%-1.80mol% or 1.65mol%-1.75mol%.

[0239] In some implementations, the lipid components in the first type of LNP are:

[0240] (a) Cationic lipids, which account for approximately 40 mol%-50 mol% of the total lipids in the first type of LNP;

[0241] (b) Helper lipids, wherein the helper lipids constitute approximately 25 mol% to 30 mol% of the total lipids in the first type of LNP;

[0242] (c) Structural lipids, wherein the structural lipids constitute approximately 20 mol%-25 mol% of the total lipids in the first type of LNP; and

[0243] (d) Amphiphilic lipids, which account for approximately 1 mol%-2 mol% of the total lipids in the first type of LNP.

[0244] In some implementations, the lipid components in the first type of LNP are:

[0245] (a) Cationic lipids, which account for approximately 35 mol% of the total lipids in the first type of LNP;

[0246] (b) Helper lipids, wherein the helper lipids constitute approximately 20 mol% of the total lipids in the first type of LNP;

[0247] (c) Structural lipids, wherein the structural lipids constitute approximately 40 mol%-45 mol% of the total lipids in the first type of LNP; and

[0248] (d) Amphiphilic lipids, which account for approximately 0.5 mol% to 2.45 mol% of the total lipids in the first type of LNP.

[0249] In some embodiments, in the nucleic acid-lipid nanoparticle composition, the lipid component of the first type of LNP consists of any one of the following:

[0250] It contains approximately 20-45 mol% cationic lipids, approximately 15-40 mol% phospholipids, approximately 23.25-58.25 mol% cholesterol or its derivatives, and approximately 1-2.5% amphiphilic lipids.

[0251] In some embodiments, in the nucleic acid-lipid nanoparticle composition, the lipid component of the first type of LNP consists of any one of the following:

[0252] It contains approximately 20-45 mol% cationic lipids, approximately 20-40 mol% phospholipids, approximately 23.25-58.25 mol% cholesterol or its derivatives, and approximately 1-2.5% amphiphilic lipids.

[0253] In some embodiments, in the nucleic acid-lipid nanoparticle composition, the lipid component of the first type of LNP consists of any one of the following:

[0254] Approximately 20 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 58.25 mol% cholesterol or its derivatives, and approximately 1.75% amphiphilic lipids;

[0255] Approximately 25 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 53.25 mol% cholesterol or its derivatives, and approximately 1.75% amphiphilic lipids;

[0256] Approximately 30 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 48.25 mol% cholesterol or its derivatives, and approximately 1.75% amphiphilic lipids;

[0257] Approximately 35-55 mol% cationic lipids, approximately 20-40 mol% phospholipids, approximately 4-44 mol% cholesterol or its derivatives, and approximately 1-3 mol% amphiphilic lipids;

[0258] Approximately 35 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 44 mol% cholesterol or its derivatives, and approximately 1 mol% amphiphilic lipids;

[0259] Approximately 35 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 44.75 mol% cholesterol or its derivatives, and approximately 1.25% amphiphilic lipids;

[0260] Approximately 35 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 43.5 mol% cholesterol or its derivatives, and approximately 1.5% amphiphilic lipids;

[0261] Approximately 35 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 43.75 mol% cholesterol or its derivatives, and approximately 1.25% amphiphilic lipids;

[0262] Approximately 35 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 43.25 mol% cholesterol or its derivatives, and approximately 1.75% amphiphilic lipids;

[0263] Approximately 35 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 43 mol% cholesterol or its derivatives, and approximately 2 mol% amphiphilic lipids;

[0264] Approximately 35 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 42 mol% cholesterol or its derivatives, and approximately 3 mol% amphiphilic lipids;

[0265] Approximately 35 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 42.5 mol% cholesterol or its derivatives, and approximately 2.5 mol% amphiphilic lipids;

[0266] Approximately 35 mol% cationic lipids, approximately 15 mol% phospholipids, approximately 48.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% amphiphilic lipids;

[0267] Approximately 35 mol% cationic lipids, approximately 25 mol% phospholipids, approximately 38.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% amphiphilic lipids;

[0268] Approximately 35 mol% cationic lipids, approximately 30 mol% phospholipids, approximately 33.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% amphiphilic lipids;

[0269] Approximately 35 mol% cationic lipids, approximately 35 mol% phospholipids, approximately 28.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% amphiphilic lipids;

[0270] Approximately 35 mol% cationic lipids, approximately 40 mol% phospholipids, approximately 23.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% amphiphilic lipids;

[0271] Approximately 40 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 38.25 mol% cholesterol or its derivatives, and approximately 1.75% amphiphilic lipids;

[0272] Approximately 45 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 33.25 mol% cholesterol or its derivatives, and approximately 1.75% amphiphilic lipids;

[0273] Approximately 45 mol% cationic lipids, approximately 40 mol% phospholipids, approximately 14 mol% cholesterol or its derivatives, and approximately 1 mol% amphiphilic lipids;

[0274] Approximately 55 mol% cationic lipids, approximately 40 mol% phospholipids, approximately 4 mol% cholesterol or its derivatives, and approximately 1 mol% amphiphilic lipids; and

[0275] It contains approximately 46 mol% cationic lipids, approximately 28 mol% phospholipids, approximately 24 mol% cholesterol or its derivatives, and approximately 2 mol% amphiphilic lipids.

[0276] In some embodiments, the nucleic acid-lipid nanoparticle composition contains a cationic lipid of C12-200 or a derivative thereof, a phospholipid of DOPE, and / or an amphiphilic lipid of DMG-PEG or DMG-PEG 2000.

[0277] In some embodiments, the nucleic acid-lipid nanoparticle composition contains a cationic lipid of C12-200 or a derivative thereof, a phospholipid of DOPE, and an amphiphilic lipid of DMG-PEG or DMG-PEG 2000.

[0278] In some embodiments, in the nucleic acid-lipid nanoparticle composition, the lipid component of the first type of LNP consists of any one of the following:

[0279] Approximately 20-45 mol% C12-200 or its derivatives, approximately 15-40 mol% phospholipids, approximately 23.25-58.25 mol% cholesterol or its derivatives, and approximately 1-2.5% PEG.

[0280] In some embodiments, in the nucleic acid-lipid nanoparticle composition, the lipid component of the first type of LNP consists of any one of the following:

[0281] Approximately 20-45 mol% C12-200 or its derivatives, approximately 20-40 mol% phospholipids, approximately 23.25-58.25 mol% cholesterol or its derivatives, and approximately 1-2.5% PEG.

[0282] In some embodiments, in the nucleic acid-lipid nanoparticle composition, the lipid component of the first type of LNP consists of any one of the following:

[0283] Approximately 20-45 mol% C12-200 or its derivatives, approximately 15-40 mol% phospholipids, approximately 23.25-58.25 mol% cholesterol or its derivatives, and approximately 1-2.5% PEG-2000.

[0284] In some embodiments, in the nucleic acid-lipid nanoparticle composition, the lipid component of the first type of LNP consists of any one of the following:

[0285] Approximately 20-45 mol% C12-200 or its derivatives, approximately 20-40 mol% phospholipids, approximately 23.25-58.25 mol% cholesterol or its derivatives, and approximately 1-2.5% PEG-2000.

[0286] In some embodiments, in the nucleic acid-lipid nanoparticle composition, the lipid component of the first type of LNP consists of any one of the following:

[0287] Approximately 20-45 mol% C12-200 or its derivatives, approximately 15-40 mol% phospholipids, approximately 23.25-58.25 mol% cholesterol or its derivatives, and approximately 1-2.5% DMG-PEG.

[0288] In some embodiments, in the nucleic acid-lipid nanoparticle composition, the lipid component of the first type of LNP consists of any one of the following:

[0289] Approximately 20-45 mol% C12-200 or its derivatives, approximately 20-40 mol% phospholipids, approximately 23.25-58.25 mol% cholesterol or its derivatives, and approximately 1-2.5% DMG-PEG. In some embodiments, in the nucleic acid-lipid nanoparticle composition, the lipid component of the first type of LNP consists of any one of the following:

[0290] Approximately 20-45 mol% C12-200, approximately 15-40 mol% DOPE, approximately 23.25-58.25 mol% cholesterol or its derivatives, and approximately 1-2.5% DMG-PEG or DMG-PEG 2000;

[0291] Approximately 20-45 mol% C12-200 or its derivatives, approximately 20-40 mol% DOPE, approximately 23.25-58.25 mol% cholesterol or its derivatives, and approximately 1-2.5% DMG-PEG 2000; approximately 35-55 mol% C12-200, approximately 20-40 mol% DOPE, approximately 4-44 mol% cholesterol or its derivatives, and approximately 1-3 mol% DMG-PEG or DMG-PEG 2000; approximately 35 mol% C12-200, approximately 20 mol% DOPE, approximately 44 mol% cholesterol or its derivatives, and approximately 1 mol% DMG-PEG or DMG-PEG 2000;

[0292] Approximately 35 mol% C12-200, approximately 20 mol% DOPE, approximately 43 mol% cholesterol or its derivatives, and approximately 2 mol% DMG-PEG or DMG-PEG 2000;

[0293] Approximately 35 mol% C12-200, approximately 20 mol% DOPE, approximately 42 mol% cholesterol or its derivatives, and approximately 3 mol% DMG-PEG or DMG-PEG 2000;

[0294] Approximately 45 mol% C12-200, approximately 40 mol% DOPE, approximately 14 mol% cholesterol or its derivatives, and approximately 1 mol% DMG-PEG or DMG-PEG 2000;

[0295] Approximately 55 mol% C12-200, approximately 40 mol% DOPE, approximately 4 mol% cholesterol or its derivatives, and approximately 1 mol% DMG-PEG or DMG-PEG 2000; and

[0296] Approximately 46 mol% C12-200, approximately 28 mol% DOPE, approximately 24 mol% cholesterol or its derivatives, and approximately 2 mol% DMG-PEG or DMG-PEG 2000.

[0297] In some embodiments, in the nucleic acid-lipid nanoparticle composition, the lipid component of the first type of LNP consists of any one of the following:

[0298] Approximately 20 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 58.25 mol% cholesterol or its derivatives, and approximately 1.75% amphiphilic lipids;

[0299] Approximately 25 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 53.25 mol% cholesterol or its derivatives, and approximately 1.75% amphiphilic lipids;

[0300] Approximately 30 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 48.25 mol% cholesterol or its derivatives, and approximately 1.75% amphiphilic lipids;

[0301] Approximately 40 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 38.25 mol% cholesterol or its derivatives, and approximately 1.75% amphiphilic lipids;

[0302] Approximately 45 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 33.25 mol% cholesterol or its derivatives, and approximately 1.75% amphiphilic lipids;

[0303] Approximately 35 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 44 mol% cholesterol or its derivatives, and approximately 1% amphiphilic lipids;

[0304] Approximately 35 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 44.75 mol% cholesterol or its derivatives, and approximately 1.25% amphiphilic lipids;

[0305] Approximately 35 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 43.5 mol% cholesterol or its derivatives, and approximately 1.5% amphiphilic lipids;

[0306] Approximately 35 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 43.25 mol% cholesterol or its derivatives, and approximately 1.75% amphiphilic lipids;

[0307] Approximately 35 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 43 mol% cholesterol or its derivatives, and approximately 2 mol% amphiphilic lipids;

[0308] Approximately 35 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 42 mol% cholesterol or its derivatives, and approximately 3 mol% amphiphilic lipids;

[0309] Approximately 35 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 42.5 mol% cholesterol or its derivatives, and approximately 2.5 mol% amphiphilic lipids;

[0310] Approximately 35 mol% C12-200 or its derivatives, approximately 15 mol% phospholipids, approximately 48.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% amphiphilic lipids;

[0311] Approximately 35 mol% C12-200 or its derivatives, approximately 25 mol% phospholipids, approximately 38.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% amphiphilic lipids;

[0312] Approximately 35 mol% C12-200 or its derivatives, approximately 30 mol% phospholipids, approximately 33.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% amphiphilic lipids;

[0313] Approximately 35 mol% C12-200 or its derivatives, approximately 35 mol% phospholipids, approximately 28.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% amphiphilic lipids;

[0314] Approximately 35 mol% C12-200 or its derivatives, approximately 40 mol% phospholipids, approximately 23.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% amphiphilic lipids;

[0315] Approximately 45 mol% C12-200 or its derivatives, approximately 40 mol% phospholipids, approximately 14 mol% cholesterol or its derivatives, and approximately 1 mol% amphiphilic lipids; or

[0316] Approximately 55 mol% C12-200 or its derivatives, approximately 40 mol% phospholipids, approximately 4 mol% cholesterol or its derivatives, and approximately 1 mol% amphiphilic lipids.

[0317] In some embodiments, in the nucleic acid-lipid nanoparticle composition, the lipid component of the first type of LNP consists of any one of the following:

[0318] Approximately 20 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 58.25 mol% cholesterol or its derivatives, and approximately 1.75% PEG2000;

[0319] Approximately 25 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 53.25 mol% cholesterol or its derivatives, and approximately 1.75% PEG2000;

[0320] Approximately 30 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 48.25 mol% cholesterol or its derivatives, and approximately 1.75% PEG2000;

[0321] Approximately 40 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 38.25 mol% cholesterol or its derivatives, and approximately 1.75% PEG2000;

[0322] Approximately 45 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 33.25 mol% cholesterol or its derivatives, and approximately 1.75% PEG2000;

[0323] Approximately 35 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 44 mol% cholesterol or its derivatives, and approximately 1% PEG2000;

[0324] Approximately 35 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 44.75 mol% cholesterol or its derivatives, and approximately 1.25% PEG2000;

[0325] Approximately 35 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 43.5 mol% cholesterol or its derivatives, and approximately 1.5% PEG2000;

[0326] Approximately 35 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 43.25 mol% cholesterol or its derivatives, and approximately 1.75% PEG2000;

[0327] Approximately 35 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 43 mol% cholesterol or its derivatives, and approximately 2 mol% PEG2000;

[0328] Approximately 35 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 42 mol% cholesterol or its derivatives, and approximately 3 mol% PEG2000;

[0329] Approximately 35 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 42.5 mol% cholesterol or its derivatives, and approximately 2.5 mol% PEG2000;

[0330] Approximately 35 mol% C12-200 or its derivatives, approximately 15 mol% phospholipids, approximately 48.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% PEG2000;

[0331] Approximately 35 mol% C12-200 or its derivatives, approximately 25 mol% phospholipids, approximately 38.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% PEG2000;

[0332] Approximately 35 mol% C12-200 or its derivatives, approximately 30 mol% phospholipids, approximately 33.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% PEG2000;

[0333] Approximately 35 mol% C12-200 or its derivatives, approximately 35 mol% phospholipids, approximately 28.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% PEG2000;

[0334] Approximately 35 mol% C12-200 or its derivatives, approximately 40 mol% phospholipids, approximately 23.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% PEG2000;

[0335] Approximately 45 mol% C12-200 or its derivatives, approximately 40 mol% phospholipids, approximately 14 mol% cholesterol or its derivatives, and approximately 1 mol% PEG2000; or

[0336] Approximately 55 mol% C12-200 or its derivatives, approximately 40 mol% phospholipids, approximately 4 mol% cholesterol or its derivatives, and approximately 1 mol% PEG2000.

[0337] In some embodiments, in the nucleic acid-lipid nanoparticle composition, the lipid component of the first type of LNP consists of any one of the following:

[0338] Approximately 20 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 58.25 mol% cholesterol or its derivatives, and approximately 1.75% DMG-PEG;

[0339] Approximately 25 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 53.25 mol% cholesterol or its derivatives, and approximately 1.75% DMG-PEG;

[0340] Approximately 30 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 48.25 mol% cholesterol or its derivatives, and approximately 1.75% DMG-PEG;

[0341] Approximately 40 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 38.25 mol% cholesterol or its derivatives, and approximately 1.75% DMG-PEG;

[0342] Approximately 45 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 33.25 mol% cholesterol or its derivatives, and approximately 1.75% DMG-PEG;

[0343] Approximately 35 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 44 mol% cholesterol or its derivatives, and approximately 1% DMG-PEG;

[0344] Approximately 35 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 44.75 mol% cholesterol or its derivatives, and approximately 1.25% DMG-PEG;

[0345] Approximately 35 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 43.5 mol% cholesterol or its derivatives, and approximately 1.5% DMG-PEG;

[0346] Approximately 35 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 43.25 mol% cholesterol or its derivatives, and approximately 1.75% DMG-PEG;

[0347] Approximately 35 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 43 mol% cholesterol or its derivatives, and approximately 2 mol% DMG-PEG;

[0348] Approximately 35 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 42 mol% cholesterol or its derivatives, and approximately 3 mol% DMG-PEG;

[0349] Approximately 35 mol% C12-200 or its derivatives, approximately 20 mol% phospholipids, approximately 42.5 mol% cholesterol or its derivatives, and approximately 2.5 mol% DMG-PEG;

[0350] Approximately 35 mol% C12-200 or its derivatives, approximately 15 mol% phospholipids, approximately 48.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% DMG-PEG;

[0351] Approximately 35 mol% C12-200 or its derivatives, approximately 25 mol% phospholipids, approximately 38.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% DMG-PEG;

[0352] Approximately 35 mol% C12-200 or its derivatives, approximately 30 mol% phospholipids, approximately 33.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% DMG-PEG;

[0353] Approximately 35 mol% C12-200 or its derivatives, approximately 35 mol% phospholipids, approximately 28.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% DMG-PEG;

[0354] Approximately 35 mol% C12-200 or its derivatives, approximately 40 mol% phospholipids, approximately 23.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% DMG-PEG;

[0355] Approximately 45 mol% C12-200 or its derivatives, approximately 40 mol% phospholipids, approximately 14 mol% cholesterol or its derivatives, and approximately 1 mol% DMG-PEG; or

[0356] Approximately 55 mol% C12-200 or its derivatives, approximately 40 mol% phospholipids, approximately 4 mol% cholesterol or its derivatives, and approximately 1 mol% DMG-PEG.

[0357] In some embodiments, the cationic lipid in the second type of LNP is SM102. In some embodiments, the accessory lipid in the LNP is a phospholipid. In some embodiments, the accessory lipid in the LNP is DSPC. In some embodiments, in the second type of LNP, the cationic lipid is SM102, the accessory lipid is DSPC, the structural lipid is cholesterol, and the amphiphilic lipid is DMG-PEG. In some embodiments, in the first type of LNP, the cationic lipid is SM102, the phospholipid is DSPC, the structural lipid is cholesterol, and the amphiphilic lipid is DMG-PEG 2000.

[0358] In some embodiments, the lipid composition of the second type of LNP is as follows: approximately 45-50 mol% cationic lipids, approximately 5-15 mol% accessory lipids, approximately 33-42 mol% structural lipids, and approximately 1-2 mol% amphiphilic lipids. In some embodiments, the lipid composition of the second type of LNP is as follows: approximately 50 mol% cationic lipids, approximately 10 mol% accessory lipids, approximately 38.5 mol% structural lipids, and approximately 1.5 mol% amphiphilic lipids. In some embodiments, the lipid composition of the second type of LNP is as follows: approximately 50 mol% SM-102, approximately 10 mol% DSPC, approximately 38.5 mol% cholesterol, and approximately 1.5 mol% DMG-PEG.

[0359] In some embodiments, in the nucleic acid-lipid nanoparticle composition, the average particle size of the LNP particles is about 50 nm to about 150 nm; optionally, about 70 nm to about 120 nm; further optionally, about 80-120 nm; and most preferably, about 100 nm. In some embodiments, in the nucleic acid-lipid nanoparticle composition, the average particle size of the first type of LNP particles is about 50 nm to about 150 nm; optionally, about 70 nm to about 120 nm; further optionally, about 80-120 nm; and most preferably, about 100 nm. In some embodiments, in the nucleic acid-lipid nanoparticle composition, the average particle size of the LNP particles is about 70 nm to 140 nm. In some embodiments, the average particle size of the LNP particles in the nucleic acid-lipid nanoparticle composition is approximately 70 nm, 71 nm, 72 nm, 73 nm, 74 nm, 75 nm, 76 nm, 77 nm, 78 nm, 79 nm, 80 nm, 81 nm, 82 nm, 83 nm, 84 nm, 85 nm, 86 nm, 87 nm, 88 nm, 89 nm, 90 nm, 91 nm, 92 nm, 93 nm, 94 nm, 95 nm, 96 nm, 97 nm, 98 nm, 99 nm, 100 nm, 101 nm, 102 nm, 103 nm, or 104 nm. m, 105nm, 106nm, 107nm, 108nm, 109nm, 110nm, 111nm, 112nm, 113nm, 114nm, 115nm, 116nm, 117nm, 118nm, 119nm, 120nm, 121nm, 122nm , 123nm, 124nm, 125nm, 126nm, 127nm, 128nm, 129nm, 130nm, 131nm, 132nm, 133nm, 134nm, 135nm, 136nm, 137nm, 138nm, 139nm or 140nm.

