Cis packaging elements for single-stranded single-polarity DNA recombinant adeno-associated virus, spaav vector, and use

Abstract

Provided are cis packaging elements for packaging a single-stranded single-polarity DNA recombinant adeno-associated virus (single polarity AAV, spAAV) vector, i.e., ITR, DtrsA'B', trsA'B', trsA', DttRR, and ttRR, and a packaged single-stranded single-polarity DNA recombinant adeno-associated virus vector. The cis packaging element comprises a Rep binding site (RBS) and a Rep cleavage site (terminal resolution site, trs); the cis packaging element can package a spAAV genome into an AAV capsid protein, namely, the cis packaging element is arranged at the 5' end of the single-stranded single-polarity DNA to be packaged, and a Rep protein packages the single-stranded single-polarity DNA into the AAV capsid protein by means of identification and cleavage. The titer and cell transfection performance of spAAV virus particles packaged by these cis packaging elements are equivalent to those of traditional rAAV virus particles.

Description

Cis packaging element of single-stranded monopolar DNA recombinant adeno-associated virus, spAAV vector and its application

[0001] This application is based on and claims priority from the Chinese patent application with application number 202310875678.3 filed on July 17, 2023, and with the invention name “cis packaging elements, spAAV vectors and applications of single-stranded monopolar DNA recombinant adeno-associated virus”. Technical Field

[0002] The present application relates to the field of gene technology, and in particular to a cis packaging element of a single-stranded monopolar DNA recombinant adeno-associated virus, a spAAV vector and applications. Background Art

[0003] Gene therapy is aimed at genetic mutations caused by gene mutations, abnormal gene expression, etc., and can be treated, prevented or alleviated by providing patients with corrective genetic material, including preventing genetic diseases caused by gene defects, abnormal regulation or abnormal expression, such as diseases caused by insufficient or overexpression of genes, malignant tumors, etc. Currently, gene delivery vectors mainly include viral gene vectors and non-viral gene vectors. Among the many available viral-derived gene vectors, such as recombinant retroviruses, recombinant lentiviruses, recombinant adenoviruses, etc., recombinant adeno-associated virus (rAAV) gene vectors are becoming increasingly popular.

[0004] Adeno-associated virus (AAV), a member of the Parvoviridae family, has become the most promising gene vector for gene therapy due to its non-pathogenicity, low immunogenicity, and broad-spectrum infectivity. AAV is a non-enveloped, single-stranded DNA virus that carries a linear, single-stranded DNA genome of approximately 4.7 kb. The AAV DNA genome is terminated by a 145-bp inverted terminal repeat (ITR) sequence at each end. The 125 bases near the end form a long palindrome that folds through complementary base pairing, giving the ITR a T-shaped hairpin structure. Both positive- and negative-strand DNA genomes carrying wild-type inverted terminal repeats are packaged into the AAV viral capsid with equal probability, resulting in the production of equal numbers of positive- and negative-strand DNA AAV viral particles. AAV viral particles infect cells, enter the nucleus, uncapsidate, and release the AAV genome into the nucleus. The ITRs at both ends of the AAV genome fold to form a T-shaped hairpin structure. The 3′ end of the ITR acts as a primer for DNA synthesis, synthesizing the second strand to form a double-stranded DNA molecule, which in turn initiates the expression of genes carried by the AAV genome. The positive and negative strands of the AAV genome can also form double-stranded DNA molecules through complementary pairing to express genes.

[0005] Currently, it is generally believed in the field that the ITRs at both ends of the AAV DNA genome contain the basic information for AAV DNA replication, packaging, integration, and rescue. Based on this, the rAAV packaging systems used all use the ITRs of AAV as the basic packaging elements, placing them at both ends of the exogenous gene DNA that needs to be packaged into the rAAV capsid, and packaging the exogenous gene DNA into the rAAV vector. Such rAAV vectors have defects in the application of gene delivery, gene manipulation, and gene editing. For example, it is impossible to deliver single-polarity single-stranded DNA to perform single-stranded DNA gene editing; it is difficult to design rAAV vectors that produce short-term gene expression for short-term gene expression, such as delivering nuclease genes for gene editing, which causes off-target risks and cytotoxicity due to long-term gene expression; it is difficult to achieve single-stranded gene pairing to express large genes; it is impossible to deliver long oligodeoxynucleotides to carry out small nucleic acid gene therapy gene regulation, etc.

[0006] Summary of the Invention

[0007] To address the technical problems existing in the prior art, the present application provides a cis packaging element for a single-stranded, monopolar DNA recombinant adeno-associated virus, a spAAV vector, and its applications. Prior to packaging, the spAAV genome has a cis packaging element for packaging AAV DNA at the 5′ end of its linear single-stranded DNA. The vector packaging the single-stranded, monopolar DNA recombinant adeno-associated virus delivers single-polarity single-stranded DNA, thereby enabling single-stranded DNA gene editing, further enabling single-stranded gene pairing to express large genes and deliver long oligodeoxynucleotides for small nucleic acid gene therapy and gene regulation.

[0008] To achieve the above objectives, the present application provides a variety of cis packaging elements (DNA cis elements) of single-stranded monopolar DNA recombinant adeno-associated viruses; the cis packaging elements contain a Rep binding site (RBS) and a Rep cleavage site (trs); the cis packaging elements are capable of packaging the spAAV genome into the AAV capsid protein, including when the cis packaging element is placed at the 5′ end of the packaged single-stranded monopolar DNA, the Rep protein packages the single-stranded monopolar DNA into the AAV capsid protein by recognizing and cleaving.

[0009] The cis packaging elements of the spAAV vector include at least one of ITR, DtrsA′B′, trsA′B′, trsA′, DttRR, and ttRR. When the cis packaging element is placed at the 5′ end of the packaging DNA, the Rep protein recognizes and cleaves it, packaging the single-stranded monopolar DNA chain into the AAV viral capsid. The spAAV genome before packaging has linear open ends (except for the ITR cis packaging element), with the 5′ end containing only the cis packaging element and the 3′ end not containing any packaging-related sequences.

[0010] That is, the spAAV vector formed by packaging with the above-mentioned cis packaging element contains only DNA molecules of a single polarity, that is, a spAAV vector of a positive-strand DNA molecule, or a spAAV vector of a negative-strand DNA molecule, and is not a mixture of the two. Only when the cis packaging element is placed at the 5' end of the upper and lower chains of the positive and negative complementary DNA at the same time, because there are cis elements upstream and downstream of the plasmid, the packaged AAV has both positive single-stranded genomes and negative single-stranded genomes, and can simultaneously package and produce a mixed product of rAAV virus particles with both positive-strand DNA molecules and rAAV virus particles with negative-strand DNA molecules. The spAAV vector can advantageously deliver single-stranded DNA with linear open ends (except for the use of ITR cis packaging elements) of the same polarity (i.e., unipolar), thereby achieving unipolar single-stranded DNA delivery (positive or negative).

[0011] Specifically, the cis packaging elements selected in this application all contain a Rep binding site and a Rep cleavage site (terminal resolution site, trs).

[0012] To achieve another object, the present application also provides a spAAV vector, which is obtained by packaging the spAAV genome using the cis packaging element provided by the above technical solution; the vector comprises an AAV capsid protein and a spAAV genome packaged by the AAV capsid protein; wherein the spAAV genome packaged by the AAV capsid protein originates from the spAAV genome before packaging; the spAAV genome comprises a spAAV vector of positive-strand DNA or a spAAV vector of negative-strand DNA, and the two are not produced at the same time. Only when the cis packaging element is simultaneously placed at the 5' end of the upper and lower strands of the positive and negative complementary DNA can a mixed product of rAAV virus particles containing both positive-strand DNA molecules and rAAV virus particles containing negative-strand DNA molecules be simultaneously packaged and produced.

[0013] Furthermore, the method for obtaining the spAAV vector includes transforming the spAAV transgenic plasmid, the Cap expression plasmid, and the Rep expression plasmid into Escherichia coli competent cells respectively; after at least one round of blue-white screening, picking out white colonies, amplifying and extracting the recombinant bacmid; using the recombinant bacmid to transfect insect cells to produce recombinant baculovirus; and, extracting the recombinant baculovirus, and infecting insect cells with the recombinant baculovirus, thereby obtaining the spAAV vector.

[0014] Based on the spAAV vector provided above, a method for delivering exogenous nucleic acid to a cell is realized, that is, the spAAV vector is contacted with a cell and infects the cell, and the genome of the spAAV vector comprises a gene expression cassette of the exogenous nucleic acid and / or optional other DNA sequences; the spAAV vector is integrated into the cell genome, homologous or non-homologous recombination occurs, and exogenous gene expression is generated;

[0015] Preferably, the genome of the spAAV vector comprises the positive-strand single-stranded DNA sequence and / or the negative-strand single-stranded DNA sequence of the exogenous gene.

[0016] Preferably, the exogenous nucleic acid comprises a nucleic acid sequence of an exogenous gene or a nucleic acid sequence of a non-exogenous gene.

[0017] The spAAV vector provided by the above technical solution can be applied to gene expression and / or gene therapy and / or gene editing and / or gene regulation or related products.

[0018] The technical effects of the technical solution of this application are:

[0019] 1. The pre-packaged spAAV genome provided by the technical solution of the present application has a cis packaging element at the 5′ end of its linear single-stranded DNA. In particular, the spAAV vector formed by the pre-packaged spAAV genome in the present application has only one polarity, that is, either a spAAV vector with positive-strand DNA or a spAAV vector with negative-strand DNA, and both cannot be produced simultaneously. In addition, the titer and cell transfection performance of the spAAV virus particles produced by packaging are comparable to those of traditional rAAV virus particles.

[0020] 2. The spAAV virus particles formed by the packaging according to the present application can advantageously deliver monopolar, single-stranded DNA of the same polarity with linear open ends (except for the use of ITR cis packaging elements). Since it is a monopolar, single-stranded DNA of the same polarity and does not have a 3′ITR structure that can be used as a DNA replication primer, spAAV DNA can only exist in the form of single-stranded DNA in cells, so it can be used for gene integration, gene editing, promoting efficient DNA pairing to express large genes and deliver long oligodeoxynucleotides, enabling it to carry out small nucleic acid gene therapy and other work.

