Method for inhibiting transcriptional activity of residual AAV vector plasmid and plasmid backbone

Abstract

Provided are a method for inhibiting the transcriptional activity of a residual AAV vector plasmid and a plasmid backbone, and a nucleic acid construct comprising a transcription-inhibitory element, a recombinant AAV vector plasmid comprising the nucleic acid construct, and an AAV product prepared by using the recombinant AAV vector plasmid. By means of inhibiting the transcriptional activity of ITR in the AAV vector plasmid, the transcription-inhibitory element is used for inhibiting transcription products generated by the residual AAV vector plasmid and the reverse packaging of the plasmid backbone.

Description

A method for inhibiting the transcriptional activity of residual AAV vector plasmid and plasmid backbone Technical Field

[0001] The present invention relates to the fields of biotechnology and biomedicine, and in particular to a method for designing an AAV vector plasmid carrying a target gene, so as to reduce the transcriptional activity of ITRs in the AAV vector plasmid on residual plasmids and plasmid backbones, thereby improving the safety of AAV products. Background Art

[0002] In recent years, gene therapy has entered a stage of rapid development, with an increasing number of gene therapy products entering the clinical research stage or being approved for marketing, involving genetic diseases, malignant tumors, cardiovascular diseases, infectious diseases, etc. Depending on the type of product, it can be divided into viral vectors and non-viral vectors. Commonly used viral vectors include adeno-associated virus (Adeno associated Virus, AAV), adenovirus (Adenovirus, AdV), herpes simplex virus (Herpes Simplex Virus, HSV), etc. Among them, AAV has good safety and high transduction efficiency and is widely recognized. As of the end of 2022, related research in the field of gene therapy has shown a continuous upward trend, and a number of cell therapy and gene therapy products have been approved for marketing. Taking recombinant adeno-associated virus (rAAV) vector gene therapy products as an example, six rAAV products have been approved for marketing worldwide, including Glybera (EMA, 2012), Luxtura (FDA, 2017), Zolgensma (FDA, 2019), Upstaza (EMA, 2022), Roctavian (EMA, 2022) and Hemgenix (FDA, 2022). There are more than 300 AAV gene therapy drugs in clinical research worldwide, of which 14 are in clinical phase 3 or above, mainly focusing on rare diseases, hemophilia, ophthalmic diseases and other fields. At the same time, domestic AAV gene therapy is also showing an explosive trend. In 2022, the CDE approved a total of 13 AAV gene therapies to enter clinical trials, of which 12 were developed by domestic companies. This also indicates that AAV therapy has entered an accelerated clinical process.

[0003] Adeno-associated virus (AAV) is a small, replication-deficient, non-enveloped virus that infects humans and some primates. Its toxic particles are approximately 20-25 nm in size and it is a member of the parvovirus family. AAV relies on co-infection with other viruses (primarily adenovirus) for replication and was first discovered as a contaminant in adenovirus preparations, hence its name. AAV has nine common serotypes (AAV 1-9), and there are also serotypes such as AAV10, AAV-DJ, and AAV-DJ / 8. More and more studies have shown that AAV gene therapy products also have the advantages of weak immunogenicity, a wide host range, stable physicochemical properties, and long-term expression of exogenous genes. It has been used as an efficient and safe delivery vector for in vivo gene therapy and is widely used in gene therapy, neural circuit labeling, in vivo imaging, gene editing, the preparation of animal models of neurological diseases, and the research and development of related drugs for rare diseases, ophthalmology, hemophilia, etc.

[0004] The AAV genome consists of a single-stranded DNA (ssDNA) molecule with a length of 4.7 kb. Between the ITR sequences (Inverted terminal repeat, ITR) at both ends, the ITR sequence is composed of 145 nucleotides in length and contains interrupted palindromic sequences. These palindromic sequences can fold into a hairpin structure that acts as a primer in the initiation of DNA replication. Between the ITR sequences is the viral coding region, which contains two open reading frames and is currently believed to express four Rep proteins, three Cap proteins and one AAP protein. ITR is the starting point of AAV DNA self-replication and the signal that triggers viral packaging. It plays a key role in the replication and packaging of the virus and is involved in the integration and escape of the viral genome from the host genome. In addition, Earley et al. [1] have shown that the ITR itself has strong promoter activity and can transcribe the gene sequence it carries. Keiser et al. [2] found that there were strong transcription signals upstream and downstream of the 3'ITR and upstream of the 5'ITR. RNA sequencing revealed that the transcribed fragments were plasmid backbone fragments containing ITRs. Subsequent in vivo experiments in crab-eating macaques found that the expression of genes related to immunity in vivo was significantly increased, which was consistent with the observed neuropathological results. This fully demonstrates that in AAV products, plasmid backbone fragments containing ITRs remain in AAV products and are transcribed, resulting in toxicity to the body. The plasmid backbone of AAV vectors generally contains elements such as antibiotic genes and bacterial replicons. Under the action of the promoter activity of the ITR, the transcription of residual plasmid DNA occurs, which may translate into unknown proteins. The transcription products of the residual plasmid DNA and the unknown proteins may both cause the body's immune response. Moreover, since the transcription start site in the ITR is difficult to confirm, the transcribed proteins are also difficult to identify.