[0360] In some embodiments, the nucleic acid-lipid nanoparticle composition further comprises an external phase buffer having a pH of about 7-9, for example, about 7-8, optionally about 7.5. In some embodiments, the external phase buffer comprises tromethamine (Tris) and / or sodium acetate (NaOAc). In some embodiments, the external phase buffer is a tromethamine (Tris)-sodium acetate (NaOAc) buffer. In some embodiments, the external phase buffer contains sucrose. In some embodiments, the external phase buffer comprises tromethamine (Tris), sodium acetate (NaOAc), and sucrose. In some embodiments, the nucleic acid-lipid nanoparticle composition further comprises an external phase buffer comprising tromethamine (Tris), sodium acetate (NaOAc), and sucrose, wherein the pH of the external phase buffer is about 7-8, optionally about 7.5.

[0361] In some embodiments, the Tris content in the outer phase buffer of the nucleic acid-lipid nanoparticle composition is about 10-30 mmol / L, optionally about 15-25 mmol / L, 15 mmol / L, 15.5 mmol / L, 16 mmol / L, 16.5 mmol / L, 17 mmol / L, 17.5 mmol / L, 18 mmol / L, 18.5 mmol / L, 19 mmol / L, 19.5 mmol / L, 20 mmol / L, 20.5 mmol / L, 21 mmol / L, 21.5 mmol / L, 22 mmol / L, 22.5 mmol / L, 23 mmol / L, 23.5 mmol / L, 24 mmol / L, 24.5 mmol / L, or 25 mmol / L, most preferably about 20 mmol / L.

[0362] In some embodiments, the NaOAc content in the outer phase buffer of the nucleic acid-lipid nanoparticle composition is selected from about 0-20 mmol / L, optionally about 5-11 mmol / L, 5 mmol / L, 5.5 mmol / L, 6 mmol / L, 6.5 mmol / L, 7 mmol / L, 7.5 mmol / L, 8 mmol / L, 8.5 mmol / L, 9 mmol / L, 9.5 mmol / L, 10 mmol / L, 10.5 mmol / L, 10.6 mmol / L, 10.7 mmol / L, 10.8 mmol / L, 10.9 mmol / L, 11 mmol / L, 11.5 mmol / L, 12 mmol / L, 12.5 mmol / L or 13 mmol / L, most preferably about 10.7 mmol / L.

[0363] In some embodiments, the concentration (w / v) of sucrose in the outer phase buffer of the nucleic acid-lipid nanoparticle composition is selected from about 5-15%, optionally about 7.5-10%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.8%, 8.9%, 9%, 9.5%, or 10%, most preferably about 8.7%.

[0364] In some embodiments, the Tris content in the external phase buffer is approximately 10-30 mmol / L, optionally approximately 15-25 mmol / L, 15 mmol / L, 15.5 mmol / L, 16 mmol / L, 16.5 mmol / L, 17 mmol / L, 17.5 mmol / L, 18 mmol / L, 18.5 mmol / L, 19 mmol / L, 19.5 mmol / L, 20 mmol / L, 20.5 mmol / L, 21 mmol / L, 21.5 mmol / L, 22 mmol / L, 22.5 mmol / L, 23 mmol / L, 23.5 mmol / L, 24 mmol / L, 24.5 mmol / L, or 25 mmol / L, most preferably approximately... 20 mmol / L; and the content of NaOAc is selected from about 0-20 mmol / L, optionally about 5-11 mmol / L, 5 mmol / L, 5.5 mmol / L, 6 mmol / L, 6.5 mmol / L, 7 mmol / L, 7.5 mmol / L, 8 mmol / L, 8.5 mmol / L, 9 mmol / L, 9.5 mmol / L, 10 mmol / L, 10.5 mmol / L, 10.6 mmol / L, 10.7 mmol / L, 10.8 mmol / L, 10.9 mmol / L, 11 mmol / L, 11.5 mmol / L, 12 mmol / L, 12.5 mmol / L or 13 mmol / L, most preferably about 10.7 mmol / L.

[0365] In some embodiments, the nucleic acid-lipid nanoparticle composition includes an external phase buffer comprising: tromethamine (Tris), sodium acetate (NaOAc), and / or sucrose, wherein the Tris content is approximately 10-30 mmol / L, optionally approximately 15-25 mmol / L, 15 mmol / L, 15.5 mmol / L, 16 mmol / L, 16.5 mmol / L, 17 mmol / L, 17.5 mmol / L, 18 mmol / L, 18.5 mmol / L, 19 mmol / L, 19.5 mmol / L, 20 mmol / L, 20.5 mmol / L, 21 mmol / L, 21.5 mmol / L, 22 mmol / L, 22.5 mmol / L, 23 mmol / L, 23.5 mmol / L, 24 mmol / L, 24.5 mmol / L, or 25 mmol / L, most preferably approximately 20 mmol / L;

[0366] The NaOAc content is selected from about 0-20 mmol / L, optionally about 5-11 mmol / L, 5 mmol / L, 5.5 mmol / L, 6 mmol / L, 6.5 mmol / L, 7 mmol / L, 7.5 mmol / L, 8 mmol / L, 8.5 mmol / L, 9 mmol / L, 9.5 mmol / L, 10 mmol / L, 10.5 mmol / L, 10.6 mmol / L, 10.7 mmol / L, 10.8 mmol / L, 10.9 mmol / L, 11 mmol / L, 11.5 mmol / L, 12 mmol / L, 12.5 mmol / L or 13 mmol / L, most preferably about 10.7 mmol / L;

[0367] The concentration (w / v) of the sucrose is selected from about 5-15%, optionally about 7.5-10%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.8%, 8.9%, 9%, 9.5% or 10%, most preferably about 8.7%.

[0368] In some embodiments, the nucleic acid-lipid nanoparticle composition includes an external phase buffer comprising: tromethamine (Tris), sodium acetate (NaOAc), and sucrose, wherein the Tris content is about 15-25 mmol / L, the NaOAc content is about 10-11 mmol / L, and the sucrose concentration (w / v) is about 8-9%.

[0369] In some embodiments, the nucleic acid-lipid nanoparticle composition includes an external phase buffer comprising: tromethamine (Tris), sodium acetate (NaOAc), and sucrose, wherein the Tris content is about 20 mmol / L, the NaOAc content is about 10.7 mmol / L, and the sucrose concentration (w / v) is about 8.7%.

[0370] In some embodiments, the nitrogen-to-phosphorus ratio of the LNPs to the nucleic acid they encapsulate in the nucleic acid-lipid nanoparticle composition is about 3:1 to 32:1; optionally, it is about 3:1 to 24:1, about 6:1 to 18:1, about 8:1 to 20:1, about 10:1 to 16:1, about 16:1 to 32:1, or about 10:1 to 32:1. In some embodiments, the nitrogen-to-phosphorus ratio of the first type of LNPs to the nucleic acid they encapsulate in the nucleic acid-lipid nanoparticle composition is about 3:1 to 32:1; optionally, it is about 3:1 to 24:1, about 6:1 to 18:1, about 8:1 to 20:1, about 16:1 to 32:1, about 10:1 to 32:1, or about 16:1. In some embodiments, the nitrogen-to-phosphorus ratio of the first type of LNPs to the nucleic acid they encapsulate in the nucleic acid-lipid nanoparticle composition is about 10:1 to 20:1. In some embodiments, the nitrogen-to-phosphorus ratio of the first type of LNP to the nucleic acid it encapsulates in the nucleic acid-lipid nanoparticle composition is about 16:1. In some embodiments, the nitrogen-to-phosphorus ratio of the second type of LNP to the nucleic acid it encapsulates in the nucleic acid-lipid nanoparticle composition is about 3:1 to 32:1; optionally, about 3:1 to 24:1, for example about 6:1 to 18:1, about 10:1 to 20:1, about 16:1 to 32:1, about 10:1 to 32:1, or about 16:1.

[0371] Furthermore, this application also provides a method for introducing a target nucleic acid sequence into a cell, the target nucleic acid sequence being inserted into the genome of the cell and / or expressed in the cell, the method comprising contacting the cell with the nucleic acid-lipid nanoparticle composition of the third aspect, the gene editing composition of the second aspect, or the linear DNA molecule of the first aspect, wherein the target nucleic acid sequence is located in the linear DNA.

[0372] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is an animal cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a blood cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is selected from one or more of the following: lymphocytes, dendritic cells, monocytes, macrophages, granulocytes, and mast cells. In some embodiments, the cell is a T cell, NK cell, and / or B cell. In some embodiments, it is a stem cell, progenitor cell, or precursor cell. In some embodiments, the cell is an activated primary T cell, optionally, the primary T cell is a human primary T cell.

[0373] In some embodiments, the target gene is inserted into the T cell receptor α chain (TRAC) or interleukin-2 receptor α (IL2RA) gene sequence of the primary cells. In some embodiments, the target nucleic acid sequence encodes a chimeric antigen receptor (CAR).

[0374] In addition, this application also provides the use of the linear DNA molecule of the first aspect, the gene editing composition of the second aspect, or the nucleic acid-lipid nanoparticle composition of the third aspect in the preparation of a medicament for treating diseases, wherein the diseases are selected from: genetic diseases, tumors, autoimmune diseases, and other diseases that require treatment by regulating gene expression.

[0375] It should be understood that the aspects and embodiments of this application described herein include those that are "comprising," "composed of," and "substantially constituted by." The alternative embodiments of this application have been described in detail above; however, this application is not limited thereto. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solution of this application, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in this application and are all within the protection scope of this application. Attached Figure Description

[0376] Figures 1A-1C: Relationship between the physicochemical properties of LNPs and different lipid components and their ratios. The LNPs contain a plasmid expressing GFP (pGC_hGAPDH_GFP DNA).

[0377] Figures 2A-2C: Relationship between transfection efficiency of Jurkat cells via LNP-delivered plasmid and different lipid group allocation ratios. The LNP carries plasmid DNA expressing GFP (pGC_hGAPDH_GFP DNA). Transfection efficiency was statistically analyzed by flow cytometry to detect GFP reporter gene expression.

[0378] Figure 3: First round of LNP delivery prescription screening. Jurkat cells were transfected with plasmids expressing GFP loaded with different LNPs, and the positive rate of GFP transfection was counted by flow cytometry (pGC_hGAPDH_GFP plasmid DNA).

[0379] Figure 4: LNP prescriptions containing DNA transfected into human primary T cells after the first round of screening. One LNP prescription contained DNA expressing GFP, and another SM102 LNP contained mRNA and sgRNA encoding Cas9, which were knocked into the IL2RA gene at a specific site. The knock-in efficiency was calculated based on the expression of the GFP reporter gene. (SpCas9-Mut-5 mRNA; sgRNA hIL2RA; DNA: hIL2RA_GFP_HA, linear double strand).

[0380] Figure 5: Second round of LNP delivery formulation screening. Different LNPs carrying DNA expressing GFP were transfected into human primary T cells. Another fixed LNP formulation, SM102, delivered mRNA and sgRNA encoding Cas9, which were knocked into the IL2RA gene at a specific site. The knock-in efficiency was calculated based on the expression of the GFP reporter gene detected by flow cytometry (SpCas9-Mut-5mRNA; sgRNA hIL2RA; DNA: hIL2RA_GFP_HA, linear double strand).

[0381] Figures 6A-6B: Cell viability (Figure 6A) and site knock-in efficiency (Figure 6B) after electroporation with different types of DNA. Human primary T cells were simultaneously electroporated with a mixture of GFP-expressing DNA template, Cas9-encoding mRNA, and sgRNA. The IL2RA gene was knocked into the plasmid template and linear DNA template expressing GFP. Flow cytometry was used to analyze GFP positivity and cell viability. Electroporation conditions were as follows: 150V voltage, 1200µs electroporation time, 3 electroporations, with an interval of 635ms between each electroporation. 20µg of nucleic acid was added to 3E6 T cells, with a mRNA:sgRNA:DNA ratio of 1:1:3 (w / w). Cell viability (Figure 6A) and GFP positivity (Figure 6B) were measured after electroporation. UNT was the untransfected control group. (SpCas9-Mut-5 mRNA; sgRNA hIL2RA; plasmid DNA: pGC_hIL2RA_GFP DNA, linear double-stranded DNA: hIL2RA_GFP_HA).

[0382] Figure 7: Effect of DNA templates carrying different nuclear sequences on site knock-in efficiency; statistical graph of GFP knock-in efficiency detected by flow cytometry. A dual LNP delivery system was used: SM102 LNPs carrying mRNA / sgRNA (SpCas9-Mut-5 mRNA; sgRNA hIL2RA), and A20 LNP formulations (C12-200 LNPs (A20)) carrying DNA. All DNA templates used in this experiment were linear double-stranded templates (see Table 8). Site knock-in was performed on the IL2RA gene in primary human T cells. UNT served as the untransfected control group. Knock-in efficiency was statistically analyzed based on the expression of the GFP reporter gene detected by flow cytometry.

[0383] Figure 8: Effect of different forms of double-stranded DNA templates on site knock-in efficiency. A dual LNP delivery system was used: SM102 LNPs carrying mRNA / sgRNA (SpCas9-Mut-5 mRNA; sgRNA hIL2RA), C12-A20 LNPs (C12-200 LNPs (A20)) carrying template DNA, N2 DNA containing the linear DNA template hIL2RA_GFP_NFAT, N2-Loop DNA containing the paired-end blocked DNA template hIL2RA_GFP_N2_Loop, and NLS-Loop DNA containing the NLS-modified paired-end blocked DNA template hIL2RA_GFP_NLS_Loop (see Table 8 for details). These were used for site knock-in into the IL2RA gene in primary human T cells. UNT represented the untransfected control group. Knock-in efficiency was calculated based on flow cytometry analysis of GFP reporter gene expression.

[0384] Figure 9: Site-directed knock-in efficiency using paired-end blocked DNA as a template is superior to that using plasmid templates. The experiment used a dual LNP delivery system: SM102 LNPs carrying mRNA and sgRNA (SpCas9-Mut-5 mRNA; sgRNA hIL2RA), and C12-B2 LNPs (C12-200 LNP(B2), i.e., LNP formulation numbered B2 in Table 2) carrying template DNA. The plasmid DNA was pGC_hIL2RA_GFP DNA, and the loop DNA was paired-end blocked DNA hIL2RA_GFP_Loop (see Table 8 for details). This was used for site-directed knock-in into the IL2RA gene in primary human T cells. UNT was the untransfected control group. Knock-in efficiency was calculated based on the expression of the GFP reporter gene detected by flow cytometry. In Figure 9, "B2" on the horizontal axis refers to LNP formulation numbered "B2" in Table 2; "C12" refers to the "C12-200 LNP (control)" formulation in Table 1.

[0385] Figure 10: Site-specific knock-in efficiency of different double-stranded DNA templates. A dual LNP delivery system was used. SM102 LNPs carried mRNA / sgRNA (SpCas9-Mut-5 mRNA; sgRNA hIL2RA), and B2 LNPs were C12-200 LNPs (B2) carrying template DNA. TF DNA was the linear DNA template hIL2RA_GFP_TF, ITR DNA was AAV2 ITR double-ended DNA hIL2RA_GFP_ITR, Loop DNA was Loop double-ended DNA hIL2RA_GFP_Loop (see Table 8 for details), and UNT was the untransfected control group. These templates were knocked into the IL2RA gene in primary human T cells. The knock-in efficiency was calculated based on the expression of the GFP reporter gene detected by flow cytometry.

[0386] Figure 11: Site-specific knock-in efficiency of linear single-stranded and paired-end blocked DNA templates. A dual LNP delivery system was used. SM102 LNPs carried mRNA / sgRNA (SpCas9-Mut-5 mRNA; sgRNA hTRAC), and B2 LNPs (C12-200 LNPs (B2)) carried template DNA. Loop DNA was paired-end blocked DNA hTRAC_GFP_Loop (see Table 8 for details), single-stranded DNA was CTS-hTRAC-GFP, and CTS single-stranded DNA was obtained by annealing CTS-hTRAC-GFP with CTS oligo. These templates were knocked into the TRAC gene in primary human T cells. The knock-in efficiency was calculated based on the expression of the GFP reporter gene detected by flow cytometry. UNT represented the untransfected control group.

[0387] Figure 12: Site knock-in efficiency mediated by different LNPs. SM102 LNPs carried three components: mRNA / sgRNA / DNA (SpCas9-Mut-5 mRNA; sgRNA hIL2RA; DNA pGC_hIL2RA_GFP), while C12 LNPs carried DNA (pGC_hIL2RA_GFP). These were used for site knock-in into the IL2RA gene in Jurkat cells. UNT represented the untransfected control group. Knock-in efficiency was calculated based on flow cytometry analysis of GFP reporter gene expression.

[0388] Figures 13A-13B: Comparison of site-specific knock-in efficiency mediated by single-LNP and dual-LNP delivery systems. Figure 13A: Site-specific knock-in of template DNA into the IL2RA gene in human primary T cells mediated by single-LNP and dual-LNP delivery systems. The single-LNP system-SM102 consists of SM102 LNPs carrying mRNA / sgRNA / DNA tricomponents; the single-LNP system-C12 consists of C12-200 LNPs (control) carrying mRNA / sgRNA / DNA tricomponents; the dual-LNP system consists of SM102 LNPs carrying mRNA / sgRNA, and C12-200 LNPs (control) carrying DNA (SpCas9-Mut-5 mRNA; sgRNA hIL2RA; hIL2RA_GFP_HA DNA). Figure 13B: Site-specific knock-in of template DNA into the IL2RA or TRAC gene in human primary T cells mediated by single-LNP and dual-LNP delivery systems. The single LNP system - C12 B2 consists of a C12-200 LNP (B2) carrying a three-component structure of mRNA / sgRNA / DNA; the dual LNP system consists of an SM102 LNP carrying mRNA / sgRNA and a C12-200 LNP (B2) carrying DNA. mRNA: SpCas9-Mut-5 mRNA; IL2RA knock-in: sgRNA hIL2RA; template DNA is loop-end-blocked DNA hIL2RA_GFP_Loop (see Table 8). TRAC knock-in: sgRNA hTRAC; template DNA is loop-end-blocked DNA hTRAC_GFP_Loop (see Table 8). Knock-in efficiency was calculated based on flow cytometry analysis of GFP reporter gene expression. UNT represents the untransfected control group.

[0389] Figure 14: Effect of different Cas9 fusion protein mRNAs on DNA knock-in efficiency. A dual LNP delivery system was used. SM102 LNPs carried mRNA / sgRNA (Cas9 Mut-5 was SpCas9-Mut-5 mRNA (abbreviated as mut5 in the x-axis of Figure 14); DrFECO Cas9 was DrFECO-Cas9-Mut-5 mRNA (abbreviated as DrFECO in the x-axis of Figure 14); EcFECO Cas9 was EcFECO-Cas9-Mut-5 mRNA (abbreviated as EcFECO in the x-axis of Figure 14); sgRNA hIL2RA). B2 LNPs carried template DNA: TF DNA was the linear DNA template hIL2RA_GFP_TF, and Loop DNA was the loop-end-closed DNA hIL2RA_GFP_Loop (see Table 8 for details). These templates were knocked into the IL2RA gene in human primary T cells. UNT was the untransfected control group. The knock-in efficiency was calculated based on the expression of the GFP reporter gene detected by flow cytometry.

[0390] Figure 15: Design of inserted sequences in homologous recombination DNA templates. DNA templates can be divided into two types: those using endogenous gene promoters (top) and those using exogenous promoters (bottom). Both templates contain homologous arms. Templates using endogenous promoters contain a 2A cleavage peptide sequence (2A), while templates using exogenous promoters contain a polyA tailing signal (pA).

[0391] Figure 16A: Schematic diagram of different DNA template structures. As shown in the figure, nuclear insertion sequences are added to both ends of the DNA insertion gene template, and circular double-ended oligonucleotide chains are connected to the nuclear insertion sequences at both ends.

[0392] Figure 16B shows the designs of the two end-cap connectors in Table 8. The upper figure uses the loop portion of the AAV ITR as the end-cap connector, and the lower figure uses a 5'-TTTT-3' single chain as the end-cap connector.

[0393] Figure 17: Schematic diagram of the preparation of double-ended DNA. Plasmid DNA is digested with enzymes to obtain linear double-stranded DNA. Oligonucleotide chains that form hairpin structures after heating and annealing are then added, and double-ended DNA is obtained under the action of DNA ligase.

[0394] Figure 18: Schematic diagram of various types of DNA templates that can be used for gene editing knock-in.

[0395] Figure 19: Schematic diagram of gene editing knock-in. Target gene knock-in can be achieved through homologous recombination (top) and non-homologous end repair (bottom).