[0021] 3. The present application can also allow two spAAV vectors containing the positive chain or negative chain of the target DNA gene to infect cells cultured in vitro separately. The genome of the spAAV vector can be integrated into the cell genome, resulting in long-term and stable gene expression; and when the spAAV vectors containing the positive chain and negative chain of the target DNA gene are co-infected with cells cultured in vitro (such as HEK 293 cells), the two can complement each other to produce double-stranded DNA molecules, which can express the target gene with higher efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is an exemplary schematic diagram of the DNA sequence structure of AAV2Flip ITR (including RBE and trs) provided in this application.

[0023] FIG2 is a schematic diagram of the packaging of the spAAV vector prepared by the insect cell baculovirus packaging system using the cis packaging element used in the examples of the present application.

[0024] FIG3 is an SDS-PAGE electrophoresis diagram of the spAAV vector capsid protein produced by packaging with cis packaging elements in the examples provided in this application.

[0025] FIG4 is a neutral agarose gel electrophoresis diagram of the spAAV vector DNA produced by packaging of cis packaging elements in the examples provided in this application.

[0026] FIG5 provides a DNA denaturing alkaline agarose gel electrophoresis diagram of the capsid protein of the spAAV vector produced by packaging the cis packaging element provided in the examples of the present application. DETAILED DESCRIPTION

[0027] To make the purpose, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application are clearly and completely described. Obviously, the described embodiments are part of the embodiments of this application, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of this application without making creative efforts are within the scope of protection of this application.

[0028] The meaning of scientific and technological terms in this application is consistent with the general understanding of those skilled in the art, unless otherwise specified. In this application, "one" or its combination with various quantifiers includes both singular and plural meanings, unless otherwise specified. In this application, for the same parameter or variable, when multiple numerical values, numerical ranges, or combinations thereof are given for description, it is equivalent to specifically revealing these numerical values, range end values, and numerical ranges formed by any combination thereof. In this application, any numerical value, whether or not it is accompanied by a modifier such as "about", covers an approximate range that can be understood by those skilled in the art, such as plus or minus 10%, 5%, etc. In this article, each "embodiment" refers equally to and covers the implementation methods of various methods and systems of this application. In this application, one or more technical features in any implementation method can be freely combined with one or more technical features in any one or more other implementation methods, and the implementation methods thus obtained also belong to the content disclosed in this application.

[0029] Adeno-associated virus (AAV), a member of the Parvoviridae family, lacks an envelope and exhibits icosahedral symmetry. Currently, over ten serotypes and over a hundred variants of AAV have been discovered, of which AAV2 is the most thoroughly studied and widely used serotype.

[0030] The AAV genome consists of a linear single-stranded DNA of approximately 4.7 kb, flanked by approximately 145 base-length ITRs. The approximately 125 bases near the ends of the ITRs contain a palindromic sequence that folds onto itself through complementary base pairing, forming a T-shaped hairpin structure. The ITRs contain a Rep protein binding site (RBE) and a terminal resolution site (TRS), which is recognized and bound by the Rep protein, producing an incision at the TRS.

[0031] The AAV genome contains two open reading frames (ORFs) between the two ITRs. These ORFs encode rep and cap, respectively. The rep gene encodes four Rep proteins: Rep78, Rep68, Rep52, and Rep40, which play a role in AAV replication, integration, rescue, and packaging. The cap gene encodes the AAV capsid proteins VP1, VP2, and VP3. VP1 is essential for the formation of infectious AAV, while VP3 is the primary protein in the AAV viral particle. The ratio of VP1, VP2, and VP3 in the AAV capsid is approximately 1:1:10.

[0032] Figure 1 shows a schematic diagram of the DNA sequence structure of the AAV2 flip ITR, which forms a T-shaped palindromic hairpin structure. As shown in Figure 1, the first 125 bases of the ITR can be divided into distinct segments: A', C', C, B', B, A. The reverse complementarity of B and B', and C and C', forms a T-shaped palindromic hairpin structure, with A and A' forming the handle of the hairpin. The A' segment is followed by the D segment. The positions of the RBE and TRS are shown in Figure 1.

[0033] Although the ITR sequences are different in the AAV genomes of various serotypes, they can all form a T-shaped palindromic hairpin structure and have RBE and trs.

[0034] During AAV viral assembly, both positive- and negative-strand DNA genomes carrying wild-type ITRs are packaged into the AAV capsid with equal probability, resulting in equal numbers of positive- and negative-strand AAV viral particles. AAV viral particles infect cells, uncapsidate, and release the AAV genome. The ITRs at either end of the AAV genome fold to form a T-shaped palindromic hairpin structure. The 3' end of the AAV genome then serves as a primer for the synthesis of a second strand, forming a double-stranded molecule that initiates expression of genes carried by the AAV genome. The positive- and negative-strand DNA molecules of the AAV genome can also form double-stranded DNA through complementary pairing to express genes.

[0035] Based on this, the present application provides a pre-packaging spAAV genome, wherein the 5′ end of the pre-packaging spAAV genome has a cis packaging element, and the 3′ end does not contain any cis packaging element sequence.

[0036] As used herein, the terms "monopolar" or "sp" refer to a single DNA polarity. That is, the spAAV vectors produced by packaging the pre-packaged spAAV genomes described herein have only one polarity: either a spAAV vector with plus-strand DNA or a spAAV vector with minus-strand DNA, but not both. The spAAV vectors produced by packaging the pre-packaged spAAV genomes described herein are also referred to as "monopolar rAAV vectors" or "spAAV vectors."

[0037] As used herein, the term "recombinant adeno-associated virus vector" or "rAAV" refers to a non-wild-type adeno-associated virus particle that serves as a gene delivery vector and comprises a recombinant AAV genome packaged in a viral capsid.

[0038] In this application, the term "pre-packaging spAAV genome" refers to the recombinant AAV genome to be packaged and provided for subsequent packaging into capsid protein.

[0039] As used herein, the term "open end" or "linear open end" refers to a 3' or 5' nucleotide sequence end that does not have any other structure (eg, hairpin structure) other than a linear straight chain.

[0040] As used herein, the term "ITR or portion thereof" refers to all or part of the DNA segment of an ITR, including "ITR components." For example, in the case of a Flip ITR (e.g., as shown in FIG1 ), the ITR components include, but are not limited to, the D region, the trs site, the RBE site, the A' region (partial), the C' region, the C region, the B' region, the B region, the A region, and the like.

[0041] It should be understood that the ITR sequences present at the ends of the AAV genome typically have the same or different segment orientations, for example, Flip / Flop, Flop / Flip, Flip / Flip, or Flop / Flop. An exemplary structural orientation of a "Flip" ITR is shown in Figure 1. An exemplary structural orientation of a "Flop" ITR is shown in Figure 1, except that the positions of the B' segment and the C' segment are swapped, and the positions of the B segment and the C segment are swapped.

[0042] In this application, the term "hairpin structure" is also called "hairpin structure", which refers to the "hairpin" shape formed by the DNA molecule folding back on itself so that some bases in the folded area are close to each other and complementary pairing, such as a T-shaped hairpin structure, as shown in Figure 1.

[0043] Different from the rAAV genome before traditional packaging, the spAAV vector formed by the spAAV genome packaged before packaging in the present application has only one polarity, that is, either a spAAV vector of positive-strand DNA or a spAAV vector of negative-strand DNA, and the two are not produced at the same time. In addition, the titer and cell transfection performance of the spAAV virus particles produced by packaging are comparable to those of traditional rAAV virus particles. The spAAV virus particles formed by the packaging according to the present application can advantageously deliver unipolar, single-stranded DNA with the same polarity of linear open ends (except utilizing ITR cis packaging elements). Since it is a unipolar, single-stranded DNA with only the same polarity and there is no 3′ITR structure that can be used as a DNA replication primer, spAAV DNA can only exist in the form of single-stranded DNA in the cell, so that it can be used for gene integration, carry out gene editing, promote efficient DNA pairing to express large genes and deliver long oligodeoxynucleotides, so that it can carry out work such as small nucleic acid gene therapy.

[0044] In some embodiments, the spAAV genome is single-stranded DNA.

[0045] In some embodiments, the 3′ end of the spAAV genome before packaging does not have an ITR or a portion thereof, and does not have other cis packaging elements.

[0046] In some embodiments, the 5′ end of the spAAV genome before packaging has the cis packaging element.

[0047] Since there is no ITR palindromic sequence at the 3′ end, the resulting spAAV vector cannot synthesize the second chain using the 3′ end as a primer and form a double-stranded DNA molecule after entering the host cell.

[0048] In some embodiments, the cis packaging element comprises trs and RBE.

[0049] In some embodiments, the cis packaging element comprises D, A', and C' segments derived from Flip ITR, for example, the corresponding structure is shown in FIG1 .

[0050] In some embodiments, the cis packaging element comprises, among other things, D, A', and B' segments derived from Flop ITR.

[0051] In some exemplary embodiments, the spAAV genome before packaging comprises, in 5′ to 3′ order: a cis packaging element, an exogenous gene expression cassette, and / or optionally other exogenous DNA sequences, wherein the cis packaging element comprises RBE and trs. In some exemplary embodiments, the spAAV genome before packaging comprises, in 5′ to 3′ order: a cis packaging element, an exogenous gene expression cassette, and / or optionally other exogenous DNA sequences, wherein the cis packaging element comprises D, A′, and C′ segments derived from Flip ITR. In some exemplary embodiments, the spAAV genome before packaging comprises, in 5′ to 3′ order: a cis packaging element, an exogenous gene expression cassette, and / or optionally other exogenous DNA sequences, wherein the cis packaging element comprises D, A′, and B′ segments derived from Flop ITR.

[0052] In some exemplary embodiments, the cis packaging element includes any one of the DNA elements ITR, DtrsA′B′, trsA′B′, trsA′, DttRR, and ttRR; wherein the 5′ end of the spAAV genome before packaging has the cis packaging element; and the 3′ end of the spAAV genome before packaging does not have all or part of the DNA sequence of ITR and does not have other cis packaging elements.