[0005] At present, the main methods for the production and preparation of AAV products are the three-plasmid transient transfection method (Helper-free AAV packaging system), the stable production cell method, and the baculovirus-infected insect cell method. The three-plasmid transient transfection method for producing AAV products mainly uses plasmids as raw materials for production. Compared with viruses, it is easier to prepare and control quality, does not require the introduction of helper viruses, and reduces safety risks. For AAV production without helper viruses, the production environment and facilities requirements are relatively low, while the production requirements for AAV using helper viruses are relatively high, such as production in a BSL2 laboratory. The three-plasmid transient transfection of suspended HEK293 cells to prepare AAV products overcomes the limitations of traditional adherent cells that cannot be mass-produced and the difficulty in achieving high titers of the final product, and has been widely used in large-scale production of rAAV. The three-plasmid transient transfection system mainly transfects HEK293 cells with the target gene vector plasmid, an adenovirus helper plasmid containing the auxiliary Rep / Cap protein expression gene, and an adeno-associated virus replication plasmid containing the Rep and Cap protein expression genes for a period of time, and then collects the cell lysate for ultracentrifugation purification to obtain high-titer virus. During the AAV production process, plasmids serve as key raw materials for rAAV production. Residual plasmid DNA and plasmid backbone DNA from AAV packaging remain as impurities throughout the AAV production process. Residual plasmid DNA in AAV production originates from plasmids added during transient transfection and plasmid DNA fragments reversely packaged during the AAV packaging process. Plasmid residues are closely linked to the GOI-AAV complex. Plasmid DNA remaining in the AAV harvest can be removed by adding certain concentrations of Benzonase and DNase I endonucleases. However, the plasmid backbone DNA encapsidated within the AAV capsid cannot be removed by nucleases due to the protection provided by the AAV capsid and remains in the AAV product. Because the vector plasmid containing the target gene contains two ITRs, which are transcriptionally active within cells, the plasmid backbone sequence containing the ITRs can be transcribed in vivo, resulting in the translation of unknown proteins. This can lead to immune responses and potential genotoxicity, seriously compromising the safety of AAV products. Chadeuf et al. [3] used different AAV packaging systems to study and showed that plasmid impurities in AAV products originated from the GOI plasmid backbone and AAV packaging plasmid. Among them, the GOI plasmid backbone residues were the main source of plasmid impurities in AAV products, accounting for about 1.3-6.1% of AAV products. Moreover, the residual plasmids are more stable in the body than AAV DNA and can express genes on the plasmids for a long time, including resistance genes. For some AAV products that are administered in large doses, the dosage is up to 1E15vg / person, such as Zolgensma, which means that 1E13vg of plasmid residues enter the human body. The potential plasmid transcription will bring great safety problems. In response to this, regulatory agencies have put forward higher requirements for the quality research and quality control of AAV products.The "Technical Guidelines for Pharmaceutical Research and Evaluation of In Vivo Gene Therapy Products" issued by the CDE in May 2022 clearly pointed out that viral vectors such as AAV that are easy to package non-vector DNA into viral particles should consider the potential risk of packaging related exogenous DNA into viral particles when selecting packaging plasmids, and the plasmid raw materials in the AAV production process need to be inspected and controlled.