[0396] Figure 20: Transfection of activated human primary T cells with LNPs encoding CD19 CAR DNA. A dual LNP delivery system was used: SM102 LNPs carrying mRNA / sgRNA (Cas9 Mut-5: SpCas9-Mut-5 mRNA; DrFECO Cas9: DrFECO-Cas9-Mut-5 mRNA; EcFECO Cas9: EcFECO-Cas9-Mut-5 mRNA; sgRNA: hTRAC), and B2 LNPs (C12-200 LNPs, B2) carrying template DNA. The 300bp homologous arm was a loop-end blocked DNA hTRAC_CD19CAR_300HA_Loop, and the 800bp homologous arm was a loop-end blocked DNA hTRAC_CD19CAR_800HA_Loop (see Table 8 for details). The above template was knocked into the TRAC gene in human primary T cells at specific sites. UNT was the untransfected control group. The expression of CD19CAR was detected by flow cytometry to calculate the knock-in efficiency.

[0397] Figure 21: Effect of DNA templates with different homologous arm lengths on CD19CAR gene editing knock-in. A dual LNP delivery system was used. SM102 LNPs carried mRNA / sgRNA (SpCas9-Mut-5 mRNA for Cas9 Mut-5; sgRNA hTRAC), and B2 LNPs were C12-200 LNPs (B2) carrying template DNA. The 300bp homologous arm was a loop-end blocked DNA hTRAC_CD19CAR_300HA_Loop, the 500bp homologous arm was a loop-end blocked DNA hTRAC_CD19CAR_500HA_Loop, and the 800bp homologous arm was a loop-end blocked DNA hTRAC_CD19CAR_800HA_Loop (see Table 8 for details). These templates were knocked into the TRAC gene in human primary T cells. UNT was the untransfected control group. Flow cytometry was used to detect CD19CAR expression and calculate the knock-in efficiency.

[0398] Figure 22: Comparison of CD19CAR gene knock-in efficiency mediated by single-LNP and dual-LNP delivery systems. The single-LNP system-SM102 consists of SM102 LNPs carrying mRNA / sgRNA / DNA tri-components (mRNA SpCas9-Mut-5; sgRNA hTRAC; template DNA hTRAC_CD19CAR_300HA_Loop). The single-LNP system-C12 B2 consists of C12-200 LNPs (B2) carrying mRNA / sgRNA / DNA tri-components. The dual-LNP system consists of SM102 LNPs carrying mRNA / sgRNA and C12-200 LNPs (B2) carrying DNA. The above templates were knocked into the TRAC gene in human primary T cells. UNT was the untransfected control group. The expression of CD19CAR was detected by flow cytometry to calculate the knock-in efficiency.

[0399] Figure 23: Comparison of knock-in efficiency of single-LNP and dual-LNP delivery systems in different types of DNA templates and Cas9 fusion protein mRNA systems. The single-LNP system-C12 B2 consists of C12-200 LNP (B2) carrying mRNA / sgRNA / DNA three components; the dual-LNP system consists of SM102 LNP carrying mRNA / sgRNA, and C12-200 LNP (B2) carrying DNA (SpCas9-Mut-5 mRNA (m5); DrFECO Cas9 is DrFECO-Cas9-Mut-5 mRNA (FECO); sgRNA is hTRAC; circular single-stranded DNA is CSS_hTRAC_CD19CAR_800HA (CSS); loop-end closed DNA is hTRAC_CD19CAR_800HA_Loop (Loop), see Table 8 for details). The above template was knocked into the TRAC gene in human primary T cells at specific sites. UNT was the untransfected control group. The expression of CD19CAR was detected by flow cytometry to calculate the knock-in efficiency.

[0400] Figure 24: Comparison of knock-in efficiency between single-LNP and dual-LNP delivery systems using endogenous and exogenous promoters in template knock-in systems. The single-LNP system-C12 B2 consists of a C12-200 LNP (B2) carrying a three-component structure of mRNA / sgRNA / DNA; the dual-LNP system consists of an SM102 LNP carrying mRNA / sgRNA and a C12-200 LNP (B2) carrying DNA (SpCas9-Mut-5 mRNA; sgRNA hTRAC; the endogenous TRAC promoter represents the DNA vector structure used, hTRAC_CD19CAR_800HA_Loop(TRAC), and the exogenous EFS promoter represents the DNA vector structure used, hTRAC_pEFS_CD19CAR_800HA_Loop(pEFS), see Table 8 for details). The templates were knocked into the TRAC gene in human primary T cells. UNT was the untransfected control group. The expression of CD19CAR was detected by flow cytometry to calculate the knock-in efficiency.

[0401] Figure 25A: Schematic diagram of pGC_hGAPDH_GFP plasmid.

[0402] Figure 25B: Schematic diagram of pGC_hIL2RA_GFP plasmid.

[0403] Figure 26: Schematic diagram of CSS_hTRAC_CD19CAR_800HA circular single-stranded DNA.

[0404] Figures 27A-27D: Different cell lines (HepG2, Jurkat, and 293T) were transfected with LNPs using different formulations, and the CD19 CAR gene was knocked into activated human primary T cells. The LNP formulations used for transfection were C12-200(B2), SM102, MC3, and ALC0315, containing a three-component package of mRNA / sgRNA / DNA (SpCas9-Mut-5 mRNA; sgRNA hTRAC; DNA vector structure: hTRAC_pEFS_CD19CAR_800HA_Loop, see Table 8 for details). The template was knocked into the TRAC gene in the cells at specific sites. UNT was the untransfected control group. Flow cytometry was used to detect CD19CAR expression and calculate the knock-in efficiency.

[0405] Figure 28: Effect of LNPs with different N / P ratios on CD19CAR gene editing knock-in. The transfected LNPs were C12-200LNP(B2), containing a three-component structure of mRNA / sgRNA / DNA (SpCas9-Mut-5 mRNA; sgRNA hTRAC; DNA vector structure hTRAC_pEFS_CD19CAR_800HA_Loop, see Table 8 for details). The template was knocked into the TRAC gene in activated primary T cells at specific sites. UNT cells served as the untransfected control group. Flow cytometry was used to detect CD19CAR expression and calculate the knock-in efficiency.

[0406] Figure 29: The effect of the ratio of the three nucleic acid components carried by the LNP on CD19CAR gene editing knock-in. The transfected LNP was a C12-200 LNP (B2), carrying a three-component structure of mRNA / sgRNA / DNA (SpCas9-Mut-5 mRNA; sgRNA hTRAC; DNA vector structure hTRAC_pEFS_CD19CAR_800HA_Loop, see Table 8 for details). The template was knocked into the TRAC gene in activated primary T cells at specific sites. UNT was the untransfected control group. Flow cytometry was used to detect CD19CAR expression and calculate the knock-in efficiency.

[0407] Figures 30A-30B: Effect of the C12-200 component ratio in the C12-200 LNP on CD19CAR gene editing knock-in. High-throughput prepared LNPs contained three components: mRNA / sgRNA / DNA (SpCas9-Mut-5 mRNA; sgRNA hTRAC; DNA vector structure: hTRAC_pEFS_CD19CAR_800HA_Loop, see Table 8 for details). MC3 LNP served as the positive control (MC3), and UNT served as the untransfected negative control. Templates were knocked into the TRAC gene in activated primary T cells at specific sites. Flow cytometry was used to detect CD19CAR expression and calculate the knock-in efficiency (Figure 30A), and the expression of the CD3 / TCR complex was detected to calculate the knockout efficiency (Figure 30B).

[0408] Figures 31A-31B: Effect of the DMG-PEG component ratio in C12-200 LNPs on CD19CAR gene editing knock-in. High-throughput prepared LNPs contained three components: mRNA, sgRNA, and DNA (SpCas9-Mut-5 mRNA; sgRNA hTRAC; DNA vector structure: hTRAC_pEFS_CD19CAR_800HA_Loop, see Table 8). MC3 LNP served as the positive control (MC3), and UNT served as the untransfected negative control. Templates were knocked into the TRAC gene in activated primary T cells at specific sites. Flow cytometry was used to detect CD19CAR expression and calculate the knock-in efficiency (Figure 31A), and the expression of the CD3 / TCR complex was detected to calculate the knockout efficiency (Figure 31B).

[0409] Figures 32A-32B: Effect of the DOPE component ratio in C12-200 LNPs on CD19CAR gene editing knock-in. High-throughput prepared LNPs contained three components: mRNA, sgRNA, and DNA (SpCas9-Mut-5 mRNA; sgRNA hTRAC; DNA vector structure: hTRAC_pEFS_CD19CAR_800HA_Loop, see Table 8). MC3 LNP served as the positive control (MC3), and UNT served as the untransfected negative control. Templates were knocked into the TRAC gene in activated primary T cells at specific sites. Flow cytometry was used to detect CD19CAR expression and calculate the knock-in efficiency (Figure 32A), and the expression of the CD3 / TCR complex was detected to calculate the knockout efficiency (Figure 32B).

[0410] Figure 33: Transfection of activated human primary T cells from different donors with LNPs encoding CD19 CAR DNA under optimized conditions (B2 formulation LNP). The transfected LNPs were C12-200 LNPs (B2), containing a three-component structure of mRNA / sgRNA / DNA (SpCas9-Mut-5 mRNA; sgRNA hTRAC; DNA vector structure hTRAC_pEFS_CD19CAR_800HA_Loop, see Table 8 for details). LNP1 and LNP2 were LNPs prepared in parallel from different batches. The template was knocked into the TRAC gene in activated human primary T cells at specific sites, and the expression of CD19CAR was detected by flow cytometry to determine the knock-in efficiency.

[0411] Figure 34: Amplification culture for CAR-T preparation via LNP transfection. The transfected LNP was C12-200 LNP (B2), containing a three-component structure of mRNA / sgRNA / DNA (SpCas9-Mut-5 mRNA; sgRNA hTRAC; DNA vector structure hTRAC_pEFS_CD19CAR_800HA_Loop, see Table 8 for details). UNT served as the control group without LNP transfection. Samples were taken at different time points during the amplification culture phase, and cell counts were performed to determine the fold increase in cell number.

[0412] Figure 35: In vitro killing effect of CD19 CAR-T target cells prepared by LNP transfection. The transfected LNP was C12-200 LNP (B2), containing a three-component structure of mRNA / sgRNA / DNA (SpCas9-Mut-5 mRNA; sgRNA hTRAC; DNA vector structure hTRAC_pEFS_CD19CAR_800HA_Loop, see Table 8 for details). UNT T cells served as the control group, containing untransfected LNP. CAR-T cells and Raji-fLuc target cells stably expressing luciferase were co-incubated for 48 h at different cell ratios (E / Tratio). Cells in the culture system were lysed, and luciferase signal was detected to calculate the CAR-T cell killing capacity.

[0413] Figure 36: In vitro killing effect of CD7 CAR-T cells transfected with LNP on target cells. The transfected LNP was C12-200 LNP (B2), containing a three-component structure of mRNA / sgRNA / DNA (SpCas9-Mut-5 mRNA; sgRNA hTRAC; DNA vector structure hTRAC_pEFS_CD7CAR_800HA_Loop, see Table 8 for details). UNT T cells served as the control group, containing untransfected LNP. CAR-T cells and Jurkat-fLuc target cells stably expressing luciferase were co-incubated for 48 h at different cell ratios (E / T ratio). Cells in the culture system were lysed, and luciferase signal was detected to calculate the CAR-T cell killing capacity.

[0414] Figure 37: In vitro killing effect of CD7 CAR-T cells transfected with LNP on target cells. The transfected LNP was C12-200 LNP (B2), containing a three-component structure of mRNA / sgRNA / DNA (SpCas9-Mut-5 mRNA; sgRNA hTRAC; DNA vector structure hTRAC_pEFS_BCMACAR_800HA_Loop, see Table 8 for details). UNT T cells served as the control group, containing untransfected LNP. CAR-T cells and MM.1S-fLuc target cells stably expressing luciferase were co-incubated for 48 h at different cell ratios (E / T ratio). Cells in the culture system were lysed, and luciferase signal was detected to calculate the CAR-T cell killing capacity.

[0415] Figures 38A-38B: In vivo efficacy of CD19 CAR-T cells transfected with LNP in tumor-bearing mice. The transfected LNP was C12-200LNP (B2), containing a three-component structure of mRNA / sgRNA / DNA (SpCas9-Mut-5 mRNA; sgRNA hTRAC; DNA vector structure hTRAC_pEFS_CD19CAR_800HA_Loop, see Table 8 for details). UNT T cells were used as the control group, consisting of untransfected LNP T cells. NCG mice (6-8 weeks old, female) were intravenously inoculated with Raji-luc cells stably expressing luciferase (5E5 / mouse). After 4 days of feeding, in vivo imaging was performed to group the mice. On the second day, after the CD19 CAR-T cells were thawed in liquid nitrogen, tumor-bearing mice were intravenously inoculated with 1E6 CAR-positive CAR-T cells. The control group was inoculated with 1E6 untransfected LNP T cells (UNT T cells) cultured and expanded under the same conditions. Five mice were in each group. During the feeding process, changes in tumor cell fluorescence signals were observed using in vivo imaging (Figure 38A), and the survival status of mice was observed and recorded (Figure 38B).

[0416] Invention Details

[0417] This application provides DNA vector molecules, gene editing compositions, and nucleic acid-lipid nanoparticle compositions that can significantly improve the efficiency of nuclear and genomic integration of target DNA sequences. Compared with viral vector-based engineered cell preparation methods, the methods using the DNA vector molecules, gene editing compositions, or nucleic acid-lipid nanoparticle compositions of this application can improve the efficiency of engineered cell preparation, and have higher safety and lower cost. Furthermore, the DNA vector molecules, gene editing compositions, and nucleic acid-lipid nanoparticle compositions provided in this application can also be used to improve the site-specific integration efficiency of large fragments (especially fragments larger than 1kb or 2kb) of target genes based on gene editing systems.

[0418] the term

[0419] As used herein, the term "linear DNA" refers to DNA molecules other than circular DNA, encompassing both uncapped and capped linear DNA. Examples of linear DNA are shown in Figure 16A as linear double-stranded DNA, double-ended double-stranded DNA, NLS-modified double-ended double-stranded DNA, and linear single-stranded DNA.

[0420] The term "circular DNA molecule" refers to a DNA molecule that contains only one ring and does not contain a free 5' phosphate group and a 3' hydroxyl group. The structures of exemplary circular DNA molecules are shown in Figure 16A for plasmid DNA and circular single-stranded DNA.

[0421] In this application, since the main function of linear DNA is to insert the target gene sequence into the cell genome as a vector, the 5' end of the linear DNA is located on one side of the 5' end at the 1 / 2 position of the target gene sequence, and the 3' end of the linear DNA is located on one side of the 3' end at the 1 / 2 position of the target gene sequence.

[0422] In this application, "5' end" and "3' end" primarily describe the relative positional relationship between nucleotides, nucleotide sequence segments, or nucleotides and nucleotide sequence segments within the same nucleic acid sequence; "5' end" and "3' end" are respectively used to describe the positions of the first and last nucleotides of a nucleic acid sequence or a segment of a nucleic acid sequence. "One side of the 5' end" describes the relative positional relationship between two sequences within the same polynucleotide sequence that do not overlap. When describing one sequence as being on the 5' end side of another sequence, it means that the first sequence is closer to the "5' end" of the polynucleotide sequence relative to the second sequence. Similarly, when describing one sequence as being on the 3' end side of another sequence, it means that the first sequence is closer to the "3' end" of the polynucleotide sequence relative to the second sequence, and the first and second sequences do not overlap. Furthermore, as used herein, "5' portion" refers to the halfway point from the "center position" of the polynucleotide sequence to the 5' end of the polynucleotide sequence. The "3' portion" refers to the halfway point from the center of the polynucleotide sequence to its 3' end. In this application, the number of nucleotides from the "center" to the 5' end and to the 3' end are equal. As used in this application, "nuclear entry sequence" refers to a short nucleotide sequence that can bind to transcription factor proteins to form a complex and be transported into the cell nucleus, including but not limited to: ETS1 nuclear entry sequence (i.e., the ETS1 gene motif recognized and bound by transcription factors), NFAT nuclear entry sequence (i.e., the NFAT gene motif recognized and bound by transcription factors), STAT2 nuclear entry sequence (i.e., the STAT2 gene motif recognized and bound by transcription factors), LEF1 nuclear entry sequence (i.e., the LEF1 gene motif recognized and bound by transcription factors), and sequences in the ITR that are recognized and bound by transcription factors, such as seq1-L and seq1-R sequences. In this application, the sequence in the ITR that is recognized and bound by transcription factors is called the "ITR nuclear entry sequence." Unless otherwise specified, "ITR nuclear entry sequence" and "AAV ITR nuclear entry sequence" have the same meaning in this application. Natural seq1-L is located upstream of the AAV structural gene, while seq1-R is located downstream. As used in this article, "primary cells" refer to single cells obtained directly from body tissues, organs, peripheral blood, and embryos and cultured in vitro. Primary cells typically have the same physiological functions as their counterparts in the body or are cells that can naturally differentiate from their counterparts in the body. Compared to their counterparts in the body, their genomic DNA remains largely unchanged. For example, primary T cells, which are T cells isolated from the body and cultured in vitro, have the same physiological functions and cell populations as T cells naturally present in the body, and have the same lifespan under the same conditions.When primary T cells are reinfused into the body, they can still perform the same functions as somatic cells present in the body. It should be understood that "primary T cells" in this application includes T cells that have the same physiological functions and genome as T cells present in the body, obtained in vitro through early cell differentiation such as stem cells or progenitor cells.

[0423] As used herein, "activated T cells" or "activated primary T cells" are used interchangeably, referring to a cluster of T cells obtained after activation, proliferation, and differentiation upon antigen stimulation, wherein the T cell cluster includes CD4+ helper T cells and CD8+ cytotoxic T cells. In some embodiments, such as the specific embodiments used in this application to obtain specific data, the method for preparing activated T cells is as follows: primary T cells are placed in a culture medium in which an appropriate amount (e.g., a supplier-recommended concentration) of CD3 / CD28 magnetic beads or antibodies are added and co-incubated in the T cell culture medium, and cultured at 37°C and 5% CO2 for 44 to 52 hours.

[0424] In this application, "BCMACAR-T" refers to CAR-T cells that have been genetically modified to express a chimeric antigen receptor that can recognize BCMA, thereby targeting and killing myeloma cells that express BCMA.

[0425] As used herein, the term "nuclear localization signal peptide" or simply "NLS" refers to a short peptide that can interact with nuclear transport vectors, such as proteins or linear DNA as described in this application, and guide said nuclear transport vectors into the cell nucleus. In nature, NLS is typically a short peptide that guides proteins into the nucleus. NLS can generally be classified into monotypes, ditypes, and non-classical proline-tyrosine types. Monotyped NLS typically consists of 4-8 amino acid residues, with a consensus sequence of KK / RXK / R, where X is any amino acid. An exemplary representation of this type is, for example, the NLS of the SV40 T antigen, with the sequence PKKKRKV (SEQ ID NO:49). The consensus sequence of dityped NLS is R / K(X). 10-12 KRXK, where X represents any amino acid residue, K / R represents lysine or arginine residues, and the number in the letter subscript indicates the number of amino acids represented by that letter, for example (X). 10-12 This represents 10-12 arbitrary amino acids. For example, the NLS nucleoplasmic protein from Xenopus laevis has the sequence KRPAATKKAGQAKKKK (SEQ ID NO:48). Non-classical proline-tyrosine NLS have a C-terminus rich in a proline-tyrosine sequence, with a consistent sequence of R / K / H-(X)2-5-PY, where X is any amino acid residue, and the number of the letter subscript indicates the number of amino acids represented by that letter, for example, (X). 2-5This indicates 2 to 5 arbitrary amino acid residues; an exemplary representation in this typing is, for example, the M9 NLS of heterologous nucleoribonucleoprotein (hnRNP)D, with the sequence FGYNNQSSNFGPMKGGNFGGRSSGPY (SEQ ID NO: 50). In this application, the NLS can be a component of linear DNA, linked to one or more nucleotides of the linear DNA to facilitate nuclear translocation of the linear DNA.

[0426] As used herein, “complementarity” of nucleic acids refers to the ability of one nucleic acid to form hydrogen bonds with another nucleic acid through conventional Watson-Crick base pairing. Percentage complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with another nucleic acid molecule (e.g., approximately 50%, 60%, 70%, 80%, 90%, and 100% complementarity out of 10, respectively). “Complete complementarity” means that all consecutive residues in a nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. As used herein, “substantially complementary” means the degree of complementarity of at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% in a region of approximately 40, 50, 60, 70, 80, 100, 150, 200, 250, or more nucleotides, or refers to two nucleic acids hybridized under stringent conditions. For a single base or nucleotide, according to the Watson-Crick base pairing rule, when A pairs with T or U, or C pairs with G or I, it is called complementary or matched, and vice versa; all other base pairings are called non-complementary. In this application, the "complementary polynucleotide sequence" of a certain polynucleotide sequence refers to a polynucleotide sequence that is completely complementary to that polynucleotide sequence.