[0053] In some exemplary embodiments, the ITR described in the present application has the following nucleotide sequence (Flip ITR):

[0054] The sequence of Flip ITR is SEQ ID NO.1:

[0055] In some exemplary embodiments, the ITR described in the present application has the following nucleotide sequence (Flop ITR):

[0056] The sequence of Flop ITR is SEQ ID NO.2:

[0057] In some exemplary embodiments, the nucleotide sequence of the ITR described herein has greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 98%, greater than 99% or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0058] In some exemplary embodiments, the nucleotide sequence of the ITR described herein is the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2.

[0059] In some exemplary embodiments, the nucleotide sequence of the RBE has the nucleotide sequence shown in SEQ ID NO: 3:

[0060] The sequence of RBE is SEQ ID NO. 3: gcgc gctc gctc gctc.

[0061] In some exemplary embodiments, the nucleotide sequence of RBE described in the present application has a sequence identity of greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 98%, greater than 99% or 100% to the nucleotide sequence shown in SEQ ID NO. 3.

[0062] In some exemplary embodiments, the nucleotide sequence of trs in the present application has the nucleotide sequence shown in SEQ ID NO. 4, SEQ ID NO. 4: agttgg.

[0063] In some exemplary embodiments, the nucleotide sequence of trs in the present application has a sequence identity of greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 98%, greater than 99% or 100% with the nucleotide sequence shown in SEQ ID NO.4.

[0064] In some exemplary embodiments, the nucleotide sequence of ITRt described in the present application has a sequence identity of greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 98%, greater than 99% or 100% with the nucleotide sequence shown in SEQ ID NO.5.

[0065] Those skilled in the art will appreciate that the ITR, RBE, and trs sequences described above are for exemplary purposes only and are not intended to be limiting, and are described based on AAV2. Those skilled in the art will be able to determine the ITR sequences and RBE and trs sequences of various types (e.g., various serotypes) of rAAV and implement the present application with appropriate adjustments without departing from the teachings of the present application.

[0066] In some exemplary embodiments, the DtrsA′B′ described in the present application has a nucleotide sequence shown in SEQ ID NO: 5:

[0067] In some exemplary embodiments, the trsA′B′ has a nucleotide sequence as shown in SEQ ID NO: 6:

[0068] In some exemplary embodiments, the trsA′ has a nucleotide sequence shown in SEQ ID NO: 7:

[0069] In some exemplary embodiments, the DttRR has a nucleotide sequence as shown in SEQ ID NO: 8:

[0070] In some exemplary embodiments, the ttRR has a nucleotide sequence as shown in SEQ ID NO: 9:

[0071] In some embodiments, the pre-packaging spAAV genome does not have a nucleotide sequence encoding a rep gene and / or a cap gene.

[0072] In some embodiments, the spAAV genome before packaging comprises an exogenous nucleic acid, for example, an exogenous gene expression cassette, between one end having the cis packaging element and the other end. The exogenous nucleic acid may have a nucleotide sequence encoding an exogenous gene.

[0073] In some embodiments, the spAAV genome before packaging comprises an optional DNA sequence between one end having the cis packaging element and the other end; wherein the optional DNA sequence can be a nucleotide sequence that does not encode an exogenous gene, for example, a stuffer sequence.

[0074] As used herein, the terms "exogenous gene," "exogenous nucleic acid," or "gene of interest" refer to genes or DNA derived from outside the organism of interest or study (e.g., an individual organism). The exogenous gene, exogenous nucleic acid, or gene of interest can be any exogenous gene or exogenous DNA that is desired to be expressed or produce a biological function in a recipient cell to which the spAAV viral particles are to be delivered.

[0075] In some embodiments, the spAAV genome comprises a positive-stranded single-stranded DNA sequence of an exogenous nucleic acid (eg, an exogenous gene expression cassette).

[0076] In some embodiments, the spAAV genome comprises a negative-sense single-stranded DNA sequence of an exogenous nucleic acid (eg, an exogenous gene expression cassette).

[0077] In some embodiments, the nucleotide sequence encoding the exogenous gene includes a forward exogenous gene expression frame.

[0078] In some exemplary embodiments, the forward exogenous gene expression frame may include a promoter, an exogenous gene open reading frame, and a poly A sequence in a 5′-3′ direction.

[0079] In some exemplary embodiments, the positive exogenous gene expression cassette may further include an enhancer and an intron.

[0080] In some exemplary embodiments, the enhancer and intron are located between the promoter and the open reading frame of the exogenous gene.

[0081] In some exemplary embodiments, the forward exogenous gene expression cassette may include a DNA sequence for gene editing or gene regulation in the 5′-3′ direction.

[0082] In some embodiments, the nucleotide sequence encoding the exogenous gene includes an inverted exogenous gene expression frame.

[0083] In some exemplary embodiments, the reverse exogenous gene expression cassette may comprise, in the 5′-3′ direction, a reverse complementary sequence of a poly A sequence, a reverse complementary sequence of an exogenous gene open reading frame, and a reverse complementary sequence of a promoter.

[0084] In some exemplary embodiments, the reverse exogenous gene expression cassette may further include an enhancer and an intron.

[0085] In some exemplary embodiments, the enhancer and intron are located between the reverse complementary sequence of the promoter and the reverse complementary sequence of the open reading frame of the exogenous gene.

[0086] In some exemplary embodiments, the reverse exogenous gene expression cassette may include a reverse complementary sequence of a DNA sequence for gene editing or gene regulation in the 5′-3′ direction.

[0087] In some embodiments, the promoter may include, for example, a CMV promoter, a CAG promoter, or a cell-specific promoter, such as a GFAP promoter, a hAAT promoter, and the like.

[0088] In some embodiments, the spAAV genome before packaging can be produced by constructing a plasmid (e.g., a spAAV transgenic plasmid). The spAAV transgenic plasmid in this application can be selected from any plasmid suitable for producing the spAAV genome before packaging described herein. Suitable spAAV transgenic plasmids can be based on, for example, but not limited to, pFastBacdual, pFastBac1 plasmids, etc.

[0089] The pre-packaging spAAV genome can subsequently be packaged into capsid protein through the spAAV vector packaging system to form a spAAV vector.

[0090] In some embodiments, the spAAV vector packaging system can include, for example, an insect cell baculovirus packaging system, a mammalian cell packaging system, and the like.

[0091] On the other hand, the present application provides a spAAV vector comprising:

[0092] Capsid protein, and spAAV genome packaged by the capsid protein;

[0093] The spAAV genome packaged by capsid protein is derived from the spAAV genome before packaging described herein.

[0094] In this application, "spAAV vector" is also referred to as "spAAV virus particle", which is produced by packaging the pre-packaging spAAV genome into AAV capsid protein (for example, using a spAAV vector packaging system).

[0095] In some embodiments, examples of the spAAV vector packaging system may include, for example, an insect cell baculovirus packaging system, a mammalian cell packaging system, and the like.

[0096] In some embodiments, the spAAV genome packaged by the capsid protein is a monopolar, single-stranded DNA.

[0097] In some embodiments, the spAAV genome packaged by the capsid protein (or may also be referred to as "packaged spAAV genome") has linear open ends at both ends (except for the use of ITR cis packaging elements), one end does not have ITR or part thereof and does not have other cis packaging elements, and the other end has any cis packaging elements.

[0098] In some embodiments, the packaged spAAV genome has a cis packaging element-derived sequence at the 5′ end.

[0099] In some embodiments, the packaged spAAV genome lacks a cis packaging element or portion thereof at the 3' end.

[0100] In some embodiments, the cis packaging element-derived sequence comprises RBE, trs.

[0101] In some embodiments, the cis packaging element-derived sequence comprises RBE and a portion of the trs sequence (the remaining trs portion after Rep cleavage).

[0102] In some embodiments, the packaged spAAV genome does not have a nucleotide sequence encoding a rep gene and / or a cap gene.

[0103] In some embodiments, the packaged spAAV genome comprises an exogenous nucleic acid, such as an exogenous gene expression cassette, between one end having a cis packaging element-derived sequence and the other end. The exogenous nucleic acid may have a nucleotide sequence encoding an exogenous gene.

[0104] In some embodiments, the nucleotide sequence encoding the exogenous gene is a monopolar, single-stranded DNA sequence.

[0105] In some embodiments, the packaged spAAV genome comprises an optional DNA sequence between one end having a cis packaging element-derived sequence and the other end; the optional DNA sequence can be a nucleotide sequence that does not encode an exogenous gene, for example, a stuffer sequence.

[0106] In some embodiments, the packaged spAAV genome comprises a positive-strand single-stranded DNA sequence of an exogenous nucleic acid (eg, an exogenous gene expression cassette).

[0107] In some embodiments, the packaged spAAV genome comprises a negative-strand single-stranded DNA sequence of an exogenous nucleic acid (eg, an exogenous gene expression cassette).

[0108] In some embodiments, the nucleotide sequence encoding the exogenous gene includes a forward exogenous gene expression frame.

[0109] In some exemplary embodiments, the positive exogenous gene expression cassette may comprise a promoter, an exogenous gene open reading frame, and a poly A sequence in the 5′-3′ direction. In some exemplary embodiments, the positive exogenous gene expression cassette may further comprise an enhancer and an intron.

[0110] In some exemplary embodiments, the enhancer and intron are located between the promoter and the open reading frame of the exogenous gene.

[0111] In some exemplary embodiments, the forward exogenous gene expression cassette may include a DNA sequence for gene editing or gene regulation in the 5′-3′ direction.

[0112] In some embodiments, the nucleotide sequence encoding the exogenous gene includes an inverted exogenous gene expression frame.

[0113] In some exemplary embodiments, the reverse exogenous gene expression cassette may comprise, in the 5′-3′ direction, a reverse complementary sequence of a poly A sequence, a reverse complementary sequence of an exogenous gene open reading frame, and a reverse complementary sequence of a promoter.