[0006] At present, the problem of AAV vector plasmid residue cannot be effectively removed through downstream processes. Therefore, the only way is to reduce the plasmid residue as much as possible from the design end, or to reduce the transcription and translation of the plasmid when the plasmid residue cannot be reduced, and further reduce the side effects that may be caused by the residual plasmid. At present, there are several main methods to reduce AAV plasmid residue: 1) remove non-essential sequences such as the backbone and resistance in the AAV vector, and only use the minicircle plasmid containing the target gene fragment [4]. However, due to the complexity of the preparation of minicircle plasmids, cumbersome operations, high technical requirements, and high prices, it has not been widely promoted; 2) optimize the transfection system and reduce the total amount of transfection plasmid used, thereby reducing the plasmid residue in AAV products. However, the total amount of transfected plasmid is often directly related to the yield of AAV. Low total amount of plasmid transfection often leads to low total amount of AAV production; 3) When the plasmid backbone is no larger than 4.7kbp, the plasmid backbone connected to the ITR is prone to reverse packaging, and the AAV vector plasmid backbone is packaged as the GOI, which not only causes the packaged AAV to be non-functional, but also transcribes unknown sequences with the ITR as the promoter, and even translates unknown proteins, thus bringing uncertain safety risks [4]; Therefore, some researchers add stuffer sequences to the AAV plasmid backbone, making the plasmid backbone length much larger than 4.7kbp, which can effectively reduce the efficiency of AAV reverse packaging [5]. Hauck et al. [6] showed that the residual plasmid DNA of AAV products mainly comes from the plasmid backbone containing ITR. As the size of the plasmid backbone increases, the plasmid residue in AAV shows a downward trend. Although these methods reduce the residual plasmid in AAV products, they still cannot prevent the transcription of residual plasmids and the possible safety risks. Therefore, when it is impossible to effectively remove plasmid residues in AAV products, how to reduce the nonspecific transcription of residual plasmids in AAV products and the translation of unknown transcription products will become an important factor affecting the safety of AAV gene therapy products.

[0007] In summary, plasmid residues in AAV products or the plasmid backbone of AAV packaging can lead to the expression of abnormal, unknown proteins, which not only affects the effectiveness of the product but also poses potential safety risks. Therefore, the field urgently needs to develop a method that can effectively solve the problem of plasmid residues in AAV products. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for effectively inhibiting the reverse packaging of the plasmid backbone and unknown transcription products and translation products produced by the plasmid DNA remaining in the AAV product and the plasmid DNA in the AAV capsid from the root.

[0009] In a first aspect of the present invention, a nucleic acid construct is provided, comprising a transcription inhibitory element, wherein the transcription inhibitory element is used to inhibit the transcriptional activity of the ITR in the AAV vector plasmid, thereby inhibiting the transcription products generated by the AAV vector plasmid residue and the reverse packaging of the plasmid backbone;

[0010] The nucleic acid construct has a structure shown in the following formula I (5' end to 3' end):

[0011] Y1-5'ITR-Y2-Z1-Y3-3'ITR-Y4 (I)

[0012] Where,

[0013] Y1, Y2, Y3, and Y4 are each independently none or a transcriptional repressor element, and Y1, Y2, Y3, and Y4 are not all none at the same time; wherein, Y2 and Y4 are forward transcriptional repressor elements, and Y1 and Y3 are reverse transcriptional repressor elements, and the reverse transcriptional repressor element is the reverse complementary sequence of the forward transcriptional repressor element;

[0014] Z1 is a target gene (GOI) expression cassette, which includes one or more promoter sequences and one or more target gene coding sequences;

[0015] "-" are each independently a linker sequence, wherein the length of the linker sequence in the "Y1-5'ITR-Y2" and "Y3-3'ITR-Y4" segments is 0-150 nt, preferably 0-100 nt;

[0016] Wherein, the transcriptional repression element is selected from the following groups: (a) a sequence containing a hairpin structure; (b) a polyA signal tailing signal sequence; (c) a polyA signal tailing signal sequence containing a stop codon; (d) a polyadenylic acid sequence, i.e., a PolyA sequence; (e) a polyadenylic acid sequence containing a stop codon; or a combination thereof.

[0017] In another preferred embodiment, the "sequence containing a hairpin structure" includes a hairpin structure formed by an inverted repeat sequence (such as shRNA) or an RNA hairpin structure formed by a bulge, or other forms of hairpin structures.

[0018] In another preferred embodiment, the transcriptional repression element is (c) a polyA tailing signal sequence containing a stop codon.

[0019] In another preferred embodiment, the "polyA signal tailing signal sequence containing a stop codon" comprises a stop codon sequence and a polyA signal tailing signal sequence.

[0020] In another preferred embodiment, the stop codon sequence contains 1-10 stop codons, preferably 3-5, more preferably 3.

[0021] In another preferred embodiment, the stop codon is selected from: TAA, TAG, TGA, or a combination thereof.

[0022] In another preferred embodiment, the polyA signal tailing signal sequence is derived from the 3'UTR sequence of a prokaryotic or eukaryotic gene, or an artificially designed and synthesized sequence having the function of a polyA signal sequence.