[0427] The term "nucleotide," in addition to referring to naturally occurring ribonucleotide or deoxyribonucleotide monomers, should also be understood herein to refer to their associated structural variants, including derivatives and analogs, which are functionally equivalent in the specific context of the use of the nucleotide, unless the context explicitly indicates otherwise. For example, "nucleotide" refers to deoxyribonucleotides or ribonucleotides. Nucleotides can be standard nucleotides (i.e., adenosine, guanosine, cytidine, thymidine, and uridine), nucleotide isomers, or nucleotide analogs. Nucleotide analogs refer to nucleotides having modified purine or pyrimidine bases or modified ribose moieties. Nucleotide analogs can be naturally occurring nucleotides (e.g., inosine, pseudouridine, etc.) or non-naturally occurring nucleotides. Non-limiting examples of modifications to the sugar or base moieties of nucleotides include the addition (or removal) of acetyl, amino, carboxyl, carboxymethyl, hydroxyl, methyl, phosphoryl, and thiol groups, as well as the substitution of the carbon and nitrogen atoms of the base by other atoms (e.g., 7-denitropurine). Nucleotide analogs also include dideoxynucleotides, 2'-O-methylnucleotides, locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and morpholino oligonucleotides. In the examples of this application, unless otherwise specified, the target gene sequence portion of the linear DNA molecule is typically composed of natural deoxyribonucleotides. It should be understood that, unless otherwise specified, the term "uridine" in this application encompasses natural uridines and their derivatives, including but not limited to: 5-methoxymethyl uridine, 5-methylthiouridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, 5-methoxy cytidine, 1-methyl-Pseudo-UTP, pseudouridine, 1-ethyl-pseudouridine, and 5-methoxy-uridine. In some other embodiments, all or part of the nucleic acid of this application may be replaced with modified bases, such as 1-methylpseuuridine or pseudouridine.

[0428] In the context of this invention, the terms "DNA" and "RNA" refer to single-stranded, double-stranded, or circular DNA and RNA molecules, respectively. Unless otherwise stated, the terms "DNA" and "DNA molecule" refer to a DNA molecule composed of A, C, G, and / or T deoxyribonucleotides, while the terms "RNA" and "RNA molecule" refer to an RNA molecule composed of A, C, G, and / or U ribonucleotides. Unless otherwise specified herein, the A, C, G, T, and U nucleotides refer to nucleotides containing adenine, guanine, cytosine, thymine, and uracil as their respective nitrogenous bases; exceptions include: when the nucleic acid sequence listed in this application is used to represent an RNA sequence, T, unless otherwise specified, refers to uracil, equivalent to U. In this application, ribonucleotides are allowed to be inserted into DNA molecules, and as long as they still function as DNA, such as being transcribed into RNA molecules that encode them, they are still called DNA molecules; conversely, deoxyribonucleotides are also allowed to be inserted into RNA molecules, and as long as they still function as RNA, such as guiding protein synthesis or serving as functional RNA, such as gRNA guiding Cas proteins to perform targeted gene editing, they are still called RNA molecules.

[0429] As used herein, “mRNA” (messenger RNA) is any RNA, naturally occurring, non-naturally occurring, or modified amino acid polymer, encoding at least one protein, and is translatable to produce the encoded protein in vitro, in vivo, in situ, or ex vivo. Those skilled in the art will appreciate that, unless otherwise stated, the polynucleotide sequence described herein may use “T” to refer to thymine when representing a DNA sequence, but when the polynucleotide sequence represents RNA (e.g., mRNA), “T” refers to “U” (uracil, encompassing uracil derivatives such as pseudouracil, methylpseudorazine, etc.). Therefore, any DNA disclosed and identified by a specific sequence number (SEQ ID NO) herein also discloses an RNA (e.g., mRNA) sequence complementary to or corresponding to said DNA, wherein each “T” in said DNA sequence is replaced by a “U”.

[0430] In this document, “coding” means i) a DNA sequence containing genetic information that can be transcribed into an RNA molecule, and / or ii) an RNA molecule containing genetic information that can be translated into an amino acid sequence. Therefore, as used herein, “coding sequence” can refer to a ribonucleotide (RNA) sequence or fragment thereof in a precursor or mature mRNA that can be translated into a protein, or to a complementary sequence or fragment thereof serving as a template for transcribing the precursor or mature mRNA.

[0431] As used herein, the term "about" refers to a typical range of error for various values ​​that is readily known to those skilled in the art. References to a value or parameter "about" herein include embodiments of that value or parameter itself. As used herein, when the term "about" precedes a numerical value, it typically indicates a range of 10% above or below that value. For example, "about 100" covers 90 and 110. In this application, "about" can be followed by an integer or a decimal. In some embodiments, "about" a number covers all values ​​that, when rounded, equal to said number; for example, about 35 mol% covers all values ​​greater than 34.4 mol% and less than 35.5 mol%; about 30 mol% covers all values ​​greater than 24 mol% and less than 35 mol%.

[0432] As used herein, the percentage of "identity," such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, and 99.5%, refers to the degree of similarity determined by sequence alignment between amino acid sequences or nucleotide sequences. For example, it is the percentage of positions with identical bases or amino acid residues out of the total number of positions, determined by introducing vacancies or other methods to make two sequences have as many identical residues as possible. The percentage of "identity" can be determined using software programs known in the art. Optionally, alignment can be performed using default parameters. Optionally, the alignment program is BLAST. Optionally, the alignment programs are BLASTN and BLASTP. Details of these programs are available on the relevant pages of the NCBI website.

[0433] As used herein, the term "gene editing molecule" refers to one or more of a protein, a nucleic acid encoding the protein, or a nucleic acid molecule (e.g., a gRNA guiding a Cas enzyme) that can guide the protein to perform site-specific modifications (e.g., cleavage) of a target sequence, wherein the protein is selected from transposases, nucleases, and helicases. Nucleases that are gene editing molecules are proteins with nuclease activity, and non-limiting examples include, but are not limited to: CRISPR-associated proteins (Cas) (e.g., CRISPR-associated protein 9 (Cas9), Cpf1, IscB, TnpB, etc.), IIS-type restriction enzymes, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), broad-spectrum nucleases, site-specific nucleases, or Cases used for inactivation of the CRISPRi or CRISPRa systems.

[0434] As used herein, "programmable DNA-binding protein" refers to a programmable DNA-binding protein, wherein "programmable" means that the target site of the DNA-binding protein can be altered by changing the sequence or structure of the target nucleic acid or target protein that binds to the DNA target site. For example, by changing the target sequence of gRNA, the target site of Cas enzyme can be changed, causing Cas enzyme to perform genetic modifications at the target site, such as cleavage (e.g., producing single-strand or double-strand breaks), deamination, or transamination. Therefore, Cas enzyme is an example of a programmable DNA-binding protein.

[0435] As used in this article, "guide RNA," also known as gRNA, is a short RNA sequence that recognizes a target sequence site through complementary hybridization and guides a programmable DNA-targeting nuclease to modify the target sequence, such as by cleaving it to produce double-strand or single-strand breaks. In the context of the CRISPR-Cas gene editing system, gRNA is the sequence that guides the Cas protein to perform gene editing. In the CRISPR-Cas9 system, gRNA contains crRNA and tracrRNA sequences. If crRNA and tracrRNA are combined into a single RNA, this gRNA can also be called a single-guide RNA (sgRNA).

[0436] As used herein, a “variant” differs from a reference amino acid sequence by at least one amino acid, for example, by the addition, insertion, deletion, or substitution of at least one amino acid. For example, the amino acid substitution may be a conserved amino acid substitution, i.e., replacing the original corresponding amino acid with an amino acid of similar properties. A “conserved substitution” may be polar to polar amino acids, such as glycine (G, Gly), serine (S, Ser), threonine (T, Thr), tyrosine (Y, Tyr), cysteine ​​(C, Cys), asparagine (N, Asn), and glutamine (Q, Gln); nonpolar to nonpolar amino acids, such as alanine (A, Ala), valine (V, Val), tryptophan (W, Trp), leucine (L, Leu), proline (P, Pro), methionine (M, Met), and phenylalanine (F, Phe); or acidic to acidic amino acids, such as aspartic acid (D, Asp) and glutamic acid (E, Gln). u); basic amino acids paired with basic amino acids, such as arginine (R, Arg), histidine (H, His), and lysine (K, Lys); charged amino acids paired with charged amino acids, such as aspartic acid (D, Asp), glutamic acid (E, Glu), histidine (H, His), lysine (K, Lys), and arginine (R, Arg); hydrophobic amino acids paired with hydrophobic amino acids, such as alanine (A, Ala), leucine (L, Leu), isoleucine (I, Ile), valine (V, Val), proline (P, Pro), phenylalanine (F, Phe), tryptophan (W, Trp), and methionine (M, Met). In some other embodiments, the variants may also contain non-conserved substitutions. In some embodiments, the "variant" of the amino acid sequence may have at least about 90%, 95%, 96%, 97%, 98%, or 99% sequence identity relative to the amino acid sequence. Compared to this amino acid sequence, a "variant" of this amino acid sequence can have an activity ranging from at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any combination of the foregoing values. As used herein, a "conserved substitution variant" of a protein, polypeptide, or amino acid sequence refers to one or more amino acid residues in which amino acid substitutions have been made without altering the overall conformation and function of the protein or enzyme. This includes, but is not limited to, substitutions of amino acids in the parent protein's amino acid sequence in the manner described above as "conserved substitutions." Therefore, two proteins or amino acid sequences with similar functions may have varying degrees of similarity. For example, similarity (identity) of 70% to 99% based on the MEGALIGN algorithm."Conservative substitution variants" also include peptides or enzymes that, as determined by BLAST or FASTA algorithms, have more than 60% amino acid identity, preferably more than 75%, ideally more than 85%, and even better if they have more than 90%, and have the same or substantially similar properties or functions as the natural or parental protein or enzyme.

[0437] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably in this document and refer to a polymeric form of amino acids of any length, which may contain coding and non-coding amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a modified peptide backbone.

[0438] Capped linear DNA molecules

[0439] A capped linear DNA molecule, also known as a ceDNA molecule, is a DNA vector having at least one covalently closed end. In some embodiments, ceDNA includes two covalently closed ends, i.e., a double-ended linear DNA molecule. ceDNA has at least one double-stranded portion, with the "covalently closed end" formed by a capping linker connecting the terminal nucleotides of the two strands at one end of the double-stranded portion. The capping linker can be a nucleic acid structure or a non-nucleic acid structure; and the "capping linker" as defined in this application does not contain nucleotides of the double-stranded portion of the linear DNA molecule, i.e., at least the first pair of nucleotides in the capping linker that are connected to each of the two strands of the double-stranded portion are not complementary. Examples of nucleic acid structures for "capping linkers" include, but are not limited to: the loop portion of an ITR that can form a T-shaped stem-loop structure with the ITR double-stranded portion, and oligonucleotides that can form a hairpin structure with the ITR double-stranded portion; examples of capping linkers are shown in the dashed box on the left side of Figure 16B. Examples of non-nucleic acid structured "terminal linkers" include, but are not limited to, combinations selected from one or more of the following structures: one or more tandem phosphate ester and analogue structures (such as thiophosphate bonds, methylthiophosphate bonds), -C1-20 alkyl- (or -C1-19 alkyl-, -C1-18 alkyl-, -C1-15 alkyl-, -C1-14 alkyl-, -C1-12 alkyl-, -C1-10 alkyl-, -C1-9 alkyl-, -C1-8 alkyl-), wherein one or more alkylene groups (-CH2-) may be replaced by one or more -O-, -NH-, -C(O)NH-, -NHC(O)-, -S-, -S2-, -C(O)O-, -OC(O)-, -S(O)2O-, -OS(O)2-, -S(O)2NH-, -NHS(O)2-, maleimide-thioether substituted -C1-20 alkyl- (or -C1-19 alkyl-, -C1-18 alkyl-, -C1-15 alkyl-, -C1-14 alkyl-, -C1-12 alkyl-, -C1-10 alkyl-, -C1-9 alkyl-, -C1-8 alkyl-), PEG structures with molecular weights between 100-2000, one or more glucose structures linked by phosphodiester bonds, peptide linkers (such as 2-10 amino acid linkers, dipeptide linkers (such as -VC-, -VA-), tripeptide linkers (such as -EVC-, -EVA-, -EGC-, -AAN-), tetrapeptide linkers (such as -GGFG-)), linkers commonly used in ADC drugs, etc.

[0440] As used herein, the term "terminal repeat" or "TR" can include any viral or synthetic terminal repeat sequence that includes at least a minimum required origin of replication and a region consisting of a palindromic hairpin structure. A Rep (i.e., replication protein) binding sequence (RBS), also known as a Rep binding element (RBE), together with a terminal resolution site (TRS), constitutes a minimum required origin of replication; therefore, a TR includes at least one RBS and at least one TRS. In a given polynucleotide sequence, mutually inversely complementary TRs are often referred to as "inverted terminal repeats" or "ITRs." In viruses, intact ITRs mediate replication, viral packaging, integration, and proviral rescue. In this application, an ITR can be an AAV ITR or a non-AAV ITR, or it can be derived from an AAV ITR or a non-AAV ITR. For example, ITRs can originate from the Parvoviridae family, which includes paraviruses and their genus (such as canine paravirus, bovine paravirus, mouse paravirus, porcine paravirus, and human paravirus B-19). Alternatively, the SV40 hairpin structure, which is the origin of SV40 replication, can also be used as an ITR. ITRs can be further modified through truncation, substitution, deletion, insertion, and / or addition. Paraviridae viruses comprise two subfamilies: the Parvovirinae subfamily, which infects vertebrates, and the Densovirinae subfamily, which infects invertebrates. The Parvovirinae subfamily includes the Adeno-Associated Virus (AAV) family, which can replicate in vertebrate hosts, including but not limited to humans, primates, cattle, dogs, horses, and sheep. For ease of description, in this application, an ITR located on one side (upstream) of the 5' end of the target gene sequence in a capped linear DNA molecule is called a "5'ITR" or "ITR-L", and an ITR located on one side (downstream) of the 3' end of the target gene sequence in a capped linear DNA molecule is called a "3'ITR" or "ITR-R". A complete ITR can also be structurally divided into a double-stranded portion and a loop portion; the double-stranded portion contains TRS and / or some or all of the RBS sequences. In the wild-type viral genome, the double-stranded portion of the ITR is the part of the ITR that is located in the same double strand as the viral structural protein gene sequence, while the loop portion is the part outside the double-stranded portion of the ITR, that is, the loop portion in the three-dimensional structure of the ITR (e.g., the hairpin structure, T-shaped structure, or Y-shaped structure in the ITR), as shown in the dashed box in Figure 16B. It is connected to the double-stranded portion of the ITR, and any double-stranded segments contained therein (e.g., the double strand formed by the inverted repeat sequence in the loop structure) are not located in the same double strand as the double-stranded portion of the ITR.In this application, "located on the same double strand" is a positional relationship between two nucleic acid segments. Both nucleic acid segments on the same double strand and the nucleic acid strand connecting the two nucleic acid segments are double strands. When the two strands of the same double strand hybridize at the maximum complementarity, there are no unpaired nucleotides in the two nucleic acid segments and the nucleic acid strand connecting the two nucleic acid segments.

[0441] In this application, the term "target gene sequence" is used to refer to a nucleic acid sequence fragment inserted into the genome, and its meaning covers both "coding sequences" and functional nucleic acids or fragments thereof, such as polynucleotide sequences that can be transcribed into miRNA, shRNA, dsRNA, guide RNA, or functional elements such as poly(A) tails, 5'UTR, 3'UTR, etc.; it also covers any nucleic acid sequence fragment that can be inserted into the genome to alter the expression of one or more genes or RNA transcription, such as one, two or more nucleotides that are not multiples of 3. In this application, when the "target gene sequence" uses an independent promoter, that is, when the promoter that initiates the transcription of the "target gene sequence" is located in the same linear DNA molecule as the target gene sequence, the linear DNA molecule may not contain a 2A peptide coding sequence; when the "target gene sequence" uses a promoter that is already present in the genome, that is, when it is inserted into the target gene and the promoter of the target gene is used to initiate the expression of the target gene sequence, then optionally, a 2A peptide coding sequence is inserted on one side of the 5' end of the target gene sequence and between the target gene sequence to minimize interference with the expression of the genome gene.

[0442] Nucleic acid-lipid nanoparticle composition

[0443] As used in this application, a "nucleic acid-lipid nanoparticle composition" comprises at least one nucleic acid molecule and one LNP, wherein at least one LNP encapsulates the aforementioned capped linear DNA molecule. Furthermore, it is not required that each LNP in the nucleic acid-lipid nanoparticle composition encapsulates a nucleic acid molecule, nor is it required that the amount and type of nucleic acid molecules encapsulated in each LNP be completely identical. In some embodiments, the loading rate of the nucleic acid-lipid nanoparticle composition of this application (i.e., the proportion of LNPs encapsulating nucleic acid molecules to the total number of LNPs in the nucleic acid-lipid nanoparticle composition) can reach 50% or more, for example, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99%.

[0444] The lipid portion of an LNP typically comprises protonable cationic lipids (or cationic lipids), structural lipids, accessory lipids, and surfactants (or amphiphilic lipids). In some embodiments, lipids are mixed with nucleic acid molecules to form lipid nanoparticles. In some embodiments, the lipid nanoparticles are initially formed as empty lipid nanoparticles and then combined or encapsulated with the nucleic acid molecules just before application (e.g., within minutes to an hour).

[0445] In some embodiments, the LNP of this application comprises 100% by molar percentage (mol%) of ionizable cationic lipids, structural lipids, auxiliary lipids, and surfactants. In some embodiments, the lipid nanoparticles contain 20-65% protonable cationic lipids (e.g., 21 mol%, 22 mol%, 23 mol%, 24 mol%, 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%). l%, 49mol%, 50mol%, 51mol%, 52mol%, 53mol%, 54mol%, 55mol%, 56mol%, 57mol%, 58mol%, 59mol%, 60mol%, 61mol%, 62mol%, 63mol% or 64mol%) ; 10-40% (such as 11mol%, 12mol%, 13mol%, 14mol%, 15mol%, 16mol%, 17mol%, 18mol%, 19mol%, 20mol%, 21mol%, 22mol%, 23mol%, 24mol%, 25mol%, 2 6 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, or 39 mol%) structural lipids; 5-50 mol% (e.g., 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, 15 mol%, 16 mol%, 17 mol%, 18 mol%, 19 mol%, 20 mol%, 21 mol%, 22 mol%, 23 mol%) structural lipids. 0.5-4 mol% (e.g., 0.6 mol%, 0.7 mol%, 0.8 mol%), ...9mol%, 1mol%, 1.1mol%, 1.2mol%, 1.3mol%, 1.4mol%, 1.5mol%, 1.6mol%, 1.7mol%, 1.8mol%, 1.9mol%, 2mol%, 2.1mol%, 2.2mol%, 2.3mol%, 2.4mol%, 2 .5mol%, 2.6mol%, 2.7mol%, 2.8mol%, 2.9mol%, 3mol%, 3.1mol%, 3.2mol%, 3.3mol%, 3.4mol%, 3.5mol%, 3.6mol%, 3.7mol%, 3.8mol%, 3.9mol%) surfactant. .

[0446] As used herein, the term “cationic lipid” has the conventional definition in the art and generally refers to a class of lipid molecules that exhibit a positive charge under certain pH conditions and are able to bind to negatively charged biomolecules (such as DNA and RNA).

[0447] As used herein, the term "phospholipid" has the conventional definition in the art, generally referring to lipids containing phosphoric acid, which are amphoteric molecules with a hydrophilic nitrogen or phosphorus head at one end and a hydrophobic (lipophilic) long hydrocarbon chain at the other end, and are generally lipid molecules composed of glycerol, two fatty acid chains and a phosphoric acid group.

[0448] As used herein, the term "amphiphilic lipid" has the conventional definition in the art and generally refers to lipid molecules having both hydrophilic and hydrophobic portions.

[0449] As used herein, the term "cholesterol derivative" refers to a class of compounds that are based on cholesterol and obtained through conventional chemical modification or by combining with other conventional chemical groups, and have the same or similar physicochemical properties as cholesterol.

[0450] In some embodiments, the cationic lipids in the first type of LNP are selected from SM102, ALC0315, DLin-MC3-DMA, C12-200, C14-4, cKK-E12, 306Oi10, Lipid 5, Lipid10, LP-01, DOTMA, DODMA, DLin-KC2-DMA, DOTAP, and DC-Chol.

[0451] The C12-200 and its isomers are amine 200. Derived amino alcohol lipid compounds. In some embodiments, the C12-200 derivative is shown in any of the following structural formulas:

[0452] 5 tails:

[0453] 4 tails:

[0454] 3 tails:

[0455] Two tails:

[0456] One tail:

[0457] In some implementation schemes, each Independently

[0458] The exemplary structural formulas of the aforementioned cationic lipids are shown below:

[0459] In this application, any LNP used to encapsulate linear double-stranded DNA, such as capped linear DNA molecules, is referred to as a Class I LNP. In some embodiments, the structural lipid in the Class I LNP is selected from cholesterol and cholesterol derivatives, and optionally, is cholesterol.