[0114] In some exemplary embodiments, the reverse exogenous gene expression cassette may further include an enhancer and an intron.

[0115] In some exemplary embodiments, the enhancer and intron are located between the reverse complementary sequence of the promoter and the reverse complementary sequence of the open reading frame of the exogenous gene.

[0116] In some exemplary embodiments, the reverse exogenous gene expression cassette may include a reverse complementary sequence of a DNA sequence for gene editing or gene regulation in the 5′-3′ direction.

[0117] In some embodiments, the promoter may include, for example, a CMV promoter, a CAG promoter, or a cell-specific promoter, such as a GFAP promoter, a hAAT promoter, and the like.

[0118] In some embodiments, the capsid protein is an AAV capsid protein. In some embodiments, the capsid protein can be a capsid protein of various AAVs selected from AAV1-AAV12 (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12).

[0119] In some embodiments, the capsid protein is an AAV capsid protein variant, such as a tyrosine single amino acid / multiple amino acid variant, a tyrosine, serine, or threonine variant, or a multi-serotype chimera such as AAV-DJ, or a polypeptide embedded variant, etc.

[0120] In some embodiments, the capsid protein can be produced from a plasmid expressing the AAV capsid protein Cap by utilizing the spAAV vector packaging system.

[0121] In some embodiments, the spAAV vector can be packaged and produced by a spAAV transgenic plasmid (producing the spAAV genome before packaging) and a plasmid expressing the AAV capsid protein Cap and the replication protein Rep (producing Cap and Rep proteins) using a spAAV vector packaging system.

[0122] In some embodiments, the AAV Cap encoding gene and the AAV Rep encoding gene can be on the same plasmid or on two different plasmids.

[0123] In some embodiments, the AAV Cap and / or AAV Rep expression plasmid may include a Cap and / or Rep gene expression cassette and desired expression elements, as well as intron sequences for enhancing expression, etc. There is no particular limitation on the plasmid used to express AAV Cap and / or Rep, and those skilled in the art can routinely use AAV Cap and / or Rep expression plasmids, see, for example, Urabe M, Ding C, Kotin RM. Insect cells as a factory to produce adeno-associated virus type 2 vectors. Hum Gene Ther. 2002 Nov 1; 13(16): 1935-43. doi: 10.1089 / 10430340260355347. PMID: 12427305.

[0124] In another aspect, the present invention also provides a spAAV transgenic plasmid for producing the spAAV genome before packaging as described herein.

[0125] The spAAV transgenic plasmid described in the present application can be selected from any plasmid suitable for producing the spAAV genome before packaging described herein, wherein the suitable spAAV transgenic plasmid can be based on, for example but not limited to, pFastBacdual plasmid, pFastBac1 plasmid, etc.

[0126] In some exemplary embodiments, the spAAV transgenic plasmid can be designed as a positive-stranded single-stranded DNA sequence encoding an exogenous gene, which may include, in 5′ to 3′ order: a cis packaging element (containing RBE and trs), a promoter (such as CMV), an exogenous gene open reading frame, and poly A.

[0127] In some exemplary embodiments, the spAAV transgenic plasmid may further include a stuffer sequence.

[0128] In some embodiments, the stuffer sequence can be present, for example, upstream of the promoter and / or downstream of the poly A.

[0129] In some exemplary embodiments, the spAAV transgenic plasmid can be designed as a negative-stranded single-stranded DNA sequence encoding an exogenous gene, which may include, in 5' to 3' order: a cis packaging element (including RBE and trs), poly A, an exogenous gene open reading frame ', and a promoter ' (such as CMV '). In some exemplary embodiments, the spAAV transgenic plasmid may also include a stuffer sequence. In some specific embodiments, the stuffer sequence may be present, for example, downstream of the promoter ' and / or upstream of the poly A '.

[0130] As used herein, the term "stuffer sequence" refers to helper DNA that is added to fill the target DNA to the AAV packaging length when the target DNA length is shorter than 4.7 kb. In some embodiments, the stuffer sequence can have different lengths, as long as the final DNA length reaches, for example, approximately 4.7 kb.

[0131] The spAAV transgenic plasmid can be packaged together with the Rep and Cap gene expression plasmids through the spAAV vector packaging system to produce the spAAV vector described herein.

[0132] On the other hand, the present application also provides a method for packaging spAAV vectors, which includes: transforming the spAAV transgenic plasmid and Cap and Rep expression plasmids into Escherichia coli competent cells respectively; after at least one round of blue-white screening, picking out white colonies, amplifying and extracting recombinant bacmids; using the recombinant bacmids to transfect insect cells to produce recombinant baculovirus; and, extracting the recombinant baculovirus and infecting insect cells with the recombinant baculovirus to obtain the spAAV vector.

[0133] In some embodiments, the E. coli competent cells are E. coli DH10Bac competent cells.

[0134] In some embodiments, the spAAV vector packaging system is an insect cell baculovirus packaging system.

[0135] In some embodiments, after at least one round of blue-white screening, white colonies are picked, amplified, and recombinant bacmids are extracted.

[0136] In some embodiments, after two or more rounds of blue-white screening, white colonies are picked, amplified, and recombinant bacmids are extracted.

[0137] In some embodiments, the recombinant bacmid is transfected into insect cells (eg, insect Sf9 cells) to produce recombinant baculovirus (eg, P3 generation recombinant baculovirus).

[0138] In some embodiments, recombinant baculoviruses (eg, two or three recombinant baculoviruses of generation P3) are infected (eg, co-infected) with insect cells (eg, Sf9 insect cells) to package and obtain spAAV vectors.

[0139] As shown in Figure 2, a schematic diagram of the packaging of spAAV vectors by cis packaging elements according to an exemplary embodiment of the present application is provided. In a specific embodiment, when the spAAV vector is packaged, the Rep protein recognizes the Rep binding site (RBE) on the recombinant baculovirus DNA, binds to the site, recognizes the terminal melting site (trs), opens the phosphodiester bond between the bases tt, and covalently binds to the 5' end, introducing the DNA into the AAV capsid for packaging. The length of the DNA packaged into the capsid is about 4.7 kb.

[0140] For methods of packaging rAAV vectors using the insect cell baculovirus packaging system, see, for example, Urabe M, Ding C, Kotin RM. Insect cells as a factory to produce adeno-associated virus type 2 vectors. Hum Gene Ther. 2002 Nov 1; 13(16): 1935-43. doi: 10.1089 / 10430340260355347. PMID: 1242730.

[0141] On the other hand, the present application also provides a method for delivering exogenous nucleic acid to a cell, comprising contacting the cell with one or more spAAV vectors described herein, wherein the genome of one or more of the spAAV vectors contains a gene expression cassette of the exogenous nucleic acid and / or optional other DNA sequences.

[0142] In some embodiments, the cell can be a eukaryotic cell.

[0143] In some embodiments, the cell can be an animal cell.

[0144] In some embodiments, the cell can be a vertebrate cell.

[0145] In some embodiments, the cell can be a mammalian cell, such as a human cell.

[0146] In some embodiments, the exogenous nucleic acid comprises the nucleic acid sequence of an exogenous gene.

[0147] In some embodiments, the exogenous nucleic acid comprises a nucleic acid sequence that is not an exogenous gene.

[0148] In some embodiments, the genome of the spAAV vector comprises a positive-stranded single-stranded DNA sequence or a negative-stranded single-stranded DNA sequence of the exogenous gene.

[0149] In some embodiments, the one or more spAAV vectors include: a spAAV vector containing only the positive-strand single-stranded DNA sequence of the exogenous nucleic acid and / or a spAAV vector containing only the negative-strand single-stranded DNA sequence of the exogenous nucleic acid.

[0150] In some embodiments, one or more of the spAAV vectors include two or more spAAV vectors, which include at least a first spAAV vector having a positive-stranded single-stranded DNA sequence of the exogenous gene and a second spAAV vector having a negative-stranded single-stranded DNA sequence of the exogenous gene.

[0151] In some embodiments, the cell is contacted with a spAAV vector having a positive-stranded single-stranded DNA sequence of the exogenous gene.

[0152] In some embodiments, the cell is contacted with a spAAV vector having a negative-sense single-stranded DNA sequence of the exogenous gene.

[0153] In a preferred embodiment, the cells are contacted with a first spAAV vector having a positive-strand single-strand DNA sequence of the exogenous gene and a second spAAV vector having a negative-strand single-strand DNA sequence of the exogenous gene.

[0154] In some embodiments, the cells are contacted simultaneously with a first spAAV vector having a positive-stranded single-stranded DNA sequence of the exogenous gene and a second spAAV vector having a negative-stranded single-stranded DNA sequence of the exogenous gene.

[0155] In some embodiments, the cells are sequentially (e.g., within 24 hours) contacted with a first spAAV vector having a positive-stranded single-stranded DNA sequence of the exogenous gene and a second spAAV vector having a negative-stranded single-stranded DNA sequence of the exogenous gene, or vice versa. It should be understood that in this application, "first spAAV vector" and "second spAAV vector" are only used to distinguish between the two and are not intended to limit the order of their use.

[0156] When cells are infected with a spAAV vector having a positive-stranded single-stranded DNA sequence of the exogenous gene, or a spAAV vector having a negative-stranded single-stranded DNA sequence of the exogenous gene, the exogenous gene carried by the vector has a greater probability (for example, 20%) of being integrated into the cell genome, resulting in long-term and stable expression of the exogenous gene.

[0157] By jointly using an spAAV vector having a positive-stranded single-stranded DNA sequence of the exogenous gene and an spAAV vector having a negative-stranded single-stranded DNA sequence of the exogenous gene, it is beneficial for the spAAV genome containing the positive and negative strands to quickly complement each other in the nucleus of the host cell to form a double-stranded molecule, thereby initiating gene expression.

[0158] In another aspect, the present invention also provides an isolated host cell comprising the spAAV vector described herein.

[0159] In some embodiments, the host cell can be a eukaryotic cell.

[0160] In some embodiments, the host cell can be an animal cell.

[0161] In some embodiments, the host cell may be a vertebrate cell. In some embodiments, the host cell may be a mammalian cell.