[0023] In another preferred embodiment, the polyA signal tailing signal sequence is derived from the 3'UTR sequence of a eukaryotic gene.

[0024] In another preferred embodiment, the polyA signal tailing signal sequence is derived from the 3'UTR sequence of a gene selected from the group consisting of SV40, bGH, hGH (artificially designed), or a combination thereof or an artificially designed sequence.

[0025] In another preferred embodiment, the polyA signal tailing signal sequence has an AATAAA common sequence and a GT-rich sequence.

[0026] In another preferred embodiment, the (c) polyA signal tailing signal sequence containing a stop codon has a structure shown in the following formula A from the 5' end to the 3' end:

[0027] SP (A)

[0028] Wherein, S is the stop codon sequence; P is the polyA signal sequence; and "-" is a phosphodiester bond.

[0029] In another preferred embodiment, the forward element (c) has a nucleotide sequence as shown in SEQ ID NO: 1.

[0030] In another preferred embodiment, the reverse element (c) has a nucleotide sequence as shown in SEQ ID NO: 2 or 18.

[0031] In another preferred embodiment, the transcription repression element plays the following roles: (1) inhibiting the transcription of the AAV vector plasmid residue; (2) inhibiting the transcription of the AAV vector plasmid after being reversely packaged into the AAV capsid; (3) inhibiting the transcription activity of the ITR in the AAV vector; (4) inhibiting the transcription of the backbone region in the AAV vector plasmid.

[0032] In another preferred embodiment, the AAV vector plasmid is an AAV vector plasmid carrying an exogenous target gene in an AAV three-plasmid packaging system.

[0033] In another preferred embodiment, the AAV vector plasmid includes: AAV9, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV10, AAV-DJ, AAV-DJ / 8 or other AAV vector plasmids with capsid mutations.

[0034] In another preferred embodiment, the AAV vector plasmid is an AAV9 vector plasmid.

[0035] In another preferred embodiment, in the nucleic acid construct represented by formula (I), Y1 and Y4 are absent, and the nucleic acid construct has a structure represented by the following formula II (5' end to 3' end):

[0036] 5'ITR-Y2-Z1-Y3-3'ITR (II)

[0037] Wherein, Y2 and Y3 are each independently None or a transcriptional repressor element, and Y2 and Y3 are not both None;

[0038] Furthermore, the transcriptional repressor element inhibits the transcriptional activity of ITR, resulting in decreased transcription of Z1, i.e., GOI.

[0039] In another preferred embodiment, in formula (II), Y2 is a forward transcriptional repressor element, and Y3 is a reverse transcriptional repressor element; preferably, wherein Y2 has a nucleotide sequence as shown in SEQ ID NO: 1, and Y3 has a nucleotide sequence as shown in SEQ ID NO: 2.

[0040] In another preferred embodiment, in formula (II), Y2 is a positive transcriptional repressor element, and Y3 is absent; preferably, wherein Y2 has a nucleotide sequence as shown in SEQ ID NO: 1.

[0041] In another preferred embodiment, in formula (II), Y2 is absent, and Y3 is a reverse transcription inhibitory element; preferably, wherein Y3 has a nucleotide sequence as shown in SEQ ID NO: 2.

[0042] In another preferred embodiment, in the nucleic acid construct represented by the nucleic acid formula (I), Y2 and Y3 are absent, and the nucleic acid construct has a structure (5' end to 3' end) as shown in the following formula III:

[0043] Y1-5'ITR-Z1-3'ITR-Y4 (III)

[0044] Wherein, Y1 and Y4 are each independently None or a transcriptional repressor element, and Y1 and Y4 are not both None;

[0045] In addition, the transcription repression element inhibits the transcription activity of ITR, resulting in decreased transcription of the AAV vector plasmid backbone region and inhibiting the reverse packaging of the plasmid backbone to produce transcription products.

[0046] In another preferred embodiment, in formula (III), Y1 is a reverse transcription repressor element, and Y4 is a forward transcription repressor element; preferably, wherein the Y4 has a nucleotide sequence as shown in SEQ ID NO: 1, and the Y1 has a nucleotide sequence as shown in SEQ ID NO: 18.

[0047] In another preferred embodiment, in formula (III), Y1 is a reverse transcription inhibitory element, and Y4 is absent; preferably, wherein said Y4 has a nucleotide sequence as shown in SEQ ID NO: 1.

[0048] In another preferred embodiment, in formula (III), Y1 is absent, and Y4 is a positive transcription inhibitory element; preferably, wherein Y1 has a nucleotide sequence as shown in SEQ ID NO: 18.