[0460] In this application, depending on the cationic lipid used, for example, when the cationic lipids used are SM102 or C12-200, the LNPs of this application can be collectively referred to as SM102 LNPs or C12-200 LNPs, respectively. In some embodiments, the C12-200 LNPs of this application can be any of the LNP formulations shown in Table 1 (numbered A1-A27), Table 2 (numbered B1-B11), and Tables 3-4 (excluding the control group). For example, "C12-A20 LNP" refers to the LNP with C12-200 as the cationic liposome, which is formulation number A20 in Table 1; "C12-B2 LNP", "C12-200LNP(B2)" and the abbreviation "B2" in the coordinates of the accompanying drawings all refer to the LNP with C12-200 as the cationic liposome, which is formulation number B2 in Table 2. The formulation of SM102 LNP used in the embodiments of this application is: SM102:DSPC:cholesterol:DMG-PEG2000 = 50:10:38.5:1.5. The C12-200 LNP (control) mentioned in this application is an LNP in the prior art, and its formulation is shown in the last row of Table 1.

[0461] In some embodiments, the accessory lipids in the first type of LNP are phospholipids. In some embodiments, the accessory lipids are selected from DSPC, DOPE, DOPC, DOPG, and DOPS, and optionally, DOPE.

[0462] In some embodiments, the polymer-modified lipid conjugates in the first type of LNP are selected from zwitterionic polymer lipids such as PEG lipids, polyglycerol (PG) lipids, polyoxazoline (POZ) lipids, polysarcosine (pSar) lipids, polyamide lipids, poly-2-methacryloyloxyethylphosphorylcholine (PMPC), cationic polymer lipids, polysaccharide-modified lipid conjugates, and mixtures thereof; the PEG lipids may alternatively be referred to as polyethylene glycol-modified lipids. PEG lipids are lipids modified with polyethylene glycol. PEG lipids may be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, DMG-PEG2000, PEG-DSPE, DTDA-PEG2000, or polyethylene glycol esterose succinate (TPGS); optionally, the PEG lipid is DMG-PEG2000. In some embodiments, the first type of LNP further encapsulates other biomolecules that can be used to insert the target gene sequence contained in the capped linear DNA into the cellular genome. These biomolecules are, for example, functional proteins or accessory nucleic acids. In some embodiments, the functional protein can create double-strand or single-strand breaks in the cellular genome, or unwind a portion of the DNA fragment in the genome. In some embodiments, the functional protein can create double-strand or single-strand breaks at specific locations in the cellular genome, or unwind DNA at those specific locations. In some embodiments, the functional protein is a programmable DNA-targeting nuclease or transposase. In some embodiments, the accessory nucleic acid comprises mRNA encoding the aforementioned functional protein. In some embodiments, the helper nucleic acid further comprises a nucleic acid molecule that hybridizes complementaryly to the target gene sequence. This complementary nucleic acid molecule can guide the aforementioned functional protein to create double-strand or single-strand breaks in the target gene sequence, or to unwind the target gene sequence. In some embodiments, the nucleic acid molecule that hybridizes complementaryly to the target gene sequence is called a guide RNA. In some embodiments, the functional protein is a transposase. In some embodiments, each type I LNP simultaneously encapsulates the aforementioned capped linear DNA molecule and the aforementioned functional protein (or helper nucleic acid). In some embodiments, only a portion of the type I LNPs simultaneously encapsulate the aforementioned capped linear DNA molecule and the aforementioned functional protein (or helper nucleic acid).

[0463] In some embodiments, the nucleic acid-lipid nanoparticle composition further comprises a second type of LNP, which has the same or different lipid composition as the aforementioned first type of LNP, and the second type of LNP at least encapsulates a biomolecule that can facilitate the insertion of the target gene sequence contained in the aforementioned capped linear DNA into the cellular genome. The biomolecule is, for example, a functional protein or a helper nucleic acid. In some embodiments, the functional protein can create double-strand or single-strand breaks in the cellular genome, or unwind a portion of the DNA fragment in the genome. In some embodiments, the functional protein can create double-strand or single-strand breaks at a specific location in the cellular genome, or unwind DNA at that specific location. In some embodiments, the functional protein is a programmable DNA-targeting nuclease or a transposase. In some embodiments, the helper nucleic acid comprises mRNA encoding the aforementioned functional protein. In some embodiments, the helper nucleic acid further comprises a nucleic acid molecule that hybridizes complementaryly to the target gene sequence, the complementary nucleic acid molecule guiding the aforementioned functional protein to create double-strand or single-strand breaks in the target gene sequence, or to unwind the target gene sequence. In some embodiments, the nucleic acid molecule that hybridizes complementaryly to the target gene sequence is called a guide RNA. In some embodiments, the functional protein is a transposase. Typically, the purpose of the second type of LNP is to introduce the aforementioned functional protein or helper nucleic acid into cells, so that it assists the aforementioned capped linear DNA molecule in inserting the target gene sequence into the cell's genome. The presence of the second type of LNP is not essential; for example, in some embodiments, the functional protein or helper nucleic acid may be simultaneously encapsulated in the first type of LNP along with the aforementioned capped linear DNA molecule.

[0464] In some embodiments, the second type of LNP has a different lipid composition from the first type of LNP, and the second type of LNP facilitates the delivery of the functional protein or mRNA expressing the functional protein. In some embodiments, the second type of LNP comprises 20-60 mol% (e.g., 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%) of cationic lipids, 25-55 mol% (e.g., 30 mol%, 35 mol%, 40 mol%, 45 mol%, or 50 mol%) of structural lipids, 5-25 mol% (e.g., 10 mol%, 15 mol%, or 20 mol%) of accessory lipids, and 0.5-15 mol% (e.g., 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, or 14 mol%) of amphiphilic lipids. In some embodiments, the cationic lipid in the second type of LNP comprises or is SM102. In some embodiments, the accessory lipid in the second type of LNP comprises or is phospholipid. In some embodiments, the accessory lipid in the second type of LNP comprises or is DSPC. In some embodiments, the structural lipid in the second type of LNP comprises or is cholesterol or a derivative thereof. In some embodiments, the amphiphilic lipid in the second type of LNP comprises or is DMG-PEG. In some embodiments, the amphiphilic lipid in the second type of LNP comprises or is DMG-PEG2000.

[0465] In some embodiments, the second type of LNP comprises 20-50 mol% ionizable cationic lipids. For example, the lipid nanoparticles may comprise 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mol% ionizable cationic lipids.

[0466] In some embodiments, the second type of LNP comprises 50-60 mol% ionizable cationic lipids. For example, the second type of LNP may comprise 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol% ionizable cationic lipids.

[0467] In some embodiments, the second type of LNP contains 5-25 mol% DSPC, optionally 2-15 mol% DSPC; for example, the lipid nanoparticles may contain 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mol% DSPC.

[0468] In some embodiments, the second type of LNP contains 25-55 mol% cholesterol, optionally 30-40 mol% cholesterol. For example, the lipid nanoparticles may contain 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 mol% cholesterol.

[0469] In some embodiments, the second type of LNP contains 0.5-15 mol% DMG-PEG, optionally 1-2 mol% DMG-PEG. For example, the lipid nanoparticles may contain 1, 1.5, or 2 mol% DMG-PEG. In some embodiments, the DMG-PEG is DMG-PEG2000.

[0470] In some embodiments, the second type of LNP comprises 50 mol% ionizable cationic lipids, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG.

[0471] In some embodiments, the second type of LNP comprises 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG. In some embodiments, the second type of LNP comprises 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG2000.

[0472] In some embodiments, the nucleic acid-lipid nanoparticle composition does not contain a second type of LNP. In some embodiments, the nucleic acid-lipid nanoparticle composition contains only one type of LNP, wherein at least part or more than 50% (e.g., more than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) of the LNP simultaneously encapsulates the aforementioned capped linear DNA and the aforementioned functional protein (or accessory nucleic acid).

[0473] In some embodiments, the N:P ratio in the LNP of the nucleic acid-lipid nanoparticle composition of this application is about 2:1 to about 30:1 (e.g., 3:1 to 24:1; 5:1 to 20:1, 7:1 to 15:1, 8:1 to 13:1, 8:1-20:1, 9:1 to 12:1, 9.5:1 to 10.5:1, 6:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, etc.).

[0474] The term "N:P ratio" as used herein, also referred to as "nitrogen-phosphorus ratio," "N / P," or "N:P" in this application, represents the molar ratio of protonable nitrogen in an ionizable cationic lipid to phosphate groups in a nucleic acid molecule (e.g., DNA, RNA). The N:P ratio describes the molar ratio of amino groups (N+) in an ionizable cationic lipid. + The cationic charge of ) and the phosphate groups (PO4) in the nucleic acid backbone - The ratio between the anionic charges of ions is the basis for the recombination of ionizable cationic lipids and nucleic acids through electrostatic interactions.

[0475] In some embodiments, the N:P ratio in the first type of LNP is the same as that in the second type of LNP. In some embodiments, the N:P ratio in the first type of LNP is different from that in the second type of LNP. In some embodiments, the N:P ratio in the first type of LNP is approximately 3:1-24:1, 6:1-18:1, 8:1-20:1, 7:1, 8:1, 9:1, 10:1, 12:1, 13:1, 14:1, 15:1, or 16:1. In some embodiments, the N:P ratio in the second type of LNP is approximately 5:1 to 20:1, 7:1 to 15:1, 8:1 to 13:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0476] In some embodiments, the LNP of this application has an average particle size of about 50-200 nm. In some embodiments, the LNP of this application has an average particle size of about 70 nm to about 120 nm. For example, about 80 nm-120 nm, about 90 nm-100 nm, about 100 nm, etc.

[0477] Encapsulating LNPs is a method known in the art. By optimizing the LNP preparation process, LNPs with selectable particle size ranges (e.g., 96nm, 97nm, 98nm, 99nm, 100nm, 101nm, 102nm, 103nm, 104nm, 105nm, 106nm, 107nm, 109nm, 110nm, 111nm, 112nm, 113nm, 114nm, 115nm, 116nm, 117nm) can be obtained. LNPs of 119 nm, 120 nm, or 119 nm, and further improvements in encapsulation efficiency (e.g., controlling the LNP encapsulation efficiency in the nucleic acid-lipid nanoparticle composition of this application to 80%, 85%, or 90% or more; or controlling the empty loading rate to no more than 10%, such as no more than 9%, no more than 8.5%, no more than 3%, or about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 7.1%, 7.6%, 7.7%, 7.9%, or 8.3%). As used in this application, "encapsulation efficiency" refers to the proportion of LNPs containing at least one nucleic acid molecule to the total number of LNPs. For example, "LNP encapsulation efficiency in the nucleic acid-lipid nanoparticle composition" refers to the proportion of LNPs containing at least one nucleic acid molecule to the total number of LNPs in the nucleic acid-lipid nanoparticle composition. When the first type of LNP and the second type of LNP encapsulate nucleic acid molecules separately and then mix them, the encapsulation efficiency of these two types of LNPs can be expressed by "first type of LNP encapsulation efficiency" and "second type of LNP encapsulation efficiency," respectively. The "encapsulation efficiency of Type I LNPs" refers to the proportion of Type I LNPs containing at least one nucleic acid molecule out of the total number of Type I LNPs. Similarly, the "encapsulation efficiency of Type II LNPs" refers to the proportion of Type II LNPs containing at least one nucleic acid molecule out of the total number of Type II LNPs. Specifically, LNP preparation can be optimized by adjusting the pH of the buffer or aqueous phase, the volume of different buffers or aqueous phases, and the composition ratio of the buffer or aqueous phase to obtain LNPs that meet expectations in terms of LNP properties, encapsulation efficiency, and stability.

[0478] In some embodiments, the nucleic acid-lipid nanoparticle composition of this application, in addition to LNP and the engineered nucleic acid molecule, also contains a buffer component and a cryoprotectant.

[0479] In some implementations, the buffer solution may be selected from: Examples of buffers include, but are not limited to, citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, glycerophosphate, calcium lactate, calcium lactobionate, propionic acid, calcium levulinate, valeric acid, calcium hydrogen phosphate, phosphoric acid, tricalcium phosphate, calcium hydrogen phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dipotassium hydrogen phosphate, diphosphate, potassium mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate, magnesium hydroxide, aluminum hydroxide, alginate, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium dodecyl sulfate, and combinations thereof.

[0480] In some implementations, the cryoprotectant may be selected from substances such as sugars / polyols, polymers, surfactants, amino acids, and salts, wherein the sugar may be selected from lactose, sucrose, trehalose, galactose, etc.

[0481] In some embodiments, the amount of the cryoprotectant is 1 to 50% w / w, such as from 2 to 50% w / w, or from 4 to 45% w / w, or from 6 to 12% w / w, or alternatively, from 6 to 10% w / w, or most preferably from 7 to 9% w / w, for example 8.7% w / w.

[0482] In some embodiments, the nucleic acid-lipid nanoparticle composition comprises a composition of the aforementioned lipid nanoparticles, nucleic acid molecules, and an external phase buffer.

[0483] In some embodiments, the external phase buffer comprises: tromethamine (Tris), sodium acetate (NaOAc), and / or sucrose, with a pH of 7-8, for example, a pH rounded to 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or 7.9.

[0484] In some embodiments, the content of the tromethamine is selected from 10-30 mmol / L, optionally 15-25 mmol / L, optionally 15 mmol / L, 15.5 mmol / L, 16 mmol / L, 16.5 mmol / L, 17 mmol / L, 17.5 mmol / L, 18 mmol / L, 18.5 mmol / L, 19 mmol / L, 19.5 mmol / L, 20 mmol / L, 20.5 mmol / L, 21 mmol / L, 21.5 mmol / L, 22 mmol / L, 22.5 mmol / L, 23 mmol / L, 23.5 mmol / L, 24 mmol / L, 24.5 mmol / L, 25 mmol / L, optionally 20 mmol / L.

[0485] In some embodiments, the sodium acetate content is selected from 0-20 mmol / L, optionally 5-11 mmol / L, optionally 5 mmol / L, 5.5 mmol / L, 6 mmol / L, 6.5 mmol / L, 7 mmol / L, 7.5 mmol / L, 8 mmol / L, 8.5 mmol / L, 9 mmol / L, 9.5 mmol / L, 10 mmol / L, 10.5 mmol / L, 10.6 mmol / L, 10.7 mmol / L, 10.8 mmol / L, 10.9 mmol / L, 11 mmol / L, 11.5 mmol / L, 12 mmol / L, 12.5 mmol / L, 13 mmol / L, and most preferably 10.7 mmol / L.

[0486] In some embodiments, the sucrose content is selected from: 5-15%, optionally 7.5-10%, more preferably 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9%, 9.5%, 10%, optionally 8.7%.

[0487] It should be understood that this application includes the various aspects, embodiments, and combinations of said aspects and / or embodiments described herein. The above description and the following examples are intended to illustrate, not limit, the scope of this application. Other aspects, improvements, and modifications within the scope of this application will be apparent to those skilled in the art to which this application pertains. Therefore, those skilled in the art should recognize that the scope of this application also includes the improvements and modifications to the said aspects and embodiments. Example

[0488] Example 1: Screening of DNA delivery LNP formulations

[0489] 1.1 First round of screening

[0490] 1.1.1 Screening based on Jurkat cell line

[0491] Different lipid mixtures were prepared according to the molar ratio formulations in Table 1 using high-throughput microfluidics. A DNA-encapsulated LNP solution was prepared by mixing the lipid working solution and DNA working solution at a volume ratio of 1:4 at a flow rate of 10 mL / min.

[0492] Table 1: Lipid formulation for first-round DNA delivery (molar percentage)

[0493] The specific steps are as follows:

[0494] (1) Prepare working solutions of different lipid contents according to the lipid prescription in Table 1, wherein the lipids are dissolved in anhydrous ethanol.

[0495] (2) Take an appropriate amount of DNA and dilute it with a citrate buffer solution containing 130mM sodium chloride (10mM, pH 4.0) to adjust the final concentration of the DNA working solution to 0.1mg / mL.

[0496] (3) Using a high-throughput microfluidic instrument, the lipid working solution and the DNA working solution were mixed at a volume ratio of 1:4 and a flow rate of 10 mL / min to prepare the LNP solution loaded with DNA.

[0497] (4) Add 9 times the volume of Tris-NaOAc-8.7% sucrose buffer solution (containing 8.7% sucrose, 20mM Tris, 10.7mM NaOAc, pH=7.5) to dilute the LNP solution and concentrate and purify it with an ultrafiltration tube to remove the ethanol solution in the system.

[0498] (5) The DNA content in the LNP solution was detected by ultraviolet spectrophotometry. The N / P ratio in the LNP was 16:1.

[0499] The physicochemical properties of the LNP formulations loaded with plasmid DNA (pGC_hGAPDH_GFP, commercially available plasmid, the map of which is shown in Figure 25A, containing the GFP coding sequence and the hGAPDH homologous arms on both sides) listed in Table 1 were analyzed, and the results are shown in Figure 1. It can be seen that the LNP loading rate tends to increase with increasing DOPE content and decreasing DMG-PEG2000 content, while the LNP particle size tends to decrease with increasing DMG-PEG2000 content.

[0500] Jurkat cell lines were transfected with the LNP formulation containing the aforementioned DNA, and the percentage of GFP-positive cells was detected after 5 days of incubation. The results are shown in Figure 2. The transfection efficiency increased with decreasing lipid content of C12-200, increasing DOPE content, and decreasing DMG-PEG2000.

[0501] Jurkat cell lines were transfected using the LNP formulations containing DNA listed in Table 1. The DNA delivery efficiency is shown in Figure 3. It can be seen that the reported DNA delivery formulation (i.e., C12-200 LNP (control) in Table 1) achieved a GFP transfection efficiency of 27.1% in Jurkat cells, while the formulation designed in this application achieves a transfection efficiency of nearly 90% (e.g., A20 reaches 82.0%, A9 reaches 85.6%, A13 reaches 91.8%, and A1, A2, A5, A23, etc., also reach or exceed 80%).

[0502] 1.1.2 Screening based on primary T cells

[0503] Five formulas (A1, A23, A20, A9, and A13) with high knock-in efficiency in the Jurkat cell line were selected for gene editing-based knock-in experiments on activated human primary T cells. The mRNA and sgRNA (SpCas9-Mut-5 mRNA and sgRNA hIL2RA; the molar ratio of each component in the SM102 LNP formula was SM102:DSPC:cholesterol:DMG-PEG2000 = 50:10:38.5:1.5, and the SM102 LNP formula in all subsequent specific examples was the same) loaded with the LNP formulas A1, A23, A20, A9, and A13 in Table 1, respectively, were simultaneously transfected into activated human primary T cells. The results, detected by flow cytometry, are shown in Figure 4. Among them, A20 had a knock-in positivity rate of 12.9% at the IL2-RA site in activated human primary T cells, and A9 had a positivity rate of 10.8%. The other three prescriptions that could efficiently transfect plasmid DNA all had knock-in positivity rates of less than 2% on T cells, while the DNA delivery prescription C12-200 LNP (control) reported in the literature had a knock-in positivity rate of only 0.95%.

[0504] As shown in Figures 3 and 4, the effect of LNP formulation on transduction efficiency changes when the DNA template or cell line used for transduction is altered. The preferred LNP formulation for Jurkat cell lines and plasmid DNA is not necessarily the preferred formulation for primary T cell and linear DNA transduction.

[0505] The delivery carrier will be further optimized based on the A20 LNP prescription, which has higher insertion and editing efficiency and is more stable.

[0506] The preparation steps of the above-mentioned SM102 LNP formulation containing mRNA and sgRNA are as follows:

[0507] (1) Accurately weigh a certain amount of SM-102 with a molar mass ratio of 50%, DSPC with a molar mass ratio of 10%, cholesterol with a molar mass ratio of 38.5%, and DMG-PEG2000 lipid with a molar mass ratio of 1.5%, and dissolve them in an appropriate amount of anhydrous ethanol to prepare a lipid working solution for later use (final concentration of lipid working solution 20 mg / mL).

[0508] (2) Prepare a citric acid buffer solution containing 130 mM sodium chloride (10 mM, pH 4.0) and a Tris-NaOAc-8.7% sucrose buffer solution (Tris: 20 mM, NaOAc: 10.7 mM, pH 7.5).

[0509] (3) Take equal amounts of mRNA and sgRNA and dilute them with the sodium chloride-citric acid buffer solution prepared above to adjust the final concentration of the working solution containing mRNA and sgRNA to 0.18 mg / mL.

[0510] (4) Using a microfluidic instrument, the lipid working solution and the RNA working solution were mixed at a volume ratio of 1:3 and a flow rate of 16 mL / min to prepare an LNP solution carrying mRNA and sgRNA.

[0511] (5) Dilute the prepared LNP solution with 9 times the volume of Tris-NaOAc-8.7% sucrose buffer solution, and concentrate and purify it with an ultrafiltration tube to remove the ethanol solution in the system.

[0512] (6) The mRNA and sgRNA content in the LNP solution was detected by ultraviolet spectrophotometry. The N / P ratio in this LNP was 6:1.