[0162] In some embodiments, the host cell can be a human cell.

[0163] In some embodiments, the host cell is obtained by contacting a eukaryotic cell (eg, an animal cell, eg, a mammalian cell, eg, a human cell) with the spAAV vector described herein.

[0164] In another aspect, the spAAV vector described herein is also provided for use in gene expression. Also provided is the use of the spAAV vector described herein in preparing a product for gene expression.

[0165] In some embodiments, the subject of gene expression is an animal, particularly a vertebrate, more particularly a mammal, and even more particularly a human.

[0166] On the other hand, the spAAV vector described in the present application is also provided, which is used for gene therapy. Specifically, the present application provides the use of spAAV in preparing products for gene therapy.

[0167] In some embodiments, the subject of gene therapy is an animal, particularly a vertebrate, more particularly a mammal, and even more particularly a human.

[0168] On the other hand, the spAAV vector described herein is also provided, which is used for gene editing. Also provided is the use of the spAAV described herein in preparing a product for gene editing.

[0169] In some embodiments, the subject of gene editing is an animal, specifically a vertebrate, more specifically a mammal, and even more specifically a human.

[0170] In another aspect, the spAAV vector described herein is also provided for use in gene regulation. Also provided is the use of the spAAV vector described herein in preparing a product for gene regulation.

[0171] In some embodiments, the subject of gene regulation is an animal, particularly a vertebrate, more particularly a mammal, and even more particularly a human.

[0172] The technical solution of the present application is further described in detail below through specific embodiments.

[0173] Example 1

[0174] This example constructs a plasmid vector and spAAV vector insect cell packaging system for spAAV vector packaging. The specific steps include:

[0175] First, the construction of plasmid vectors for spAAV vector packaging;

[0176] Second, the preparation of recombinant Bacmid;

[0177] Third, preparation of baculovirus;

[0178] Fourth, spAAV vector packaging;

[0179] Finally, the spAAV vector is detected and identified; this step includes first purification using iodixanol density gradient ultracentrifugation, then detecting the titer of the spAAV vector by fluorescent quantitative PCR, SDS-PAGE electrophoresis analysis of the spAAV vector capsid protein, identification of the spAAV vector genome, DNA neutral agarose gel electrophoresis analysis, DNA alkaline agarose gel electrophoresis analysis, annealing of the spAAV genome containing the positive and negative strands of the target gene DNA to identify the monopolar genome, S1 enzyme digestion to identify the single-stranded DNA genome of the spAAV vector, and gene expression in HEK293 cells infected with the spAAV vector.

[0180] The following describes the above steps in detail.

[0181] First, construction of plasmid vectors for spAAV vector packaging

[0182] In this embodiment, the rAAV transgenic plasmid containing the DtrsA′B′ related sequence only at one end of the exogenous gene encoding expression frame is also called a “monopolar” plasmid, wherein the main plasmid involved is the spAAV DNA genomic plasmid.

[0183] The construction method specifically includes:

[0184] 1.spAAV-EGFP(+)DNA genomic plasmid

[0185] The plasmid DNA contains D sequence, trs sequence, A′B′ sequence, 1.3kb stuff1 stuffer sequence, forward EGFP gene expression frame (CMV-EGFP-polyA) sequence, 1.3kb stuff2 stuffer sequence, and the entire packaging frame is 5.0kb.

[0186] The construct used pFastBacdual-ITR-EGFP as the vector.

[0187] Among them, the DtrsA′B′ sequence (5′ to 3′ direction) is SEQ ID NO: 5, specifically

[0188] 5′-AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAA-3′.

[0189] The sequence of the EGFP gene expression cassette is promoter CMV, enhancer, intron, EGFP open reading frame, and polyA sequence;

[0190] The first step was to construct pFastBacdual-DtrsA′B′-CMV-EGFP-polyA-RHstuff.

[0191] DtrsA′B′ is obtained by denaturing and annealing artificially synthesized primer 1 and primer 2.

[0192] Primer 1 and primer 2 were synthesized by Sangon Biotech (Shanghai) Co., Ltd.; wherein, the sequence of primer 1 is SEQ ID NO.6:

[0193] 5′-CAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAA-3′;

[0194] The sequence of primer 2 is SEQ ID NO.7:

[0195] 5′-CGCGTTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGTAC-3′.

[0196] The denaturation and annealing steps included: 5 μL each of Primer 1 and Primer 2 oligonucleotides (200 μM concentration), 4 μL of 5× annealing buffer, and DNase- and RNase-free deionized water to a final volume of 20 μL. Annealing was performed in a PCR instrument at 95°C for 4 minutes. The reaction tubes were then removed from the laboratory and allowed to cool to room temperature over 5-10 minutes to form oligo-dsDNA with KpnI / MluI sticky ends. This oligo-dsDNA was then inserted into the KpnI / MluI-digested pFastBacdual-ITR-EGFP vector, resulting in the vector pFastBacdual-DtrsA′B′-CMV-EGFP-polyA.

[0197] RHstuff was obtained by denaturing and annealing artificially synthesized primers 3 and 4, both of which were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0198] The sequence of primer 3 is SEQ ID NO. 8: 5′-GACCGATAATCTCTAGATTGAGGAACAA-3′;

[0199] The sequence of primer 4 is SEQ ID NO. 9: 5′-AGCTTTGTTCCTCAATCTAGAGATTATCG-3′.

[0200] Primers 3 and 4 were denatured and annealed using the same steps as above to form oligo-dsDNA with RsrII / HindIII sticky ends, which were then inserted into the RsrII / HindIII double-digested pFastBacdual-DtrsA′B′-CMV-EGFP-polyA vector to obtain the pFastBacdual-DtrsA′B′-CMV-EGFP-polyA-RHstuff vector.

[0201] The second step was to construct pFastBacdual-DtrsA′B′-1.3kb stuff1-CMV-EGFP-polyA-1.3kb stuff2.

[0202] First, 1.3 kb stuff1 was fused with a CMV fragment by PCR and cloned to obtain a 1.3 kb stuff1-CMV fragment. The primers included primer 5, primer 6, primer 7, and primer 8, all of which were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0203] The sequence of primer 5 is SEQ ID NO.10:

[0204] 5′-TCACTGACGCGTCGGAAGGTCGGTATCCACGGA-3′.

[0205] The sequence of primer 6 is SEQ ID NO.11:

[0206] 5′-CTTCAACCGGCTGAAGCTGGCTAGTTATTAATAGTAATCA-3′;

[0207] The sequence of primer 7 is SEQ ID NO.12:

[0208] 5′-TGATTACTATTAATAACTAGCCAGCTTCAGCCGGTTGAAG-3′.

[0209] The sequence of primer 8 is SEQ ID NO.13:

[0210] 5′-CCCGGGGAATTCAATCGATGTTCGAATCCCAAT-3′.

[0211] The PCR reaction system consisted of 2.5 μL of KOD-Plus-Neo, 5 μL of 10x PCR Buffer, 5 μL of 25 mM MgSO4, 5 μL of 25 mM MgSO4, 5 μL of DMSO, 0.6 μL each of primer 5 and primer 6 (10 uM), 1-50 ng of gene template saCas9, and HO was added to make up to 50 μL.

[0212] The PCR reaction program was: 95°C for 4 min; 95°C for 10 s, 56°C for 30 s, and 72°C for 3 min, for 25 cycles.

[0213] A 1.3 kb stuff1 fragment was obtained by PCR amplification; a CMV fragment was obtained by amplification using primers 7 and 8 and template pFastBacdual-ITR-EGFP; a 1.3 kb stuff1-CMV fragment was obtained by fusion PCR amplification using primers 5 and 8 and templates 1.3 kb stuff1 and CMV.

[0214] A 1.3 kb stuff1-CMV fragment was obtained by fusion PCR amplification and inserted into the pFastBacdual-DtrsA′B′-CMV-EGFP-polyA-RHstuff vector by MluI / EcoRI double enzyme digestion to obtain the pFastBacdual-DtrsA′B′-1.3 kb stuff1-CMV-EGFP-polyA-RHstuff vector.

[0215] A 1.3 kb stuff2 fragment was obtained by PCR cloning. The primers included primer 9 and primer 10, both of which were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0216] The sequence of primer 9, SEQ ID NO: 14, is:

[0217] 5′-TTTGTAGGTAACCTCAAGAAGCACATCCTGAATC-3′.

[0218] The sequence of primer 10, SEQ ID NO: 15, is:

[0219] 5′-GCACGTGGTTACCTTGCCCAGAATGTCTGTGCTG-3′.

[0220] The PCR reaction system consisted of 2.5 μL of KOD-Plus-Neo, 5 μL of 10x PCR Buffer, 5 μL of 25 mM MgSO4, 5 μL of 25 mM MgSO4, 5 μL of DMSO, 0.6 μL each of primers 9 and 10 (10 μM), 1-50 ng of gene template saCas9, and HO was added to make up to 50 μL.

[0221] The PCR reaction program was as follows: 95°C for 4 min; 95°C for 10 s, 56°C for 30 s, and 72°C for 1.5 min, for 25 cycles.

[0222] The 1.3kb stuff2 fragment obtained by PCR amplification was inserted into the pFastBacdual-DtrsA′B′-1.3kb stuff1-CMV-EGF-polyA-RHstuff vector by single enzyme digestion with BstEII to obtain the pFastBacdual-DtrsA′B′-1.3kb stuff1-CMV-EGFP-polyA-1.3kb stuff2 vector.

[0223] So far, we have obtained

[0224] pFastBacdual-DtrsA′B′-1.3kb stuff1-CMV-EGF-polyA-1.3kb stuff2 (spAAV-CMV-EGFP(+)) vector, that is, spAAV-CMV-EGFP(+) genome packaging plasmid.

[0225] The construction of the EGFP expression frame in this embodiment is a prior art. For details, please refer to the literature: Li Taiming et al., Preparation of AAV-ITR Gene Expression Microcarriers in Insect Cells, Chinese Journal of Biotechnology, 2015, 31(8), page 1232, "Construction of pFastBacdual-ITR-EGFP Plasmid" in method 1.2.1.