[0049] The second aspect of the present invention provides a recombinant AAV vector plasmid, which contains the nucleic acid construct as described in the first aspect of the present invention.

[0050] In another preferred embodiment, the AAV vector plasmid is an AAV vector plasmid carrying an exogenous target gene in an AAV three-plasmid packaging system.

[0051] In another preferred embodiment, the AAV vector plasmid includes: AAV9, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV10, AAV-DJ, AAV-DJ / 8 or other AAV vector plasmids with capsid mutations.

[0052] In another preferred embodiment, the AAV vector plasmid is an AAV9 vector plasmid.

[0053] In a third aspect, the present invention provides a method for inhibiting ITR transcriptional activity in an AAV vector plasmid, the method comprising adding a transcriptional repressor element upstream of the 5'ITR, downstream of the 5'ITR, upstream of the 3'ITR, and / or downstream of the 3'ITR of the target AAV vector plasmid, or constructing the nucleic acid construct as described in the first aspect of the present invention in the target AAV vector plasmid;

[0054] Wherein, the transcriptional repression element is selected from the following groups: (a) a sequence containing a hairpin structure; (b) a polyA signal tailing signal sequence; (c) a polyA signal tailing signal sequence containing a stop codon; (d) a polyadenylic acid sequence, i.e., a PolyA sequence; (e) a polyadenylic acid sequence containing a stop codon; or a combination thereof.

[0055] In another preferred embodiment, the method comprises adding the transcriptional repression element downstream of the 5'ITR and / or upstream of the 3'ITR of the target AAV vector plasmid.

[0056] In another preferred embodiment, the method comprises adding a forward element (c) downstream of the 5'ITR, and / or adding a reverse element (c) upstream of the 3'ITR;

[0057] Preferably, a forward element (c) is added downstream of the 5' ITR, and a reverse element (c) is added upstream of the 3' ITR; or

[0058] An inverted element (c) was added upstream of the 3' ITR.

[0059] In another preferred embodiment, the method comprises adding the nucleotide sequence shown in SEQ ID NO: 1 downstream of the 5' ITR, and adding the nucleotide sequence shown in SEQ ID NO: 2 upstream of the 3' ITR.

[0060] In another preferred embodiment, the method comprises adding a nucleotide sequence as shown in SEQ ID NO: 2 upstream of the 3' ITR.

[0061] In another preferred embodiment, the method comprises adding the transcriptional repression element upstream of the 5'ITR and / or downstream of the 3'ITR of the target AAV vector plasmid.

[0062] In another preferred embodiment, the method comprises adding a reverse element (c) upstream of the 5'ITR, and / or adding a forward element (c) downstream of the 3'ITR;

[0063] Preferably, the reverse element (c) is added upstream of the 5' ITR and the forward element (c) is added downstream of the 3' ITR.

[0064] In another preferred embodiment, the method comprises adding the nucleotide sequence shown in SEQ ID NO: 18 upstream of the 5' ITR, and adding the nucleotide sequence shown in SEQ ID NO: 1 downstream of the 3' ITR.

[0065] In another preferred embodiment, the AAV vector plasmid is an AAV vector plasmid carrying an exogenous target gene in an AAV three-plasmid packaging system.

[0066] In another preferred embodiment, the AAV vector plasmid includes: AAV9, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV10, AAV-DJ, AAV-DJ / 8 or other AAV vector plasmids with capsid mutations.

[0067] In another preferred embodiment, the AAV vector plasmid is an AAV9 vector plasmid.

[0068] The fourth aspect of the present invention provides a method for preparing an AAV product, the method comprising: using the recombinant AAV vector plasmid as described in the second aspect of the present invention to perform AAV packaging, and harvesting the packaged AAV for use in preparing an AAV product.

[0069] In another preferred embodiment, the method comprises the following steps:

[0070] (i) constructing the recombinant AAV vector plasmid as described in the second aspect of the present invention;

[0071] (ii) using the recombinant AAV vector plasmid constructed in step (i) and a three-plasmid packaging system to transfect host cells;

[0072] (iii) After transfection, the host cells are cultured for 48-72 hours, and the cell culture fluid containing the virus (referred to as the virus fluid) is harvested and purified for use in preparing the AAV product.

[0073] In another preferred embodiment, the host cells are selected from: HEK293T cells, suspension HEK293, and Hela cells.

[0074] In another preferred embodiment, the AAV vector plasmid is an AAV vector plasmid carrying an exogenous target gene in an AAV three-plasmid packaging system.