[0513] In the specific embodiments of this application, the sgRNA is a covalently linked product of crRNA and tracrRNA. Approximately 20 bases at the 5' end of the sgRNA form a complementary pairing sequence with the genome, while 21-100 nt of bases form the sequence that interacts with Cas9. The chemically synthesized sgRNA nucleotide sequence in the specific embodiments of this application includes the following modifications: thiomodification and 2'-O-methoxymodification (2'-OMe). Specifically, the first 1-3 nucleotides from the 5' end and the first 1-3 nucleotides from the 3' end of the sgRNA are modified with 2'-OMe, and the bonds between the first 1-3 nucleotides from the 5' end and the first 1-3 nucleotides from the 3' end are phosphate thioester bonds. These modifications further enhance sequence stability and prevent degradation. Additionally, for example, the 2'-F of the nucleotides also possesses this function. Furthermore, in this application, when co-transfecting sgRNA, Cas9 mRNA, and DNA template, the mass ratio of the three is 1:1:3.

[0514] In all specific embodiments of this application, the Cas9 protein mRNA was prepared by in vitro transcription (IVT), and the specific preparation method is as follows:

[0515] (1) According to the instructions of the IVT kit (Novoprotein), the IVT reaction system was prepared by mixing 10x Transcription Buffer, ATP, GTP, CTP, PseudoUTP, CleanCap, water for injection, plasmid template and Enzyme Mix respectively.

[0516] (2) The plasmid template is a linearized plasmid encoding a specific Cas9 protein with a T7 promoter.

[0517] (3) The mixed reaction system was placed at 37°C for 40 min.

[0518] (4) Add the appropriate proportion of DNase I to terminate the reaction, and separate and recover the mRNA.

[0519] 1.2 Second Round of Screening

[0520] Based on the lipid proportions of each component in the C12-200 LNP (A20) formulation obtained from the first round of screening, a second round of DNA delivery LNP formulation optimization screening was conducted. Using high-throughput microfluidics, different DNA-loaded LNP solutions were prepared according to the molar ratios in Table 2, following the same procedures as in 1.1.

[0521] Table 2: Results of the second round of DNA delivery lipid formulation and screening

[0522] Knock-in experiments were conducted on activated primary human T cells by simultaneously adding SM102 LNP-coated mRNA and sgRNA (SpCas9-Mut-5 mRNA and sgRNA hIL2RA) and LNP-coated linear DNA (hIL2RA_GFP_HA DNA) prepared according to the molar ratios in Table 2. The results are shown in Figure 5 and Table 2. The second round of screening yielded five formulations (B1, B2, B3, B6, and B9) that achieved positive rates for site-specific knock-in of GFP linear template DNA on primary T cells comparable to or higher than those of A20, with the highest positive rate reaching 13%. This represents a significant improvement compared to the reported LNP delivery efficiency, which is typically below 5%. Furthermore, the loading rates of these formulations were also higher than those of A20.

[0523] Example 2: Template DNA Optimization Design

[0524] 2.1 Optimization of template DNA under electroporation conditions

[0525] DNA templates expressing the GFP reporter gene with different structures were co-introduced into activated human primary T cells via electroporation along with Cas9 mRNA (SpCas9-Mut-5 mRNA) and sgRNA (sgRNA hIL2RA). Cell viability and the insertion of the GFP reporter gene at the IL2RA site were detected by flow cytometry. Electroporation was performed using the H1 electroporator from Suzhou Yida Biotechnology, with the following parameters: 150V voltage, 1200μs electroporation time, 3 electroporations, and an interval of 635ms between each electroporation. 20μg of nucleic acid was added to every 3E6 T cells, with an mRNA:sgRNA:DNA ratio of 1:1:3 (w / w). UNT was the untransfected control group. The results are shown in Figure 6, illustrating cell viability (Figure 6A) and GFP positivity rate (Figure 6B) after electroporation. It is evident that the cell viability and knock-in efficiency of the gene-edited knock-in group using linear double-stranded DNA (hIL2RA_GFP_HA, linear DNA) were superior to those using plasmid DNA (pGC_hIL2RA_GFP, structure shown in Figure 25B).

[0526] 2.2 Optimization of Template DNA

[0527] 2.2.1 DNA Template Element Optimization

[0528] Linear double-stranded uncapped DNA templates expressing GFP reporter genes with different nuclear sequences were packaged using A20 LNPs (A20 in Table 1). These templates included hIL2RA_GFP_TF DNA (FadR-TF in Figure 7), hIL2RA_GFP_ETS1 DNA (ETS1 in Figure 7), hIL2RA_GFP_NFAT DNA (NFAT in Figure 7), hIL2RA_GFP_STAT1 DNA (STAT1 in Figure 7), hIL2RA_GFP_LEF1 DNA (LEF1 in Figure 7), and hIL2RA_GFP_HA DNA (none in Figure 7). The specific structures are shown in Table 8. These templates were then simultaneously transfected with activated human primary T cells along with the mRNA (SpCas9-Mut-5 mRNA) and sgRNA (sgRNA hIL2RA) packaged using SM102 LNPs in section 1.1.2. UNT was the untransfected control group. Flow cytometry was used to analyze the efficiency of IL2RA site-targeted editing and knock-in of the GFP reporter gene in transfected T cells. The results are shown in Figure 7, indicating that the hIL2RA_GFP_TF DNA template containing the FadR-TF nuclear insertion sequence had the highest nuclear insertion efficiency.

[0529] 2.2.2 Comparison of linear double-stranded and double-ended blocked DNA templates

[0530] DNA templates expressing the GFP reporter gene (N2 DNA, i.e., linear double-stranded unclosed hIL2RA_GFP_NFAT; N2_Loop DNA, i.e., double-ended closed DNA template hIL2RA_GFP_N2_Loop; or NLS_Loop DNA, i.e., NLS-modified double-ended closed DNA template hIL2RA_GFP_NLS_Loop, the specific structures of which are shown in Table 8) were packaged in C12-A20 LNPs (i.e., NLS-modified double-ended closed DNA template hIL2RA_GFP_NLS_Loop, the specific structures of which are shown in Table 8) were simultaneously transfected into activated human primary T cells along with the mRNA (SpCas9-Mut-5 mRNA) and sgRNA (sgRNA hIL2RA) packaged in SM102 LNPs used in section 1.1.2 above. UNT served as the untransfected control group. Flow cytometry was used to analyze the efficiency of IL2RA site-targeted editing and knock-in of the GFP reporter gene in transfected T cells. As shown in Figure 8, it can be seen that, under the same LNP system, capped DNA has better transduction efficiency than uncapped linear DNA, and linking to the nuclear localization signal peptide NLS can lead to higher editing insertion efficiency or higher final protein expression levels.

[0531] 2.2.3 Comparison between circular plasmids and paired-end blocked DNA templates

[0532] Using C12 LNPs (specifically, the C12-200 LNP (control) formulation in Table 1, abbreviated as C12 on the horizontal axis of Figure 9) or C12-B2 LNPs (i.e., LNPs with formulation number B2 in Table 2, abbreviated as B2 on the horizontal axis of Figure 9), DNA templates expressing the GFP reporter gene with different structures were packaged (plasmid DNA: pGC_hIL2RA_GFP DNA; or loop DNA: loop-terminated DNA hIL2RA_GFP_Loop; as shown in Table 8). C12 LNPs, along with the mRNA (SpCas9-Mut-5 mRNA) and sgRNA (sgRNA hIL2RA) packaged in SM102 LNPs used in section 1.1.2 above, were simultaneously transfected into activated human primary T cells. UNT served as the untransfected control group. Flow cytometry was used to analyze the efficiency of IL2RA site-targeted editing and knock-in of the GFP reporter gene in transfected T cells. The results are shown in Figure 9. It can be seen that, when the DNA-encapsulated LNP formulation is consistent (both are C12-B2 LNPs), the double-ended DNA template has a better GFP knock-in efficiency than the plasmid DNA template. When the DNA template is plasmid DNA, the knock-in efficiency mediated by C12-200 LNP (control) is better than that mediated by C12-B2 LNP. However, when the DNA template is double-ended linear double-stranded DNA (Loop DNA), the knock-in efficiency mediated by C12-B2 LNP is better than that mediated by C12-200 LNP (control). This further confirms that different LNPs are suitable for delivering different DNA structures; that is, the preferred LNP formulation for plasmid DNA delivery differs from that for linear double-stranded DNA (e.g., double-ended linear double-stranded DNA).

[0533] 2.2.4 Further optimization of DNA template structure

[0534] Optionally, a B2 LNP (i.e., the LNP with prescription number B2 in Table 2) was used to encapsulate a DNA template expressing the GFP reporter gene (TF DNA, i.e., linear DNA template hIL2RA_GFP_TF; ITR DNA, i.e., AAV2 ITR paired-end blocked DNA hIL2RA_GFP_ITR; Loop DNA, i.e., Loop paired-end blocked DNA hIL2RA_GFP_Loop; specific structures are shown in Table 8). This template, along with the mRNA (SpCas9-Mut-5 mRNA) and sgRNA (sgRNA hIL2RA) encapsulated in the SM102 LNP used in section 1.1.2 above, was simultaneously transfected into activated human primary T cells. UNT served as the untransfected control group. Flow cytometry was used to analyze the efficiency of IL2RA site-targeted editing and knock-in of the GFP reporter gene in transfected T cells. The results are shown in Figure 10.

[0535] Furthermore, the DNA template expressing the GFP reporter gene (Loop DNA, i.e., loop-end-closed DNA hTRAC_GFP_Loop; single-stranded DNA (i.e., ssDNA in the horizontal axis of Figure 11), i.e., CTS_hTRAC_GFP; CTS partial single-stranded DNA (i.e., CTSDNA in the horizontal axis of Figure 11, with a double-stranded region less than 5% of the full length), i.e., the DNA template with partial double strands obtained by annealing CTS_hTRAC_GFP and CTS oligo; the specific structure is shown in Table 8) was simultaneously transfected with the mRNA (SpCas9-Mut-5 mRNA) and sgRNA (sgRNA hIL2RA) packaged in the SM102 LNP used in the aforementioned section 1.1.2, and UNT served as the untransfected control group. The results are shown in Figure 11. It is evident that, under the same LNP conditions and with the same linear DNA template, the double-stranded linear DNA template with closed ends has a higher knock-in efficiency than other DNA template structures (such as single-stranded and a small number of double-stranded DNA templates) (see Figures 10 and 11). Among the double-stranded DNA structures with closed ends, the hIL2RA_GFP_Loop structure is superior to the hIL2RA_GFP_ITR structure (see Figure 10). In particular, the double-stranded DNA loaded with C12-B2 LNP can achieve a knock-in positivity rate of 24% in activated primary T cells.

[0536] The method for preparing double-ended blocked DNA in this application embodiment is as follows:

[0537] The target gene sequence, flanked by BsaI restriction sites, was cloned into plasmid DNA and amplified by E. coli fermentation before extraction. Oligonucleotide chains (with capping linkers and, after annealing, double-stranded with BsaI restriction sites) were denatured by heating and then rapidly cooled and annealed. These were added to a reaction system of plasmid DNA, BsaI enzyme, and T4 DNA ligase at a 10:1 ratio. The reaction was carried out at 37°C for 3 hours, followed by denaturation at 75°C for 30 minutes. Linear DNA was digested with DNA exonuclease III and purified by ethanol-isopropanol precipitation or column chromatography. Specific methods are shown in Figure 17 and WO2023122303.

[0538] In the specific embodiments used in this application, the method for linking the NLS polypeptide to linear DNA is as follows: DBCO-dT (i.e., dibenzocyclooctyne-modified deoxythymidine nucleotide, the structure of which is shown in the following formula) located in the linear DNA sequence is linked to the azide ((N3)-)-modified NLS polypeptide via ring strain-promoted click chemistry. In the specific embodiments used in this application, the DBCO-dT is the third T in the capping linker 5'-TTTT-3' of the capping linear DNA (i.e., one side of the 5' end of the target gene sequence), which is linked to LYS(N3) at the C-terminus of the NLS polypeptide. The azide-modified NLS polypeptide is shown below: PKKKRKVEDPYC{LYS(N3)} (SEQ ID NO:40), that is, the NH2 of the Lys residue at the C-terminus of this NLS polypeptide is replaced with N3 relative to the natural Lys.

[0539] Example 3: Optimization of CRISPR-Cas9 gene editing compositions

[0540] 3.1 Jurkat cell transfection

[0541] Jurkat cell lines were transfected with plasmid DNA templates (pGC_hIL2RA_GFP) loaded with LNPs from different formulations (SM102 and C12, with the SM102 LNP formulation being the same as in Example 1, and the C12 LNP formulation being shown in Table 1 as C12-200 LNP (control)). The expression levels of the GFP reporter gene, which was knocked into the IL2RA site, were analyzed by flow cytometry in the Jurkat cell lines. UNT was the untransfected control group. The results are shown in Figure 12. It can be seen that in the Jurkat cell lines, both SM102 LNP and C12-200 LNP (control) mediated plasmid DNA templates achieved high levels of insertion editing efficiency, with C12-200 LNP (control) showing only slightly better performance.

[0542] 3.2 Primary T cells transfected with knock-in GFP

[0543] Different formulations of LNPs were used to introduce a gene knock-in triad into activated primary T cells: mRNA (SpCas9-Mut-5 mRNA, SEQ ID NO:37), sgRNA (sgRNA hIL2RA, SEQ ID NO:35), and linear DNA (hIL2RA_GFP_HA DNA). Flow cytometry was used to analyze the efficiency of IL2RA site-targeted editing and knock-in of the GFP reporter gene. The results are shown in Figure 13A. It can be seen that for the insertion of shorter gene fragments such as the GFP reporter gene, the SM102 LNP RNA+C12 LNP DNA dual LNP delivery system (i.e., SM102 LNP carrying mRNA and sgRNA in Example 1, and C12 LNP carrying DNA as C12-200 LNP (control)) is significantly better than the SM102-carried triad delivery system, and slightly better than the C12 LNP-carried triad delivery system. The SM102 LNP formulation is the same as in Example 1, and the C12 LNP formulation is shown in Table 1 as C12 C12-200 LNP (control). UNT is the untransfected control group.

[0544] Furthermore, it is noteworthy that, using the same SM102-loaded DNA template and incorporating the CRISPR-Cas9 system, the GFP reporter gene knock-in rate in primary cells was significantly lower than that in Jurkat cells. This demonstrates that a DNA delivery system suitable for targeted editing and knock-in in Jurkat cell lines is not necessarily suitable for primary cells.

[0545] Different formulations of LNPs were used to introduce a gene knock-in triad (i.e., mRNA (SpCas9-Mut-5 mRNA), sgRNA (sgRNA hIL2RA (SEQ ID NO:35) or sgRNA hTRAC (SEQ ID NO:36)) and DNA (hIL2RA_GFP_Loop DNA (SEQ ID NO:64) or hTRAC_GFP_Loop (SEQ ID NO:67)) into activated primary T cells, and the efficiency of targeted editing of the knock-in GFP reporter gene was analyzed by flow cytometry. The results are shown in Figure 13B, where the SM102 LNP RNA+C12 LNP (B2) DNA dual LNP delivery system (i.e., the SM102 LNP of Example 1 carrying mRNA and sgRNA, and C12-200 in Table 2) was used. The LNP(B2)-encapsulated DNA delivery system, which encapsulates three components, achieves good knock-in efficiency (i.e., knock-in efficiencies exceeding 11% and 15% respectively) regardless of the gene insertion site. This means that the gene insertion site does not affect the DNA delivery preference for LNP formulations.

[0546] 3.3 Selection of Cas9 fusion protein

[0547] The mRNA / sgRNA was packaged using SM102 LNPs (where the mRNA is Cas9 Mut-5, i.e., SpCas9-Mut-5mRNA (SEQ ID NO:37); DrFECO Cas9, i.e., DrFECO-Cas9-Mut-5mRNA (SEQ ID NO:39); or EcFECO Cas9, i.e., EcFECO-Cas9-Mut-5mRNA (SEQ ID NO:38); and the sgRNA is the sgRNA targeting hIL2RA (SEQ ID NO:35)). The template DNA (TF DNA, i.e., linear double-stranded DNA template hIL2RA_GFP_TF; or Loop DNA, i.e. Loop double-ended DNA hIL2RA_GFP_Loop, see Table 8 for details) was packaged using B2 LNPs (as shown in Table 2). FECO is an L2 cyclic peptide of approximately 20 amino acids that can bind to single-stranded DNA (ssDNA). It originates from RecA family DNA recombinases and promotes ssDNA integration. Ec and Dr indicate the source of FECO, with Ec originating from E. coli and Dr originating from Deinococcus radiodurans Bacterium. The sequences of SpCas9-Mut-5 mRNA, EcFECO-Cas9-Mut-5, and DrFECO-Cas9-Mut-5 are shown in the sequence listing at the end of this article.

[0548] The knock-in efficiency of the GFP reporter gene was analyzed using flow cytometry. A dual LNP delivery system was used: B2LNPs (i.e., C12-200 LNPs (B2)) carried different types of template DNA, and SM102 LNPs carried different types of mRNA encoding Cas9 fusion proteins. Both LNPs were simultaneously transfected into activated human primary T cells to allow the GFP gene to be inserted at the IL2RA site. UNT served as the untransfected control group. The results are shown in Figure 14. Under the same conditions, Cas9 Mut-5 protein exhibited superior knock-in editing performance on activated human primary T cells compared to other Cas9 variants. Similarly, under the same conditions, Loop DNA (dual-ended linear double-stranded DNA) achieved superior knock-in editing performance on activated human primary T cells compared to other linear double-stranded DNAs.

[0549] 3.4 Primary T cell transfection with CD19 CAR knock-in to prepare CAR-T cells

[0550] 3.4.1 Optimization of Homologous Arms

[0551] The gene knock-in triad, consisting of mRNA (SpCas9-Mut-5 mRNA (mut5) or DrFECO-Cas9-Mut-5 mRNA or EcECO-Cas9-Mut-5 mRNA), sgRNA (sgRNA hTRAC), and different types of CD19 CAR-expressing double-ended DNA templates, such as a loop with a 300bp homologous arm (hTRAC_CD19CAR_300HA_Loop), a loop with a 500bp homologous arm (hTRAC_CD19CAR_500HA_Loop), a loop with an 800bp homologous arm (hTRAC_CD19CAR_800HA_Loop), and an 800bp homologous arm loop modified with NLS peptide (hTRAC_CD19CAR_800HA_NLS_Loop), was introduced into activated primary T cells using flow cytometry. The efficiency of targeted TRAC editing to knock into the CD19 CAR gene was analyzed. The results are shown in Figures 20-21, where the Cas protein used in Figure 21 is SpCas9-Mut-5, and UNT is the untransfected control group. It can be seen that using the SM102 LNP RNA+C12 LNP(B2)DNA dual LNP delivery system (i.e., SM102 LNP carrying mRNA and sgRNA, and C12 LNP (i.e., C12-200 LNP(B2)) carrying DNA) with both homologous arms of the DNA being 800 bp in length, a good knock-in effect was achieved (reaching a knock-in efficiency of over 9.8%).

[0552] 3.4.2 Optimization of LNP Encapsulation Combination

[0553] Different formulations of LNP were used to introduce three gene knock-in components (i.e., mRNA (SpCas9-Mut-5 mRNA), sgRNA (sgRNA hTRAC), and DNA (Loop double-ended DNA hTRAC_CD19CAR_300HA_Loop)) into activated primary T cells, and the efficiency of targeted editing of the knock-in CD19 CAR reporter gene was analyzed by flow cytometry. UNT was the untransfected control group. As shown in Figure 22, in the comparison of the SM102 LNP RNA+C12 LNP(B2) DNA dual LNP delivery system (i.e., SM102 LNP carrying mRNA and sgRNA, and C12-200 LNP(B2) carrying DNA in Example 1), the SM102 LNP carrying three components single LNP delivery system, and the C12 LNP(B2) carrying three components single LNP delivery system, the C12 LNP(B2) carrying three components single LNP delivery system had the best delivery effect (achieving an insertion editing efficiency of over 9%, which is significantly higher than the dual LNP system and the SM102 carrying three components system).

[0554] Example 4: Comparative Tests of More DNA Structures

[0555] 4.1 Influence of DNA Structure

[0556] LNP was used to introduce a gene knock-in triad into activated primary T cells, namely mRNA (SpCas9-Mut-5 mRNA (SEQ ID NO:37, referred to as m5 in the horizontal axis of Figure 23) or DrFECO-Cas9-Mut-5 mRNA (SEQ ID NO:39, referred to as FECO in the horizontal axis of Figure 23)), sgRNA (sgRNA hTRAC), and DNA (circular single-stranded DNA CSS_hTRAC_CD19CAR_800HA (CSS, SEQ ID NO.73), Loop double-end blocked DNA hTRAC_CD19CAR_800HA_Loop (Loop)). UNT was the untransfected control group. The efficiency of targeted editing and knock-in of the CD19 CAR gene was analyzed by flow cytometry. The results are shown in Figure 23. Regardless of whether the SM102 LNP RNA + C12 LNP(B2) DNA dual-LNP delivery system (i.e., SM102 LNP carrying mRNA and sgRNA, and C12-200 LNP(B2) carrying DNA as in Example 1) or the single-LNP delivery system C12 LNP(B2) carrying the three components was used, the knock-in efficiency of loop-end-closed DNA was superior to that of circular single-stranded DNA. Furthermore, for target gene sequences larger than 1KB (such as CAR genes, which are typically longer than 2KB with homologous arm sequences), the single-LNP system showed higher insertion / editing efficiency compared to the dual-LNP system.