[0226] 2. pAAV-EGFP(-)DNA genomic plasmid

[0227] The plasmid DNA 5' to 3' direction contains D sequence, trs sequence, A'B' sequence, and reverse EGFP gene expression cassette sequence

[0228] The DtrsA′B′ sequence is SEQ ID NO: 5, including

[0229] 5′-AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAA-3′.

[0230] The reverse EGFP gene expression frame sequence, namely the polyA sequence, the reverse complementary sequence of the EGFP open reading frame, introns, enhancers, promoters, and downstream Rep recognition and cleavage sequences.

[0231] The reverse EGFP expression cassette was inserted into the

[0232] Using pFastBacdual-DtrsA′B′-1.3kb stuff1-CMV-EGF-polyA-1.3kb stuff2 as a template, a 1.3kb stuff2′-polyA′-EGFP′-CMV′-1.3kb stuff1′ fragment was cloned by PCR. The target fragment was amplified using primers 11 and 12. Both primers 11 and 12 were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0233] The sequence of primer 11, SEQ ID NO: 16, comprises:

[0234] 5′-TCACTGACGCGTCACGTGGTTACCTTGCCCAGA-3′;

[0235] The sequence of primer 12, SEQ ID NO: 17, comprises:

[0236] 5′-CAAGATCGGTCCGCGGAAGGTCGGTATCCACGGA-3′.

[0237] PCR fragments were inserted into the

[0238] pFastBacdual-DtrsA′B′-CMV-EGFP-polyA-RHstuff vector, which is obtained

[0239] pFastBacdual-DtrsA′B′-1.3kb stuff2′-polyA′-EGFP′-CMV′-1.3kb stuff1′ (spAAV-EGFP(-)) genome packaging plasmid.

[0240] Second, preparation of recombinant Bacmid.

[0241] Specifically, the spAAV vector packaging plasmid prepared by the above steps and the three recombinant plasmids were used to prepare recombinant Bacmids respectively, and the recombinant method was as follows:

[0242] 1. Slowly thaw 100ul DH10Bac competent cell culture medium on ice.

[0243] 2. Add 500 ng of plasmid DNA and mix gently.

[0244] 3. Place on ice for 30 minutes, heat shock at 42°C for 90 seconds, and immediately place on ice for 2 minutes.

[0245] 4. Add 900 μL LB medium and culture in a shaking incubator at 37°C and 225 rpm for 2-3 hours.

[0246] 5. Add 40 μL of 2% (20 mg / mL) galactoside X-gal and 20 μL of 20% (200 mg / ml) isopropyl-β-d-thiogalactoside IPTG to the center of a pre-made 90 mm agar plate containing 50 μg / mL kanamycin Kan, 7 μg / mL gentamicin Gen, and 10 μg / mL tetracycline Tet.

[0247] Use a sterile applicator to spread the solution over the entire surface of the culture plate and incubate at room temperature until all the liquid disappears.

[0248] 6. Dilute the cells 10-fold with LB medium (10 -1 , 10 -2 ), 100 μL of each gradient was applied to LB plate.

[0249] 7. Incubate at 37°C for 48 hours, pick two white colonies, streak them onto a new LB agar plate (same resistance as above), and incubate at 37°C overnight.

[0250] 8. Pick two single plaques for PCR identification; PCR identification of Bacmid, the primers used are target gene F / R and PUC M13 F / R.

[0251] 9. Remove the correctly identified bacmid plaque and inoculate it into 10 mL of LB (Kan+, Gen+, Tet+). Shake the culture for 12 hours. Isolate the recombinant bacmid DNA using the OMEGA kit according to the instructions in the OMEGA kit. Measure the bacmid concentration, aliquot, and freeze at -20°C. This will yield the recombinant bacmid.

[0252] Third, preparation of baculovirus.

[0253] The specific steps for preparing baculovirus include:

[0254] 1. Prepare transfection reagent

[0255] 1×HBS 200mL preparation

[0256] Hepes 0.954g

[0257] NaCl 1.754g

[0258] 150 mL sterile ddH2O

[0259] Adjust the pH to 7.4 with 1M NaOH, dilute to 200 mL, filter aseptically in a safety cabinet, and store at 4°C

[0260] PEI 20mL preparation method includes

[0261] PEI 0.043g

[0262] 1 mL of anhydrous ethanol

[0263] After fully dissolved, dilute to 20 mL with 1× HBS, freeze and thaw three times (freeze at -20°C, thaw at room temperature), and store at -20°C.

[0264] 2. Cell Plating

[0265] Plating: Take a 6-well plate, aspirate the suspended culture cells and count them so that the cell density is 2x10 6 cells / mL, 2mL per well, and the cell survival rate is above 95%.

[0266] 3. Transfection (amount per well)

[0267] Solution A: PEI: Add 6 μL PEI and 94 μL 1× HBS, mix well, and let stand for 4 minutes.

[0268] Solution B: Take 3 μg of bacmid DNA (inactivate the bacmid at 65°C for 30 minutes in advance), add 1× HBS to make the final volume 100 μL, and mix gently.

[0269] Take 100 μL of solution A and add solution B, mix well, and incubate at room temperature for 30 minutes. Add to the wells with cells and culture for 96 hours.

[0270] 4. Virus Amplification

[0271] 1) Separation of P1

[0272] After confirming that the cells were in the late infection stage (96 h), 2 mL of virus-containing culture medium was collected from each well into a sterile 15 mL centrifuge tube and centrifuged at 500 g for 5 minutes to remove cell debris.

[0273] Transfer the supernatant to a sterile EP tube and store at 4°C in the dark. For long-term storage, aliquot and freeze at -80°C.

[0274] 2) Amplify the virus to obtain P2

[0275] Take 8 mL of suspension cultured cells with a multiplicity of infection (MOI) of 0.1 and a density of 2×10 6 1 cell / mL, calculate the required volume of P1 to be 1.6 mL. Incubate at 27°C for 72 h. Collect the suspended cells in a sterile 15 mL centrifuge tube and centrifuge at 500 g for 5 minutes. Aliquot the supernatant into sterile EP tubes. This viral supernatant is P2 and stored at 4°C in the dark. Alternatively, aliquots can be frozen at -80°C to extend storage time.

[0276] 3) Amplify the virus to obtain P3

[0277] P3 can be obtained by amplification according to the above method, with an MOI of 0.1 and a density of 2×10 6 cells / mL, 200 μL P2 storage solution was added to 10 mL of suspended culture cells, and P3 was collected after culture for 72 h.

[0278] The P1 virus titer usually obtained is 1×10 6 -1×10 7 The P2 titer is between 1×10 7 ~1×10 8 between.

[0279] 4) Virus packaging

[0280] Take 100 mL of suspension cultured cells at an MOI of 1 and a density of 5 × 10 6cells / mL, add 5 mL each of P3 generation Rep, Cap, and target gene, culture for 72 hours, and collect the cells.

[0281] Fourth, preparation of spAAV vector.

[0282] The spAAV vector packaging system involved in this application can be an insect cell baculovirus packaging system or a mammalian cell packaging system. The insect cell is Sf9 cell.

[0283] This embodiment uses the insect cell baculovirus packaging system as an example for description, and the specific steps include:

[0284] First, one or two recombinant plasmids expressing the AAV replication protein Rep and the AAV capsid protein Cap (the Rep and Cap genes are on one or two separate plasmids), and another plasmid containing the spAAV DNA genome, are transformed into E. coli DH10Bac competent cells. After two rounds of blue-white screening, colonies containing recombinant bacmids are white, while colonies without recombination are blue. White colonies are selected for amplification and the recombinant bacmid is extracted.

[0285] Next, using an insect cell transfection reagent, the two or three recombinant bacmids were transfected into Sf9 insect cells. After 4-5 days, the cell supernatant was collected to obtain P1 insect cell recombinant baculovirus. The P1 recombinant baculovirus was amplified by infecting Sf9 insect cells twice to obtain P3 recombinant baculovirus. The titer of the P3 baculovirus was determined using the plaque assay: virus titer (pfu / mL) = 1 / dilution factor × number of plaques × 1 / inoculation volume per well.

[0286] Finally, two or three recombinant baculoviruses of generation P3 were co-infected into Sf9 insect cells to package and obtain spAAV vector virus particles. For the procedure, please refer to the literature: Urabe M, Ding C, Kotin RM. Insect cells as a factory to produce adeno-associated virus type 2 vectors. Hum Gene Ther. 2002 Nov 1; 13(16): 1935-43. doi: 10.1089 / 10430340260355347. PMID: 12427305.

[0287] Refer to Figure 2, which is a schematic diagram of the packaging of the spAAV vector prepared using the insect cell baculovirus packaging system in this example. In the figure, during spAAV vector packaging, the Rep protein recognizes and binds to the Rep binding site (RBE) on the recombinant baculovirus DNA, identifies the terminal melting site (trs), breaks the phosphodiester bond between the tt bases, and covalently binds to the 5′ end, introducing the DNA into the AAV capsid. The length of the DNA packaged into the capsid is approximately 4.7 kb.

[0288] Finally, detection and identification analysis

[0289] 1. Virus Purification

[0290] The purification was performed by iodixanol density gradient ultracentrifugation, and the specific steps included:

[0291] 1. Obtaining Cell Lysate

[0292] 1) 72 hours after co-transfection, cells were collected by centrifugation at 1000g for 5 minutes;

[0293] 2) Discard the supernatant and resuspend the cells in 10 mL of Lysis Buffer;

[0294] Lysis Buffer(1L):NaCl 8.766g

[0295] Tris 6.055g

[0296] DH2O 950ml

[0297] Adjust pH to 8.5 with 5M HCl and make up to 1L

[0298] Sterile filtration in a safety cabinet and stored at 4°C

[0299] 3) Freeze in liquid nitrogen and thaw at 37°C, repeat 3-4 times until clear;

[0300] 4) Centrifuge at 5000 g for 30 minutes at 4°C to collect the supernatant; resuspend the pellet in 4-5 mL of PBS and centrifuge again to collect the supernatant;

[0301] 5) Add DNase (10 μL / mL) and RNase (1 μL / mL) and digest in a 37°C water bath for 1 h for purification;

[0302] 2. Virus Purification by Iodixanol Density Gradient Ultracentrifugation

[0303] 1) Dilute 60% iodixanol to 15%, 25%, 40%, and 58% with PBS-MK (1×PBS, 1 mM MgCl2, 2.5 mM KCl). Add solid NaCl to the 15% phase to a final concentration of 1 M. 2) Add 0.5% phenol red solution to 1.5 μL / mL for the 15% and 25% phases, and to 0.5 μL / mL for the 58% phase.