[0075] In another preferred embodiment, the AAV vector plasmid includes: AAV9, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV10, AAV-DJ, AAV-DJ / 8 or other AAV vector plasmids with capsid mutations.

[0076] In another preferred embodiment, the AAV vector plasmid is an AAV9 vector plasmid, and the AAV with the serotype of AAV9 is prepared.

[0077] In another preferred embodiment, the three-plasmid packaging system further comprises a helper plasmid for assisting the recombinant AAV vector plasmid in AAV packaging;

[0078] The auxiliary plasmids include: an adenovirus auxiliary plasmid containing auxiliary Rep / Cap protein expression genes, and an adeno-associated virus replication plasmid containing Rep and Cap protein expression genes.

[0079] The fifth aspect of the present invention provides an AAV product, which is prepared using the method described in the fourth aspect of the present invention.

[0080] In another preferred embodiment, the AAV product is used as a gene therapy drug for gene therapy.

[0081] In another preferred embodiment, the AAV product further comprises a pharmaceutically acceptable carrier.

[0082] In another preferred embodiment, the AAV product contains significantly reduced AAV vector plasmid residues and transcription products generated by reverse packaging of the AAV vector plasmid backbone.

[0083] The sixth aspect of the present invention provides a use of the recombinant AAV vector plasmid as described in the second aspect of the present invention for preparing AAV products.

[0084] In another preferred embodiment, the AAV product is used as a gene therapy drug for gene therapy.

[0085] The seventh aspect of the present invention provides an AAV packaging system, which includes the recombinant AAV vector plasmid as described in the second aspect of the present invention.

[0086] In another preferred embodiment, the packaging system further comprises: an auxiliary plasmid for assisting the recombinant AAV vector plasmid in AAV packaging.

[0087] In another preferred embodiment, the AAV packaging system is a three-plasmid packaging system.

[0088] In another preferred embodiment, the three-plasmid packaging system includes: the recombinant AAV vector plasmid and the auxiliary plasmid as described in the second aspect of the present invention, and the auxiliary plasmid includes: an adenovirus auxiliary plasmid containing auxiliary Rep / Cap protein expression genes, and an adeno-associated virus replication plasmid containing Rep and Cap protein expression genes.

[0089] In an eighth aspect of the present invention, a kit is provided, comprising:

[0090] (C1) the recombinant AAV vector plasmid according to the second aspect of the present invention;

[0091] (C2) a helper plasmid, used to assist the recombinant AAV vector plasmid in AAV packaging;

[0092] and (C3) instructions or a label, wherein the instructions or the label indicate that the kit is used for AAV virus packaging.

[0093] In another preferred embodiment, the helper plasmid includes: an adenovirus helper plasmid containing helper Rep / Cap protein expression genes, and an adeno-associated virus replication plasmid containing Rep and Cap protein expression genes.

[0094] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1. Schematic diagram of AAV9 vectors containing the 3×Stop codon-bGH polyA signal sequence. (A) Schematic diagram of the AAV9 plasmid AAV9-Vector structure; (B) Schematic diagram of the AAV9-Vector-5Pn3 structure; (C) Schematic diagram of the AAV9-Vector-5nrP3 structure; (D) Schematic diagram of the AAV9-Vector-5PnrP3 structure.

[0096] Figure 2. Detection and analysis of AAV vector ITR transcription activity in HEK293T cells.

[0097] Figure 3. Schematic diagram of vectors constructed by adding a 3×Stop codon-polyA signal sequence to the backbone region of the AAV9 vector. (A) Schematic diagram of the AAV9-Vector structure (without the 3×Stop codon-polyA signal sequence); (B) Schematic diagram of the AAV9-Vector-5'PolyA structure; (C) Schematic diagram of the AAV9-Vector-3'PolyA structure; (D) Schematic diagram of the AAV9-Vector-bPolyA structure.

[0098] Figure 4. Analysis of transcriptional activity of the constructed AAV vector backbone region.

[0099] Figure 5. Analysis of ITR transcriptional activity in AAV products in mice. DETAILED DESCRIPTION

[0100] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that introducing transcriptional repression elements upstream and downstream of the ITR of the AAV vector plasmid can effectively inhibit the transcriptional activity of the ITR. By adding a PolyA signal tailing signal sequence with a hairpin structure or a combination of it and a stop codon sequence to the AAV plasmid backbone, the inventors achieved effective inhibition of the ITR transcription effect, significantly reducing the production of unknown transcription products of residual plasmid DNA and / or AAV-packaged plasmid backbone DNA. On the other hand, the use of the stop codon sequence can effectively inhibit the protein translation of unknown transcription products, which helps to further improve the safety of AAV products.