[0557] 4.2 Exogenous promoters vs. endogenous promoters

[0558] Different formulations of LNP were used to introduce three gene knock-in components into activated primary T cells: mRNA (SpCas9-Mut-5 mRNA), sgRNA (sgRNA hTRAC), and loop-terminated DNA (hTRAC_CD19CAR_800HA_Loop (TRAC) using the TRAC endogenous promoter, and template DNA hTRAC_pEFS_CD19CAR_800HA_Loop (pEFS, see Table 8 and SEQ ID NO:71) with the EFS exogenous promoter). UNT was the untransfected control group. The efficiency of targeted editing and knock-in of the CD19 CAR gene was analyzed by flow cytometry. As shown in Figure 24, when the target gene sequence is a large fragment of more than 1KB, such as a CAR coding sequence, the single LNP delivery system with C12 LNP(B2) loaded with three components was significantly superior to the SM102 LNP RNA+C12 LNP(B2) DNA dual LNP delivery system in comparison with the single LNP delivery system with C12 LNP(B2) loaded with three components. In addition, under the same conditions, the exogenous promoter was more effective than the endogenous promoter. However, the promoter did not affect the preference of linear double-stranded DNA for the LNP delivery formula.

[0559] The specific embodiment of this application shows the following method for preparing LNPs containing mRNA, sgRNA, and DNA:

[0560] (1) As needed, according to the component ratio in Table 2, accurately weigh a certain amount of C12-200, DOPE, cholesterol and DMG-PEG2000 lipids in the required molar mass ratio and add an appropriate amount of anhydrous ethanol to dissolve them and prepare lipid working solution for later use.

[0561] (2) Take an appropriate amount of mRNA, sgRNA and DNA, and dilute them with sodium chloride-citric acid buffer solution (sodium chloride: 130mM, citric acid: 10mM, pH 4.0) to adjust the final concentration of the nucleic acid working solution to 0.1mg / mL.

[0562] (3) Using a microfluidic instrument, the lipid working solution and the nucleic acid working solution were mixed at a volume ratio of 1:3 and a flow rate of 16 mL / min to prepare an LNP solution containing the three components.

[0563] (4) Add 9 times the volume of Tris-NaOAc-8.7% sucrose buffer solution (containing 8.7% sucrose, 20mM Tris, 10.7mM NaOAc, pH=7.5) to dilute the LNP solution and concentrate and purify it with an ultrafiltration tube to remove the ethanol solution in the system.

[0564] (6) The nucleic acid content in the LNP solution was detected by ultraviolet spectrophotometry.

[0565] Example 5: Testing of different cell types

[0566] Different formulations of LNP were used to introduce three components of gene knock-in nucleic acid into HepG2, Jurkat, 293T cell lines and activated human primary T cells. These components were mRNA (SpCas9-Mut-5 mRNA), sgRNA (sgRNA hTRAC), and loop-terminated DNA (hTRAC_pEFS_CD19CAR_800HA_Loop, see Table 8 and SEQ ID NO:71). After 7 days of incubation, the efficiency of targeted editing and knock-in of the CD19 CAR gene was analyzed by flow cytometry. UNT was the untransfected control group. The results are shown in Figures 27A, 27B, 27C, and 27D. When the target cells are HepG2, Jurkat, and 293T cell lines, among the delivery systems that encapsulate the three components in SM102 LNP, MC3 LNP, ALC0315 LNP, and C12-B2 LNP (abbreviated as B2, as shown in Table 2), the MC3 LNP delivery system is the best. However, when the target cells are activated human primary T cells, the C12-B2 LNP (abbreviated as B2) is the best.

[0567] The preparation steps for the LNP formulations containing mRNA, sgRNA, and DNA are as follows:

[0568] (1) Accurately weigh a certain amount of lipids and dissolve them in an appropriate amount of anhydrous ethanol to prepare lipid working solutions for later use (lipid working solution concentration 20 mg / mL). Among them, the proportion of each component in the ALC0315 LNP formulation is ALC-0315:DSPC:cholesterol:PEG = 46.3:9.4:42.7:1.6; the proportion of each component in the MC3 LNP formulation is D-Lin-MC3-DMA:DSPC:cholesterol:PEG-DMG2000 = 50:10:38.5:1.5; the molar ratio of each component in the SM102 LNP formulation is SM102:DSPC:cholesterol:DMG-PEG2000 = 50:10:38.5:1.5; the components of C12-B2 LNP (abbreviated as B2) are shown in Table 2.

[0569] (2) Prepare a citric acid buffer solution containing 130 mM sodium chloride (10 mM, pH 4.0) and a Tris-NaOAc-8.7% sucrose buffer solution (Tris: 20 mM, NaOAc: 10.7 mM, pH 7.5).

[0570] (3) Take an appropriate amount of template DNA, Cas9 mRNA and sgRNA and mix them in a mass ratio of 5:2:2. Dilute them with the sodium chloride-citric acid buffer solution prepared above and adjust the concentration of the three-component nucleic acid working solution to 0.18 mg / mL.

[0571] (4) Using a microfluidic instrument, the lipid working solution and the RNA working solution were mixed at a volume ratio of 1:3 and a flow rate of 16 mL / min to prepare an LNP solution containing the three components of nucleic acid.

[0572] (5) Dilute the prepared LNP solution with 9 times the volume of Tris-NaOAc-8.7% sucrose buffer solution, and concentrate and purify it with an ultrafiltration tube to remove the ethanol solution in the system.

[0573] (6) The total nucleic acid content in the LNP solution was determined by ultraviolet spectrophotometry. The N / P ratio in each LNP was 6:1. The formulations of ALC0315, MC3, and SM102 LNPs in all subsequent specific examples are the same as those here.

[0574] Example 6: Optimization of the proportion of nucleic acid components in LNP

[0575] 6.1 N / P ratio screening of C12-200 LNP(B2) formulation

[0576] Keeping the lipid components in the formulation constant, C12-200 LNP(B2) with different N / P ratios were prepared. These were then introduced into activated human primary T cells, introducing three components of knock-in nucleic acid: mRNA (SpCas9-Mut-5 mRNA), sgRNA (sgRNA hTRAC), and loop-terminated DNA (hTRAC_pEFS_CD19CAR_800HA_Loop, see Table 8 and SEQ ID NO:71). After 7 days of incubation, the efficiency of targeted editing and knock-in of the CD19 CAR gene was analyzed by flow cytometry. UNT was the untransfected control group. The results are shown in Figure 28. When the N / P ratio was below 16, the knock-in efficiency of CD19CAR initially increased synchronously with increasing N / P; however, when the N / P ratio was above 16, further increases in N / P resulted in little difference in knock-in efficiency.

[0577] 6.2 Proportion of the three nucleic acid components loaded in the C12-200 LNP (B2) formulation

[0578] Using C12-200 LNP(B2) to package three components of nucleic acid in different proportions, gene knock-in three components of nucleic acid were introduced into activated human primary T cells, namely mRNA (SpCas9-Mut-5 mRNA), sgRNA (sgRNA hTRAC), and loop-terminated DNA (hTRAC_pEFS_CD19CAR_800HA_Loop, see Table 8 and SEQ ID NO:71). After 7 days of incubation, the efficiency of targeted editing and knock-in of the CD19 CAR gene was analyzed by flow cytometry. UNT was the untransfected control group. The results are shown in Figure 29. The knock-in efficiency of CD19CAR was good in the range of DNA:mRNA:sgRNA = 3:2:2 to 8:2:2, with the optimal ratio of three components being 5:2:2.

[0579] Example 7: Optimization of the formulation range of a three-component LNP formulation for primary T cell delivery of nucleic acids

[0580] 7.1 Optimization of the component range of LNP formulation C12-200 - Target gene CAR

[0581] Different lipid mixtures were prepared according to the molar ratio formulations in Table 3 using high-throughput microfluidic control. A three-component LNP solution containing nucleic acid was prepared by mixing the lipid working solution and nucleic acid working solution at a volume ratio of 1:4 at a flow rate of 10 mL / min.

[0582] Table 3: Optimization of Component Ranges (Molar Percentage) for LNP Formulation C12-200

[0583] The specific steps are as follows:

[0584] (1) Prepare working solutions of different lipid contents according to the lipid prescription in Table AA, wherein the lipids are dissolved in anhydrous ethanol.

[0585] (2) Take an appropriate amount of template DNA, Cas9 mRNA (SpCas9-Mut-5 mRNA), and sgRNA and mix them in a mass ratio of 5:2:2. Dilute them with a citrate buffer solution containing 130mM sodium chloride (10mM, pH 4.0) to adjust the final concentration to 0.1mg / mL and prepare the nucleic acid working solution.

[0586] (3) Using a high-throughput microfluidic instrument, the lipid working solution and the nucleic acid working solution were mixed at a volume ratio of 1:4 and a flow rate of 10 mL / min to prepare an LNP solution containing the three components of nucleic acid.

[0587] (4) Add 9 times the volume of Tris-NaOAc-8.7% sucrose buffer solution (containing 8.7% sucrose, 20mM Tris, 10.7mM NaOAc, pH=7.5) to dilute the LNP solution and concentrate and purify it with an ultrafiltration tube to remove the ethanol solution in the system.

[0588] (5) The nucleic acid content in the LNP solution was detected by ultraviolet spectrophotometry. The N / P ratio in C12-200 LNP was 16:1, and the N / P ratio in MC3 LNP was 6:1.

[0589] The physicochemical properties of LNP formulations containing three nucleic acid components—mRNA (SpCas9-Mut-5 mRNA), sgRNA (sgRNA hTRAC), and loop-terminated DNA (hTRAC_pEFS_CD19CAR_800HA_Loop2, sequence SEQ ID NO.80, which differs from hTRAC_pEFS_CD19CAR_800HA_Loop (see Table 8 and SEQ ID NO:71) in that it lacks a nuclear sequence; the sequences other than the end-capped DNA are shown in the sequence listing at the end of this document)—were analyzed. The results are shown in Table 3. It can be seen that the LNP loading rate tends to decrease with increasing C12-200 content, while the LNP particle size tends to increase with increasing C12-200 content.

[0590] Activated human primary T cells were transfected using the LNP formulation described above. After 7 days of incubation, the percentage of T cells positive for CD19 CAR and CD3 / TCR complex expression was measured. UNT served as the untransfected control group. The results are shown in Figures 30A and 30B. Both the CD19 CAR knock-in efficiency and the CD3 / TCR complex knockout efficiency initially increased synchronously with the lipid content of C12-200, and then decreased. LNP formulations with a C12-200 lipid content in the range of 20-45% showed superior efficacy compared to the previously disclosed MC3 LNP formulation.

[0591] 7.2 Optimization of DOPE and PEG component range in LNP formulation - Target gene CAR

[0592] Table 4: Optimization of DOPE and PEG component ranges (molar percentage) in LNP formulation

[0593] Using high-throughput microfluidics, LNP solutions of different nucleic acid tricomponents were prepared according to the molar ratio prescriptions in Table 4, with the specific methods and steps being the same as in 7.1.

[0594] The physicochemical properties of the LNP formulations containing the three nucleic acid components—mRNA (SpCas9-Mut-5mRNA), sgRNA (sgRNA hTRAC), and loop-terminated DNA (hTRAC_pEFS_CD19CAR_800HA_Loop2 (SEQ ID NO:80), same as in 7.1)—were analyzed, and the results are shown in Table 4. It can be seen that the LNP loading rate and particle size tend to decrease with increasing PEG-DMG2000 content, while initially increasing and then decreasing with increasing DOPE content.

[0595] Activated human primary T cells were transfected using the LNP formulation described above. After 7 days of incubation, the percentage of T cells positive for CD19CAR and CD3 / TCR complex expression was measured. UNT cells served as the untransfected control group. The results are shown in Figures 31A, 31B, 32A, and 32B. Both the CD19CAR knock-in efficiency and the CD3 / TCR complex knockout efficiency initially increased and then decreased with increasing PEG-DMG2000 and DOPE ratios. LNP formulations with DOPE ratios of 15-40% (especially 20%-40%) and PEG-DMG2000 ratios of 1-2.5% showed superior efficacy compared to the previously disclosed MC3 LNP formulation.

[0596] Based on the experimental results in 7.1 and 7.2, it can be seen that for LNP formulations containing three nucleic acid components, when the DNA template is linear double-stranded DNA (especially double-ended blocked DNA), the proportions of each component of LNP are as follows: C12-200 20-45%, DOPE 20-40%, cholesterol 23.25-58.25%, and PEG 1-2.5%, which is superior to the published MC3 LNP formulation.

[0597] Example 8: Preparation and efficacy evaluation of LNP formulation for CAR-T

[0598] 8.1 Effects of different donor sources on the preparation of LNP-transfected CAR-T cells

[0599] C12-B2 LNPs (B2) containing three nucleic acid components—mRNA (SpCas9-Mut-5 mRNA), sgRNA (sgRNA hTRAC), and CD19 CAR-expressing loop double-ended DNA (hTRAC_pEFS_CD19CAR_800HA_Loop, see Table 8 and SEQ ID NO:71)—were transfected into activated human primary T cells from different donor sources. After 7 days of incubation, the percentage of CD19CAR-positive T cells was measured. The results are shown in Figure 33 (including two batches of LNPs: LNP1 and LNP2). The percentage of CD19CAR-positive T cells varied to some extent among cells from different donor sources, but most achieved a positive rate of around 20-30%. The T cell culture conditions were Lonza x-vivo15 medium supplemented with 4% inactivated fetal bovine serum (FBS, Gibco), 100 IU / mL human interleukin-2 (IL-2, Gibco), 5 ng / mL human interleukin-7 (IL-7, Gibco), and 5 ng / mL human interleukin-15 (IL-15, Gibco). Unless otherwise specified, the T cell culture and transfection conditions (including the LNP formulation) in all subsequent specific examples are the same as those described here.

[0600] 8.2 LNP transfection for CAR-T amplification culture

[0601] Activated human primary T cells were transfected with LNP (B2) to prepare CD19 CAR-T cells. The CAR-T cell density was maintained at 0.5E6 to 1E6 cells / mL for 7 days of cell proliferation and expansion culture. Cell counts were performed at different culture time points. The fold increase in cell count at different time points during the culture phase is shown in Figure 34. The growth rate of LNP-transfected CAR-T cells (LNP-CD19CAR) was similar to that of untransfected T cells (UNT).

[0602] 8.3 Phenotypic Analysis of CAR-T Cells Prepared by LNP Transfection

[0603] Flow cytometry phenotypic analysis was performed on cells cultured for 7 days. The results are shown in Tables 5-7. CAR-T cells prepared by LNP transfection expressing different types of CARs (i.e., CD19 CAR, BCMACAR, and CD7 CAR sequences are shown in the sequence listing at the end of the article, and the encapsulated capped linear double-stranded DNA sequences are shown in SEQ ID NO. 71, 81, and 83, respectively) all showed a CAR positivity rate (CAR+) higher than 25%, while the CD3 / TCR+ ratio was lower than 5%. This demonstrates that the optimized LNP and gene editing composition used in this project is suitable for the delivery and expression of various CARs.

[0604] Furthermore, the CAR-T cells prepared by LNP showed similarities to untransfected T cells (UNT) in CD4 / CD8 typing and T cell differentiation typing, indicating that the method of preparing CAR-T cells by LNP does not significantly change the T cell typing.

[0605] Table 5: Flow cytometry typing of CD19 CAR-T cells

[0606] Table 6: Flow cytometry typing of CD7 CAR-T cells

[0607] Table 7: Flow Cytometry Classification of BCMACAR-T Cells

[0608] 8.4 LNP transfection for in vitro CAR-T cell killing

[0609] CD19 CAR-T in vitro killing

[0610] CD19 CAR-T cells (CD19 CAR-T) prepared by LNP transfection, untransfected T cells (UNT T), and Raji-fLuc target cells stably expressing luciferase (Luciferase-labeled human Burkitt's lymphoma cells, highly expressing CD19, Raji-fLuc, Nanmo Biotechnology, catalog number: NM-B07-1) were co-incubated for 48 h at different effector cell / target cell ratios (E / T ratio). The cells in the culture system were then lysed, and the luciferase signal was detected to calculate the CAR-T cell killing ability. The culture conditions were RPMI 1640 medium (Gibco) supplemented with 10% inactivated fetal bovine serum (FBS, Gibco). The culture conditions for the in vitro CAR-T cell killing experiments in all subsequent specific examples were the same as those described here. As shown in Figure 35, CD19 CAR-T cells transfected with LNP showed significantly higher cytotoxicity against Raji-fLuc target cells than untransfected T cells (UNT).

[0611] CD7 CAR-T in vitro killing

[0612] CD7 CAR-T cells (CD7 CAR-T, CAR sequence SEQ ID NO.84) prepared by LNP transfection, untransfected LNP T cells (UNT T), and Jurkat-fLuc target cells stably expressing luciferase (as before) were co-incubated for 48 h at different effector cell / target cell ratios (E / T ratio). Cells were then lysed, and the luciferase signal in the culture system was detected to calculate the CAR-T cell killing ability. As shown in Figure 36, the cytotoxicity of CD7 CAR-T cells prepared by LNP transfection against Jurkat-fLuc target cells was significantly higher than that of untransfected LNP T cells (UNT T).

[0613] BCMACAR-T in vitro killing

[0614] LNP-transfected BCMACAR-T cells (BCMACAR-T), untransfected LNP T cells (UNT T), and MM.1S-fLuc target cells stably expressing luciferase (as before) were co-incubated for 48 h at different effector / target cell ratios (E / T ratio). After cell lysis, the luciferase signal in the culture system was detected and the CAR-T cell killing ability was calculated, as shown in Figure 37. The killing ability (cytotoxicity) of LNP-transfected BCMACAR-T cells against MM1S-fLuc target cells was significantly higher than that of untransfected LNP T cells (UNT T).

[0615] The efficacy of CAR-T cells prepared by 8.5LNP transfection in mice

[0616] The efficacy of CD19 CAR-T in tumor-infected mouse models

[0617] CD19 CAR-T cells prepared by LNP transfection were collected after 7 days of expanded culture and then cryopreserved in liquid nitrogen for later use. NCG mice (6-8 weeks old, female) were inoculated via tail vein with Raji-luc cells overexpressing luciferase (5E5 / mouse, as before), and grouped for in vivo imaging after 4 days of feeding. On the second day, after the CD19 CAR-T cells were thawed in a water bath, tumor-bearing mice were inoculated via tail vein with 1E6 CAR-positive CAR-T cells, while the control group was inoculated with 1E6 untransfected LNP T cells (UNT T) cultured and expanded under the same conditions, with 5 mice in each group. During the feeding process, changes in tumor cell fluorescence signals were observed via in vivo imaging, and the survival status of the mice was simultaneously monitored and recorded to evaluate the tumor-suppressive effect of CAR-T drugs in mice. As shown in Figures 38A and 38B, compared with mice inoculated with untransfected LNP T cells (UNT T), mice inoculated with CD19 CAR-T cells (CAR-T) showed a slower rate of increase in tumor cell fluorescence signal (see Figure 38A) and a significantly longer survival time (see Figure 38B), indicating that CD19 CAR-T cells exhibit better tumor killing and inhibition effects in mice.

[0618] In all embodiments of this application, the DNA template design scheme is shown in Table 8, and the Cas9 variant and fusion protein scheme is shown in Table 9.

[0619] Table 8: DNA template design scheme. DNA template sequences (sequences of plasmids excluding the backbone, and sequences of capped double-stranded linear DNA excluding the capping linkers) are shown in SEQ ID NO: 57-73, 80, 81, and 83.

[0620] Note: seq1-L and seq1-R are truncated versions of AAV ITR, containing the nuclear insertion sequence. seq1-L and seq1-R are located at the 5' and 3' ends of the linear nucleic acid strand, respectively, i.e., on one side of the 5' end and the other side of the 3' end of the target gene sequence. DNA structure diagrams with different structural features are shown in Figures 16A and 16B. GFP indicates that the target gene sequences to be inserted are all GFP (fluorescent protein) gene sequences; CD19 CAR indicates that the target gene sequences to be inserted are all CAR gene sequences targeting CD19. Genes listed under each gene homology arm column indicate that the homology arm sequence originates from that gene sequence.

[0621] Table 9: Cas9 protein variants and fusion protein design schemes (amino acid sequences are shown in SEQ ID NO:42-44 at the end of the document; nucleotide sequences are shown in SEQ ID NO:37-39 at the end of the document).