[0304] 3) Using a 1.27 x 120 mm flat-blade spinal puncture needle, add 8 mL of 15%, 6 mL of 25%, 8 mL of 40%, and 5 mL of 58% iodixanol diluent in that order through the bottom of a 39 mL quick-seal tube, avoiding the formation of bubbles.

[0305] 4) Carefully overlay the cell lysate on the iodixanol density gradient solution and fill the tube (to the top line). If necessary, add Lysis Buffer to fill the tube to the top. Balance the tube and centrifuge at 69,000 rpm at 18°C ​​for 1.5 h (BECKMAN COULTER 70Ti rotor).

[0306] 5) Pierce the quick-seal tube from the bottom, discard the first 4 mL (corresponding to the 58% phase), and collect 6 mL of the solution in the tube;

[0307] 6) Ultrafiltration was performed using ultrafiltration tubes (Amicon Uitra-4 Centrifugai Filters, Ultracel-100k) at 3000 g for 5 minutes. Repeated washing with PBS (1× PBS, Solebol, supplemented with 5M NaCl to a final concentration of 350 mM) was performed until the residual iodixanol concentration was <0.1%. The final volume was reduced to approximately 200 μL per 100 mL packaging volume.

[0308] 7) Add glycerol to a final concentration of 5%, sterile filter, and store in aliquots at -80°C;

[0309] 2. Fluorescence quantitative detection

[0310] The method for quantitatively detecting the titer of spAAV vector using fluorescent PCR (RT-PCR method) specifically includes:

[0311] 1. Dilute the plasmid ITR-EGFP 10 times, then perform gradient dilution, and make a standard curve with 5 dilution gradients.

[0312] 2. Dilute the virus 10 times, then perform gradient dilution, dilute 4 gradients

[0313] 3. Buffer preparation (protect from light):

[0314] 2xSuperpreMix Plus(SYBR Green) 10μL

[0315] Upstream primer, 5'-TCCGCGTTACATAACTTACGG-3' (SEQ ID NO: 22; synthesized by Sangon Biotech (Shanghai) Co., Ltd.), 0.3 μL.

[0316] Downstream primer, 5'-GGGCGTACTTGGCATATGAT-3' (SEQ ID NO: 23; synthesized by Sangon Biotech (Shanghai) Co., Ltd.), 0.3 μL.

[0317] DD H2O 4.4μL

[0318] 4. Sample addition, 20 μL system: 5 μL template + 15 μL Buffer, two replicates for each gradient

[0319] 5. RT-PCR (Roche LightCycler) procedure:

[0320] 3. Analysis of spAAV vector capsid protein by SDS-PAGE electrophoresis

[0321] 1. Glue making:

[0322] 1) Separating Gel (10%): To a separating gel beaker, add the following ingredients (9.093 mL): 3.69 mL of double-distilled water, 2.97 mL of 30% acrylamide gel preparation solution (acrylamide: methylenebisacrylamide = 29:1), 2.25 mL of 2M Tris, pH 8.8, 90 μL of 10% ammonium persulfate, 90 μL of 10% SDS, and 3 μL of TEMED. Mix thoroughly and pipette the separating gel between two glass plates until the liquid level reaches 1 cm below the bottom edge of the comb. Slowly add 75% ethanol using a pipette and allow to stand. After the separating gel polymerizes, discard the 75% ethanol layer.

[0323] 2) Stacking Gel (5%): To a stacking gel beaker, add the following ingredients (2.357 mL): 1.83 mL of double-distilled water, 0.39 mL of 30% acrylamide gel preparation solution (acrylamide:methylenebisacrylamide = 29:1), 0.75 mL of 0.5 M Tris, pH 6.8, 30 μL of 10% ammonium persulfate, 30 μL of 10% SDS, and 2 μL of TEMED. Mix thoroughly and immediately use a pipette to add stacking gel to the separating gel between two glass plates until full. Gently insert a comb and allow to stand until it solidifies. This forms a gel slab.

[0324] 2. Sample processing:

[0325] Virus sample: 5X loading buffer = 2:1 ratio was mixed, and the mixture was placed in a boiling water bath for 10 minutes. After boiling, the mixture was centrifuged immediately and all samples were subjected to electrophoresis.

[0326] 3. Electrophoresis

[0327] 1) Install the gel plate made of two glass plates vertically on the power supply rack in the electrophoresis tank, so that the concave edge of the gel plate is close to the power supply rack.

[0328] 2) Fix the gel plate and power supply rack in the power supply slot as required, add electrophoresis buffer as required, and gently remove the comb in the gel plate.

[0329] 3) Take the treated sample solution and use a microinjector to draw 15 μL of the sample solution and slowly add it to the notch in the gel plate (the sample entry point).

[0330] 4) During electrophoresis, control the voltage to 90V for the stacking gel and 120V for the separation gel. Stop electrophoresis when the bromophenol blue streak in the gel is observed to be 1 cm from the bottom.

[0331] 4. Stain. Use a blade or thin plate to gently pry apart the two glass plates outside the gel. Run a blade along the gel at the junction of the separating gel and the stacking gel to cut the separating gel. Then, carefully transfer the separating gel to a staining dish, add 100 mL of staining solution, cover, and stain for 1-3 hours.

[0332] 5. Destain. Pour off the staining solution. Rinse the stained gel several times with water, place it in clean water until just above the gel, and microwave on high for 2 minutes. Remove and shake slowly, repeating this process until protein bands are clearly visible.

[0333] Figure 3 shows the SDS-PAGE electrophoresis results of capsid proteins of spAAV vectors packaged with cis packaging elements. Lane MW: protein molecular weight standard; Lane 1: spAAV vector packaged with the ITR cis element; Lane 2: spAAV vector packaged with the DtrsA′B′ cis element; Lane 3: spAAV vector packaged with the trsA′B′ cis element; Lane 4: spAAV vector packaged with the trsA′ cis element; Lane 5: spAAV vector packaged with the DttRR cis element; Lane 6: spAAV vector packaged with the ttRR cis element.

[0334] As can be seen from the figure, the capsid protein composition of the spAAV vector produced by packaging multiple cis packaging elements is no different from that of the ordinary rAAV capsid protein. They are all composed of three proteins: Vp1, Vp2, and Vp3, and the composition of the three proteins is basically 1:1:10.

[0335] 4. spAAV vector genome identification

[0336] First, perform DNA neutral agarose gel electrophoresis analysis. The specific steps include:

[0337] 1. Prepare agarose solution by adding the correct amount of agarose powder and the correct amount of 1X TAE electrophoresis buffer to a conical flask or glass bottle.

[0338] 2. Loosely plug the neck of the flask with Kimwipes. If using a glass flask, loosen the stopper. Heat the suspension in a boiling water bath or microwave until the agarose dissolves.

[0339] 3. Cool the clear solution to 40-50°C and add 4S red staining solution to quickly cast the gel. When the gel is completely solidified, place it in the electrophoresis tank and add freshly prepared 1X TAE electrophoresis buffer until the gel is just covered.

[0340] 4. Prepare 10 μL of virus, add 1.1 μL 10X PCR Buffer, place in PCR instrument, 95℃ for 5 minutes, 72℃ for 5 minutes, 55℃ for 5 minutes, 37℃ for 5 minutes, 80℃ for 5 minutes, 72℃ for 5 minutes, 55℃ for 5 minutes, and 37℃ for 15 minutes.

[0341] 5. Add 0.2 volumes of 6X gel loading buffer to the renatured sample.

[0342] 6. Add all samples dissolved in 6X loading buffer to the sample wells and run electrophoresis at 100 V for 50 minutes.

[0343] 7. Imaging: Place the gel in a gel imager and take photos.

[0344] Figure 4 shows the results of neutral agarose gel electrophoresis of spAAV vectors packaged with cis packaging elements. Lane MW: DNA molecular weight standard; Lane 1: spAAV vector packaged with the ITR cis element; Lane 2: spAAV vector packaged with the DtrsA′B′ cis element; Lane 3: spAAV vector packaged with the trsA′B′ cis element; Lane 4: spAAV vector packaged with the trsA′ cis element; Lane 5: spAAV vector packaged with the DttRR cis element; Lane 6: spAAV vector packaged with the ttRR cis element.

[0345] As can be seen from the figure, the spAAV vector DNA produced by packaging various cis packaging elements is a monopolar single-stranded DNA. When analyzed by neutral agarose gel electrophoresis, its DNA will appear as a DNA band much smaller than its molecular weight and often appears diffuse.

[0346] Then, DNA alkaline agarose gel electrophoresis analysis is performed, and the specific steps include:

[0347] 1. Preparation of Alkaline Agarose Gel: Prepare the agarose solution by adding 0.36 g agarose powder and 27 mL distilled water to a conical flask or glass bottle. Gently plug the neck of the flask with Kimwipes. If using a glass bottle, loosen the stopper. Heat the suspension in a microwave oven on medium heat until the agarose dissolves. Allow the clear solution to equilibrate in a 56°C water bath for 5 minutes. Add 3 mL of 10X alkaline gel electrophoresis buffer (equilibrate in a 56°C water bath for 2 minutes), mix thoroughly, and quickly cast the gel. Once the gel has completely solidified, place it in an electrophoresis tank and add freshly prepared 1X electrophoresis buffer (4°C) until the gel is just covered.