[0101] On this basis, the present invention was completed.

[0102] the term

[0103] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0104] The term "about" can refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0105] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0106] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the range and, where appropriate, fractional values ​​thereof (e.g., tenths and hundredths of an integer).

[0107] As used herein, the term "AAV" includes naturally occurring adeno-associated viruses and recombinant forms of adeno-associated viruses (rAAV), and includes mutant forms of AAV.

[0108] As used herein, "therapeutically effective amount" refers to a dose that produces an effect on a subject after administration. The exact dose depends on the purpose of the treatment and can be determined by those skilled in the art using known techniques.

[0109] As used herein, "storage" or "preservation" means that the formulation is not administered to a subject immediately after preparation, but is placed under specific conditions (eg, a specific temperature, etc.) for a period of time before use.

[0110] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0111] Transcriptional repressor elements

[0112] The present invention provides a nucleic acid construct comprising a transcription inhibitory element, which is used to inhibit the transcriptional activity of ITR in the AAV vector, thereby inhibiting the transcription products generated by the AAV vector plasmid residues and the reverse packaging of the plasmid backbone in the AAV product.

[0113] As used herein, the term "transcription repression element" refers to a specific nucleotide sequence that functions as a transcriptional terminator and is added upstream and / or downstream of the ITR of an AAV vector plasmid to inhibit transcription initiated by the promoter activity of the ITR. Transcription repression elements include naturally occurring and synthetic nucleotide sequences; any nucleotide sequence capable of terminating transcription can be used as a transcription repression element.

[0114] In an embodiment of the present invention, the transcriptional repression element is selected from the following group: (a) a sequence containing a hairpin structure; (b) a polyA signal tailing signal sequence; (c) a polyA signal tailing signal sequence containing a stop codon; (d) a polyadenylic acid sequence, i.e., a PolyA sequence; (e) a polyA sequence containing a stop codon; or a combination thereof. These transcriptional repression elements include naturally occurring and artificially synthesized ones. For example, naturally occurring polyA signal sequences derived from the 3'UTR sequence of prokaryotic or eukaryotic genes include (but are not limited to) polyA signal sequences derived from the 3'UTR sequence of SV40 and bGH genes; and artificially synthesized sequences with the function of a polyA signal sequence. Furthermore, a sequence containing a stop codon is tandemly connected to a polyA signal sequence to form a polyA signal tailing signal sequence containing a stop codon, which can also serve as a transcriptional repression element.

[0115] In some embodiments of the present invention, a polyA signal tailing signal sequence or a polyA signal tailing signal sequence containing a termination codon is used as a transcription inhibition element and added upstream and / or downstream of the ITR of the AAV vector plasmid. During transcription, the polyA signal tailing signal sequence is used to add a polyA tail, thereby terminating transcription.

[0116] In other embodiments of the present invention, the transcription repression element is a polyadenylation sequence or a polyadenylation sequence containing a stop codon directly added upstream and / or downstream of the ITR of the AAV vector plasmid, which also plays a role in transcription termination.

[0117] As used herein, the term "hairpin-containing sequence" refers to a sequence that forms an RNA hairpin structure (e.g., shRNA) through inverted repeats or through a bulge. Hairpin structures can be formed in two ways: 1. A sequence consisting of three or more nucleotide sequences that interact with each other to form a multi-layered structure, thereby generating an RNA hairpin. 2. A bulge is formed, which can interact with a U-rich sequence in the corresponding mRNA molecule to form a hairpin structure.

[0118] As used herein, the term "AAV vector plasmid backbone reverse packaging" refers to the packaging of the AAV vector plasmid backbone portion rather than the GOI sequence in the AAV packaging; wherein the plasmid backbone portion generally contains elements such as antibiotic genes and bacterial replicons.