[0622] The sequences used in the above embodiments of this application are shown in the following sequence listing. It should be understood that the following sequences are merely exemplary sequences for the embodiments of this application and are not intended to limit the scope of this application. The nucleic acid sequences in the following sequence listing may represent DNA or RNA sequences. When representing an RNA sequence, "T" indicates uridine. In this application, uridine encompasses uridine analogues (e.g., 1-methylpseudouridine). Unless otherwise specified, all uridine in the mRNA in the specific embodiments of this application is 1-methylpseudouridine.

[0623] sequence list

Claims

1. A nucleic acid-lipid nanoparticle composition comprising a linear double-stranded DNA molecule and a lipid nanoparticle composition for encapsulating the nucleic acid molecule, wherein the linear double-stranded DNA molecule comprises a complementary double-stranded portion of a target gene, and the nanoparticle composition comprises: (a) Cationic lipids, which account for about 30 mol% to 55 mol% of the total lipids in the composition; (b) Phospholipids, which comprise about 5 mol% to 50 mol% of the total lipids in the composition, for example 10 mol% to 45 mol%; (c) Structural lipids, comprising approximately 20 mol% to 50 mol% of the total lipids in the composition; and (d) Amphiphilic lipids, which account for about 1 mol% to 3 mol% of the total lipids in the composition.

2. The nucleic acid-lipid nanoparticle composition according to claim 1, wherein: The cationic lipid is selected from at least one of SM102, ALC0315, DLin-MC3-DMA, C12-200, C14-4, 306Oi10, Lipid 5, LP-01, DOTMA, DODMA, DLin-KC2-DMA, DOTAP and DC-Chol or a derivative thereof. The phospholipid is selected from at least one of DOPE, DSPC, DPPC, DOPC, DPPG, POPC, POPE, DPPE, DMPE, DSPE, SOPC and SOPE or lipids modified by anionic or cationic modifying groups. The structural lipids are selected from cholesterol and its derivatives. The amphiphilic lipids are selected from at least one of DMG-PEG, PEG-c-DMG, PEG-C14, PEG-c-DMA, PEG-DSPE, ALC0159, PEG-PE, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, Tween-20, Tween-80, PEG-DPG, PEG-s-DMG, DAA, PEG-c-DOMG, and GalNAc-PEG-DSG.

3. The nucleic acid-lipid nanoparticle composition according to claim 1 or 2, wherein, The cationic lipid is C12-200 or a derivative thereof, the phospholipid is DOPE, the structural lipid is cholesterol, and / or the amphiphilic lipid is DMG-PEG, optionally DMG-PEG2000.

4. The nucleic acid-lipid nanoparticle composition according to any one of claims 1-3, wherein the lipid component is composed of any one of the following: It contains approximately 20-45 mol% cationic lipids, approximately 15-40 mol% phospholipids, approximately 23.25-58.25 mol% cholesterol or its derivatives, and approximately 1-2.5% amphiphilic lipids.

5. The nucleic acid-lipid nanoparticle composition according to any one of claims 1-4, wherein the lipid component is composed of any one of the following: Approximately 20 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 58.25 mol% cholesterol or its derivatives, and approximately 1.75% amphiphilic lipids; Approximately 25 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 53.25 mol% cholesterol or its derivatives, and approximately 1.75% amphiphilic lipids; Approximately 30 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 48.25 mol% cholesterol or its derivatives, and approximately 1.75% amphiphilic lipids; Approximately 40 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 38.25 mol% cholesterol or its derivatives, and approximately 1.75% amphiphilic lipids; Approximately 45 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 33.25 mol% cholesterol or its derivatives, and approximately 1.75% amphiphilic lipids; Approximately 35 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 44 mol% cholesterol or its derivatives, and approximately 1% amphiphilic lipids; Approximately 35 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 44.75 mol% cholesterol or its derivatives, and approximately 1.25% amphiphilic lipids; Approximately 35 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 43.5 mol% cholesterol or its derivatives, and approximately 1.5% amphiphilic lipids; Approximately 35 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 43.75 mol% cholesterol or its derivatives, and approximately 1.25% amphiphilic lipids; Approximately 35 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 43.25 mol% cholesterol or its derivatives, and approximately 1.75% amphiphilic lipids; Approximately 35 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 43 mol% cholesterol or its derivatives, and approximately 2 mol% amphiphilic lipids; Approximately 35 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 42 mol% cholesterol or its derivatives, and approximately 3 mol% amphiphilic lipids; Approximately 35 mol% cationic lipids, approximately 20 mol% phospholipids, approximately 42.5 mol% cholesterol or its derivatives, and approximately 2.5 mol% amphiphilic lipids; Approximately 35 mol% cationic lipids, approximately 15 mol% phospholipids, approximately 48.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% amphiphilic lipids; Approximately 35 mol% cationic lipids, approximately 25 mol% phospholipids, approximately 38.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% amphiphilic lipids; Approximately 35 mol% cationic lipids, approximately 30 mol% phospholipids, approximately 33.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% amphiphilic lipids; Approximately 35 mol% cationic lipids, approximately 35 mol% phospholipids, approximately 28.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% amphiphilic lipids; Approximately 35 mol% cationic lipids, approximately 40 mol% phospholipids, approximately 23.25 mol% cholesterol or its derivatives, and approximately 1.75 mol% amphiphilic lipids; Approximately 45 mol% cationic lipids, approximately 40 mol% phospholipids, approximately 14 mol% cholesterol or its derivatives, and approximately 1 mol% amphiphilic lipids; or It contains approximately 55 mol% cationic lipids, approximately 40 mol% phospholipids, approximately 4 mol% cholesterol or its derivatives, and approximately 1 mol% amphiphilic lipids.

6. The nucleic acid-lipid nanoparticle composition according to any one of claims 1-5, wherein the 5' end of one or both strands of the linear double-stranded DNA molecule is connected to the 3' end of its complementary strand by a capping linker, wherein the capping linker is a nucleic acid structure or a non-nucleic acid structure.

7. The nucleic acid-lipid nanoparticle composition according to any one of claims 1-6, wherein the linear double-stranded DNA molecule further comprises an adeno-associated virus (AAV) inverted terminal repeat (ITR) nuclear insertion sequence located at the 5' end and / or 3' end of the target gene, wherein the AAV ITR nuclear insertion sequence comprises the complete D region of the AAV ITR but does not contain the BB' and CC' regions of the AAV ITR.

8. The nucleic acid-lipid nanoparticle composition according to any one of claims 1-7, wherein, The AAV ITR nuclear sequence: (1) Contains a complete AAV terminal break site (TRS) sequence or its core region sequence; (2) A Rep binding element (RBE) sequence containing the complete AAV ITR or its core region sequence; (3) It does not contain a complete TRS sequence or its core region sequence; (4) Does not contain a complete RBE sequence or its core region sequence; or (5) It does not contain a complete TRS sequence or its S core region sequence, nor does it contain a complete RBE sequence or its core region sequence.

9. The nucleic acid-lipid nanoparticle composition according to any one of claims 7-8, wherein the linear DNA comprises an AAV ITR truncated form, and the AAV ITR nuclear insertion sequence is located in the AAV ITR truncated form.

10. The nucleic acid-lipid nanoparticle composition according to any one of claims 7-9, wherein the AAV ITR is the ITR of AAV2.

11. The nucleic acid-lipid nanoparticle composition according to any one of claims 7-10, comprising one, two or more AAV ITR nuclear insertion sequences.

12. The nucleic acid-lipid nanoparticle composition according to any one of claims 7-11, wherein the AAV ITR nuclear insertion sequence comprises one or more base sequences selected from the following: SEQ ID NO:25 to 26, SEQ ID NO:53 to 56 and SEQ ID NO:74-77.

13. The nucleic acid-lipid nanoparticle composition according to any one of claims 7-12, further comprising one or more nuclear insertion sequences selected from the group consisting of: FadR nuclear insertion sequence, ETS1 nuclear insertion sequence, NFAT nuclear insertion sequence, STAT2 nuclear insertion sequence and LEF1 nuclear insertion sequence; optionally, comprising a nuclear insertion sequence selected from one or more of the following nucleotide sequences: SEQ ID NO:3-7.

14. The nucleic acid-lipid nanoparticle composition according to any one of claims 7-13, wherein the 5' end and 3' end of the target gene contain an AAV ITR nuclear insertion sequence, preferably, the AAV ITR nuclear insertion sequence on the 5' end and 3' end of the target gene contains or is SEQ ID NO:25 or SEQ ID NO:26, or contains or is SEQ ID NO:26 and SEQ ID NO:25, respectively.

15. The nucleic acid-lipid nanoparticle composition according to any one of claims 7-14, wherein the end-capping linker is composed of 1-200 nucleotides, for example, 1-100, 2-60, 3-40, 4-30, or 5-20 nucleotides.

16. The nucleic acid-lipid nanoparticle composition according to any one of claims 7-15, wherein the end-capping linker comprises an inverted repeat sequence.

17. The nucleic acid-lipid nanoparticle composition according to any one of claims 6-16, wherein the end-capping linker comprises or is the base sequence shown below: 5'-TTTT-3'.

18. The nucleic acid-lipid nanoparticle composition according to any one of claims 1-17, further comprising one or more nuclear localization signal peptides (NLS); optionally, the NLS is linked to the capping linker; optionally, the NLS comprises a polynucleotide sequence as shown in SEQ ID NO:40, or the amino acid sequence of the NLS is as shown in SEQ ID NO:

40.

19. The nucleic acid-lipid nanoparticle composition according to any one of claims 7-18, wherein the double-stranded portion comprises an AAV ITR nuclear insertion sequence, a target gene, and a homologous arm, said double-stranded portion comprising a structure selected from any one of the following: (1) -AAV ITR nuclear insertion sequence L-homologous arm L-target gene sequence-homologous arm R-AAV ITR nuclear insertion sequence R-; (2) -AAV ITR nuclear insertion sequence L-homologous arm L-target gene sequence -AAV ITR nuclear insertion sequence R-; or (3)-AAV ITR nuclear sequence L-target gene sequence-homologous arm R-AAV ITR nuclear sequence R-.

20. The nucleic acid-lipid nanoparticle composition according to any one of claims 6-19, wherein the 5' end or 3' end of the target gene further comprises one or more selected from: a 2A peptide coding sequence, a regulatory sequence, and a tailing signal.

21. The nucleic acid-lipid nanoparticle composition according to claim 19 or 20, wherein the length of the homologous arm is about 100 to 2000 bp, for example about 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1600 bp or 1800 bp.

22. The nucleic acid-lipid nanoparticle composition according to any one of claims 19-21, wherein the homologous arm is capable of homologous recombination with a sequence selected from the following target genes: human GAPDH gene, human IL2RA gene and human TRAC gene.

23. The nucleic acid-lipid nanoparticle composition according to any one of claims 1-22, wherein the length of the target gene sequence is about 500-5000 bp, for example 1000-4500 bp, 1500-4000 bp, 1000-3500 bp, or 1000-3000 bp.

24. The nucleic acid-lipid nanoparticle composition according to any one of claims 1-23, wherein the target gene encodes a chimeric antigen receptor (CAR).

25. The nucleic acid-lipid nanoparticle composition according to any one of claims 1-24, wherein the nitrogen-to-phosphorus ratio of the lipid nanoparticle composition to the lipid nanoparticles (LNPs) formed by the linear nucleic acid molecules is about 3:1 to 32:1; optionally, about 6:1 to 18:1, 8:1 to 20:1, or 16:1 to 32:

1.

26. The nucleic acid-lipid nanoparticle composition according to claim 25, wherein the lipid nanoparticle composition and the lipid nanoparticles (LNPs) formed therefrom have an average particle size of about 50 nm to about 150 nm; about 70 nm to about 120 nm; about 80-120 nm; or about 100 nm.

27. The nucleic acid-lipid nanoparticle composition according to any one of claims 1-26, further comprising: A composition of a gene-editing molecule encoding a nucleic acid and lipid nanoparticles encapsulating the encoding nucleic acid; optionally, the nucleic acid encoding the gene-editing molecule includes nucleic acids encoding nucleases, helicases, transposases, and / or guide RNA (gRNA).

28. The nucleic acid-lipid nanoparticle composition according to claim 27, wherein the nuclease comprises: Transcription activation-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), CRISPR-associated proteins (Cas), Ago (Argonaute) proteins, a wide range of nucleases, and megaTAL; optionally, the Cas includes Cas9 or a Cas9 variant thereof; optionally, the Cas9 variant contains the following mutations compared to a Cas9 reference sequence: K526A, R691A, Q695A and H698A; The Cas9 reference sequence is shown in SEQ ID NO:41; Optionally, the amino acid sequence of the Cas contains or is as shown in SEQ ID NO:42, or the mRNA sequence of the Cas contains or is as shown in SEQ ID NO:

37.

29. The nucleic acid-lipid nanoparticle composition according to claim 28, wherein the nucleic acid encoding the gene editing molecule comprises mRNA encoding Cas and guide RNA (gRNA); optionally, the guide RNA is sgRNA (single guide RNA).

30. The nucleic acid-lipid nanoparticle composition according to any one of claims 27-29, wherein the lipid nanoparticle composition encapsulating linear nucleic acid molecules and the lipid nanoparticle composition encapsulating gene editing molecules encoding nucleic acids are the same or different.

31. The nucleic acid-lipid nanoparticle composition of claim 30, wherein the lipid nanoparticle composition encapsulating the nucleic acid encoded by the gene editing molecule comprises the following lipid components: about 50 mol% SM-102, about 10 mol% DSPC, about 38.5 mol% cholesterol and about 1.5 mol% DMG-PEG.

32. The nucleic acid-lipid nanoparticle composition of claim 31, wherein the nitrogen-to-phosphorus ratio of the lipid nanoparticles (LNPs) encapsulating linear nucleic acid molecules is about 16:1, and the nitrogen-to-phosphorus ratio of the lipid nanoparticles (LNPs) encapsulating gene editing molecules encoding nucleic acids is about 6:1 or 10:

1.

33. An engineered linear DNA molecule comprising a complementary double-stranded portion of a target gene, the double-stranded portion further comprising an adeno-associated virus (AAV) inverted terminal repeat (ITR) nuclear insertion sequence located on one side of the 5' end and / or one side of the 3' end of the target gene, wherein the 5' end of one or both strands of the double-stranded portion of the linear DNA molecule is connected to the 3' end of its complementary strand by a capping linker, the capping linker being a nucleic acid structure or a non-nucleic acid structure, and the AAV ITR nuclear insertion sequence comprising the complete D region of the AAV ITR but excluding the BB' and CC' regions of the AAV ITR.

34. The engineered linear DNA molecule according to claim 33, wherein, The AAV ITR nuclear sequence: (1) Contains a complete AAV terminal break site (TRS) sequence or its core region sequence; (2) A Rep binding element (RBE) sequence containing the complete AAV ITR or its core region sequence; (3) It does not contain a complete TRS sequence or its core region sequence; (4) Does not contain a complete RBE sequence or its core region sequence; or (5) It does not contain a complete TRS sequence or its S core region sequence, nor does it contain a complete RBE sequence or its core region sequence.

35. The engineered linear DNA molecule according to any one of claims 33-34, wherein the linear DNA comprises an AAV ITR truncated form, and the AAV ITR nuclear sequence is located in the AAV ITR truncated form.

36. The engineered linear DNA molecule according to any one of claims 33-35, wherein the AAV ITR is the ITR of AAV2.

37. The engineered linear DNA molecule according to any one of claims 33-36, comprising one, two or more AAV ITR nuclear insertion sequences.

38. The engineered linear DNA molecule according to any one of claims 33-37, wherein the AAV ITR nuclear insertion sequence comprises one or more base sequences selected from: SEQ ID NO:25 to 26, SEQ ID NO:53 to 56 and SEQ ID NO:74-77.

39. The engineered linear DNA molecule according to any one of claims 33-38, further comprising one or more nuclear insertion sequences selected from the group consisting of: FadR nuclear insertion sequence, ETS1 nuclear insertion sequence, NFAT nuclear insertion sequence, STAT2 nuclear insertion sequence and LEF1 nuclear insertion sequence, preferably comprising a nuclear insertion sequence selected from one or more of the following nucleotide sequences: SEQ ID NO: 3-7.

40. The engineered linear DNA molecule according to any one of claims 33-39, wherein the 5' end and 3' end of the target gene contain AAV ITR nuclear insertion sequences, preferably, the AAV ITR nuclear insertion sequences on the 5' end and 3' end of the target gene respectively contain or are SEQ ID NO:25 or SEQ ID NO:26, or contain or are SEQ ID NO:26 and SEQ ID NO:25 respectively.

41. The engineered linear DNA molecule according to any one of claims 33-40, wherein the end-capping linker consists of 1-200 nucleotides, for example, 1-100, 2-60, 3-40, 4-30, or 5-20 nucleotides.

42. The engineered linear DNA molecule according to any one of claims 33-41, wherein the end-capping linker comprises an inverted repeat sequence.

43. The engineered linear DNA molecule according to any one of claims 33-42, wherein the end-capping linker comprises or is the base sequence shown below: 5'-TTTT-3'.

44. The engineered linear DNA molecule according to any one of claims 33-43, further comprising one or more nuclear localization signal peptides (NLS), optionally, the NLS being linked to the capping linker, optionally, the NLS comprising a polynucleotide sequence as shown in SEQ ID NO:40, or the amino acid sequence of the NLS being shown in SEQ ID NO:

40.

45. The engineered linear DNA molecule according to any one of claims 33-44, wherein the double-stranded portion comprises an AAV ITR nuclear insertion sequence, a target gene, and a homologous arm, said double-stranded portion comprising a structure selected from any one of the following: (1) -AAV ITR nuclear insertion sequence L-homologous arm L-target gene sequence-homologous arm R-AAV ITR nuclear insertion sequence R-; (2) -AAV ITR nuclear insertion sequence L-homologous arm L-target gene sequence -AAV ITR nuclear insertion sequence R-; or (3)-AAV ITR nuclear sequence L-target gene sequence-homologous arm R-AAV ITR nuclear sequence R-.

46. ​​The engineered linear DNA molecule according to any one of claims 33-45, wherein the 5' end and / or 3' end of the target gene further comprises one or more of the following: a 2A peptide coding sequence, a regulatory sequence, and a tailing signal.

47. The engineered linear DNA molecule according to any one of claims 33-46, wherein the length of the homologous arm is about 100 to 2000 bp, for example about 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1600 bp or 1800 bp.

48. The engineered linear DNA molecule according to any one of claims 33-47, wherein the homologous arm undergoes homologous recombination with a sequence selected from the group consisting of: Human GAPDH gene, human IL2RA gene, or human TRAC gene.

49. The engineered linear DNA molecule according to any one of claims 33-48, wherein the length of the target gene sequence is about 500-5000 bp, for example about 1000-4500 bp, about 1500-4000 bp, about 1000-3500 bp, or about 1000-3000 bp.

50. The engineered linear DNA molecule according to any one of claims 33-49, wherein the target gene encodes a chimeric antigen receptor (CAR).

51. Lipid nanoparticles comprising an engineered linear DNA molecule of any one of claims 33-50.

52. A gene editing composition for site-directed insertion of a target gene, comprising: (1) an engineered linear DNA molecule according to any one of claims 33-50, and (2) Gene editing molecules or their encoding nucleic acid molecules, Optionally, the gene-editing molecule is selected from one or more of the following: nuclease, helicase, transposase, nuclease mRNA, helicase mRNA, transposase mRNA, and guide RNA (gRNA).

53. The gene editing composition of claim 52, wherein the nuclease is a programmable DNA-binding protein, optionally, the programmable DNA-binding protein is selected from any one or more of the following: transcription activation-like effector nuclease (TALEN), zinc finger nuclease (ZFN), CRISPR-associated protein (Cas), Ago (Argonaute) protein, a wide range of nucleases, and megaTAL.

54. Lipid nanoparticles comprising the gene editing composition of claim 52 or 53.

55. A method for introducing a target nucleic acid sequence into primary cells, said target nucleic acid sequence being inserted into the genome of said primary cells and / or expressed in said primary cells, said method comprising contacting said primary cells with an engineered linear DNA molecule according to any one of claims 33-50, a gene editing composition according to claim 52 or 53, or a nucleic acid-lipid nanoparticle composition according to any one of claims 1-32.

56. The method of claim 55, wherein the primary cell is a T cell; optionally, an activated primary T cell; optionally, the primary T cell is a human primary T cell.

57. The method according to claim 55 or 56, wherein the target gene is inserted into the T cell receptor α (TRAC) or interleukin 2 receptor α (IL2RA) gene sequence of the primary cells.

58. The method according to any one of claims 55-57, wherein the target nucleic acid sequence encodes a chimeric antigen receptor (CAR).

59. Use of the engineered linear DNA molecule according to any one of claims 33-50, the gene editing composition according to claim 52 or 53, or the nucleic acid-lipid nanoparticle composition according to any one of claims 1-32 in the preparation of a medicament for treating a disease selected from: genetic diseases, tumors, autoimmune diseases, and other diseases requiring treatment by regulating gene expression.

Images