[0348] 2. Sample preparation: Take 30 μl of virus sample, add 3 μl of proteinase K (20 mg / mL), digest at 65°C for 15 minutes, centrifuge at 12000 g for 5 minutes, take 30 μl of supernatant, and add 6 μl of 6X alkaline gel loading buffer.

[0349] 3. Electrophoresis: Add all DNA samples to the sample wells and start electrophoresis at a voltage of <3.5V / cm (horizontal electrophoresis tank JY-SPAT 27V electrophoresis for 3h).

[0350] 4. Elution: Place the gel in 400 mL of water for injection and elute on a horizontal shaker at 56 rpm for 1 hour, changing the water every 30 minutes.

[0351] 5. Staining: Use 1X TAE staining solution containing 0.5 μg / mL EB (ethidium bromide) to stain the washed gel.

[0352] 6. Imaging: Place the gel in a gel imager and take photos.

[0353] Figure 5 shows the results of alkaline agarose gel electrophoresis of spAAV vectors. Lane MW: DNA molecular weight standard; Lane 1: spAAV vector packaged with the ITR cis element; Lane 2: spAAV vector packaged with the DtrsA′B′ cis element; Lane 3: spAAV vector packaged with the trsA′B′ cis element; Lane 4: spAAV vector packaged with the trsA′ cis element; Lane 5: spAAV vector packaged with the DttRR cis element; Lane 6: spAAV vector packaged with the ttRR cis element.

[0354] As can be seen from the figure, the spAAV vector DNA produced by packaging various cis packaging elements is single-stranded, monopolar DNA. When analyzed by alkaline agarose gel electrophoresis, it presents a single DNA band with a single-stranded DNA molecular weight of 4.7 kb.

[0355] The above are merely preferred embodiments of the present application and are not intended to limit the scope of protection of the present application. Persons skilled in the art will readily appreciate that the present application may be modified and altered in various ways. Any changes, modifications, substitutions, integrations, or parameter changes to these embodiments, which fall within the spirit and principles of the present application and achieve the same functionality through conventional substitutions, without departing from the principles and spirit of the present application, fall within the scope of protection of the present application.

Claims

1. A plurality of cis packaging elements for packaging single-stranded monopolar DNA recombinant adeno-associated viruses, wherein the cis packaging elements comprise a Rep binding site (RBS) and a Rep cleavage site (trs); The cis packaging element is capable of packaging its downstream spAAV genome into the AAV capsid protein, including when the cis packaging element is placed at the 5′ end of the packaged single-stranded monopolar DNA, the Rep protein packages the single-stranded monopolar DNA into the AAV capsid protein through recognition and cleavage.

2. The cis packaging element for packaging a single-stranded monopolar DNA recombinant adeno-associated virus according to claim 1, characterized in that: The cis packaging element includes any one of the DNA elements of ITR, DtrsA′B′, trsA′B′, trsA′, DttRR, and ttRR; The 5′ end of the spAAV genome before packaging has the cis packaging element; The 3′ end of the spAAV genome before packaging does not have all or part of the DNA sequence of ITR and does not have other cis packaging elements.

3. The cis packaging element for packaging a single-stranded monopolar DNA recombinant adeno-associated virus according to claim 2, characterized in that: The spAAV genome before packaging comprises the cis packaging element, exogenous nucleic acid and / or other exogenous DNA sequence in 5′ to 3′ order; The cis packaging element comprises RBE and trs; and / or, the cis packaging element comprises D, A′ and C′ segments derived from Flip ITR; and / or, the cis packaging element comprises D, A′ and B′ segments derived from Flop ITR; And / or, the Flip ITR has the nucleotide sequence shown in SEQ ID NO.1; And / or, the Flop ITR has the nucleotide sequence shown in SEQ ID NO.2; And / or, the RBE has the nucleotide sequence shown in SEQ ID NO.3; And / or, the trs has the nucleotide sequence shown in SEQ ID NO.4; and / or, the DtrsA′B′ has the nucleotide sequence shown in SEQ ID NO: 5; and / or, the trsA′B′ has the nucleotide sequence shown in SEQ ID NO: 6; and / or, the trsA′ has the nucleotide sequence shown in SEQ ID NO: 7; and / or, the DttRR has the nucleotide sequence shown in SEQ ID NO: 8; And / or, the ttRR has the nucleotide sequence shown in SEQ ID NO:

9.

4. The cis packaging element for packaging a single-stranded monopolar DNA recombinant adeno-associated virus according to claim 3, characterized in that: The exogenous nucleic acid is located downstream of the 5′ cis packaging element of the spAAV genome before packaging, and may have a nucleotide sequence for encoding an exogenous gene; Preferably, the exogenous nucleic acid comprises an exogenous gene expression cassette.

5. A spAAV vector, obtained by packaging a spAAV genome using the cis packaging element according to any one of claims 1 to 5; The spAAV includes an AAV capsid protein and a spAAV genome packaged by the AAV capsid protein; in, The spAAV genome packaged by the AAV capsid protein is derived from the spAAV genome before packaging; The spAAV genome comprises a spAAV vector of positive-strand DNA or a spAAV vector of negative-strand DNA, and the two are not produced at the same time.

6. The spAAV vector according to claim 5, characterized in that The 5′ end of the packaged spAAV genome in the spAAV vector has a cis packaging element; The exogenous nucleic acid is placed downstream of the 5′ end cis packaging element; And / or, the exogenous nucleic acid comprises a nucleotide sequence that may not encode an exogenous gene and / or has a nucleotide sequence for encoding an exogenous gene; And / or, the nucleotide sequence encoding the exogenous gene is an exogenous gene expression frame; And / or, the nucleotide sequence encoding the exogenous gene is a monopolar single-stranded DNA sequence; And / or, the nucleotide sequence not encoding the exogenous gene includes a stuffing sequence; the stuffing sequence may have different lengths, filling the final DNA length to reach 4.7 kb.

7. The spAAV vector according to claim 6, characterized in that: The cis packaging element comprises an RBE sequence and a trs sequence, wherein the 5′ end sequence packaged into the spAAV virus capsid is the trs sequence portion remaining after Rep cleavage; And / or, the exogenous gene expression frame includes a promoter, an exogenous gene open reading frame and a poly A sequence in the 5′-3′ direction; Preferably, enhancers and introns may also be included between the promoter and the exogenous gene open reading frame; And / or, the exogenous gene expression frame includes a forward exogenous gene expression frame and a reverse exogenous gene expression frame; Preferably, the forward exogenous gene expression frame and the reverse exogenous gene expression frame are complementary sequences.

8. The spAAV vector according to claim 6, characterized in that The AAV capsid protein may be any one selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12; and / or, the AAV capsid protein is an AAV capsid protein variant, including any one of a tyrosine single amino acid / multiple amino acid variant, a tyrosine variant, a serine variant, a threonine variant, an AAV-DJ multi-serotype chimera, or a polypeptide embedded variant; And / or, the AAV capsid protein can be produced by utilizing a plasmid expressing the AAV capsid protein Cap.

9. The spAAV vector according to any one of claims 5 to 8, characterized in that The spAAV vector can be produced by packaging the spAAV transgenic plasmid and the plasmid expressing the AAV capsid protein Cap and the replication protein Rep through an AAV vector packaging system, including an insect cell packaging system and a mammalian cell packaging system; The spAAV transgenic plasmid can be selected as the plasmid of the spAAV genome before packaging; Preferably, the spAAV transgenic plasmid is a pFastBacdual plasmid or a pFastBac1 plasmid; The spAAV transgenic plasmid can be designed as a positive-strand single-stranded DNA sequence or a negative-stranded DNA sequence encoding a foreign gene; the positive-stranded DNA sequence or the negative-stranded DNA sequence includes a cis packaging element, a promoter, a foreign gene open reading frame, and poly A in 5′ to 3′ order; And / or, the spAAV transgenic plasmid can produce the spAAV genome before packaging.

10. A method for obtaining a spAAV vector as described in any one of claims 5 to 9, comprising transforming a spAAV transgenic plasmid, a Cap expression plasmid, and a Rep expression plasmid into competent E. coli cells respectively; selecting white colonies after at least one round of blue-white screening, amplifying and extracting recombinant bacmids; transfecting insect cells with the recombinant bacmids to produce recombinant baculoviruses; and extracting the spAAV transgenic, Cap expression, and Rep expression recombinant baculoviruses, and co-infecting Sf9 cells with the recombinant baculoviruses to obtain the spAAV vector; in, The spAAV vector packaging system is an insect cell baculovirus packaging system or a mammalian cell packaging system.

11. A method for delivering exogenous nucleic acid to a cell, comprising contacting a cell with one or more spAAV vectors as described in any one of claims 5 to 9 and infecting the cell, wherein: The genome of the spAAV vector comprises the gene expression frame of the exogenous nucleic acid and / or other optional DNA sequences; the spAAV vector is integrated into the cell genome, homologous or non-homologous recombination occurs, and the exogenous gene expression is generated; The genome of the spAAV vector comprises the positive-strand single-stranded DNA sequence and / or the negative-strand single-stranded DNA sequence of the exogenous gene; The exogenous nucleic acid comprises a nucleic acid sequence of an exogenous gene or a nucleic acid sequence of a non-exogenous gene; Preferably, the spAAV vector comprises two or more spAAV vectors, which at least comprise a first spAAV vector having a positive-strand single-stranded DNA sequence of the exogenous gene and a second spAAV vector having a negative-strand single-stranded DNA sequence of the exogenous gene; Preferably, the first spAAV vector and the second spAAV vector are used alone or in combination; Preferably, the combined use comprises contacting the cell with the first spAAV vector and the second spAAV vector in sequence, so that the first spAAV vector and the second spAAV vector containing the positive-strand and negative-strand spAAV genomes, respectively, are rapidly complementary and paired in the nucleus of the host cell to form a double-stranded molecule, thereby initiating gene expression; And / or, the cell is a eukaryotic cell; preferably, the cell is an animal cell; more preferably, the cell includes a vertebrate cell or a mammalian cell.

12. Use of the cis packaging element according to any one of claims 1 to 4 in gene expression and / or gene therapy and / or gene editing and / or gene regulation and its products.

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