[0119] PolyA signal tailing signal sequence

[0120] DNA is the carrier of genetic information. Genetic information is transferred from DNA to RNA through transcription and then translated into protein. DNA transcription requires RNA polymerase to strictly recognize the start sequence (promoter) and the end sequence (terminator) of the gene. Studies by You et al. have shown that the RNA hairpin structure after transcription in bacteria can enter the interior of RNA polymerase after folding, inducing its structural changes, releasing RNA, and terminating transcription [7]. There are three types of RNA polymerases in eukaryotes, and their transcription termination methods are also different. The transcription termination of Pol I requires the transcription termination factor TTF-1 to bind to the terminator downstream of the rRNA gene, causing the polymerase to pause. The genes transcribed by Pol III are relatively small, and there is a section of oligo (dA) (poly A) in its template chain. After the poly U is transcribed, the binding between the two is weak, making the complex unstable. The transcription termination of Pol II has been studied the most and is a poly A-dependent transcription termination method. More than 90% of the modifications of the 3' end of eukaryotic mRNA are achieved by nuclease cleavage and addition of poly A. In mammalian cells, this modification reaction depends on the AAUAAA sequence upstream of the cleavage site. The AAUAAA hexamer is the first discovered and recognized polyadenylation signal of mRNA, with a GU or U-rich sequence downstream of the cleavage site and a stimulatory sequence upstream of the AAUAAA sequence. Endonuclease cleavage occurs between the AAUAAA sequence and the downstream U-rich sequence, resulting in an upstream sequence with a 3'OH end and a downstream sequence with a 5' phosphate end. The upstream sequence is polyadenylated at the end, a poly A tail is added, and the downstream sequence is degraded. Therefore, the poly A tail does not exist in the gene, but is the end product of the polyadenylation reaction after internal cleavage at the 3' end of the pre-mRNA. In eukaryotic cells, the tail length can range from 90 adenylate nucleotides in yeast to approximately 250 adenylate nucleotides in mammals [8]. The main function of the poly A tail is to maintain the activity of mRNA as a translation template and increase the stability of the mRNA itself. It is often used in plasmid construction for gene and protein expression. Analysis revealed that the polyA signal is composed of a hairpin structure and a specific 5'-AAUAAA-3'. It is speculated that it may be recognized by the RNA polymerase transcription complex, redirecting the RNA polymerase structure under the action of the hairpin structure, thereby terminating transcription.

[0121] As used herein, the terms "polyA signal tailing signal sequence," "polyA signal sequence," and "tailing signal sequence" are used interchangeably to refer to a signal sequence located 3' to the gene stop codon that controls the addition of a polyA tail to mRNA during gene transcription. A typical polyA signal tailing signal sequence is shown in nucleotides 12-236 of SEQ ID NO:1, which contains a conserved AATAAA sequence.

[0122] PolyA tail signal sequence containing a stop codon

[0123] As used herein, the terms "polyA signal tailing signal sequence containing a stop codon", "polyA signal sequence containing a stop codon", and "Stop codon-polyA signal sequence" can be used interchangeably to refer to a nucleotide sequence containing a stop codon sequence and a polyA signal tailing signal sequence.

[0124] The Stop codon-polyA signal sequence has the structure shown in Formula A from the 5' end to the 3' end:

[0125] SP(A)

[0126] Wherein, S is the stop codon sequence; P is the polyA signal sequence. The stop codon sequence contains 1-10 stop codons, preferably 3-5, more preferably 3. Suitable codons are selected from the following group:

[0127] In some embodiments of the present invention, a sequence containing three stop codons is concatenated with one polyA signal sequence to form a "3×Stop codon-polyA signal sequence". In one specific embodiment of the present invention, three sequences containing stop codons are concatenated with a polyA signal sequence derived from the bGH gene to form a 3×Stop codon-bGH polyA signal sequence having a nucleotide sequence as shown in SEQ ID NO:1; the reverse complementary sequence of the sequence has a nucleotide sequence as shown in SEQ ID NO:2. In another specific embodiment of the present invention, three sequences containing stop codons are concatenated with a polyA signal sequence derived from the SV40 gene to form a 3×Stop codon-SV40 polyA signal sequence.

[0128] In an embodiment of the present invention, a polyA tailing signal sequence containing a termination codon is added as a transcriptional repression element to the upstream and / or downstream of the ITR of the AAV vector plasmid, wherein the polyA tailing signal sequence plays a transcription termination role, and further, the termination codon plays a translation termination role, which can effectively reduce the residual plasmid using ITR as a promoter for transcription and unknown protein expression, thereby reducing potential safety risks.

[0129] Recombinant AAV vector plasmid of the present invention

[0130] The nucleic acid construct of the present invention is used to construct a recombinant AAV vector plasmid. Therefore, the present invention provides a recombinant AAV vector plasmid containing the following nucleic acid structure (5' end to 3' end):

[0131] Y1-5'ITR-Y2-Z1-Y3-3'ITR-Y4 (I)

[0132] Where,

[0133] Y1, Y2, Y3, and Y4 are each independently none or a transcriptional repressor element, and Y1, Y2, Y3, and Y4 are not all none at the same time; wherein, Y2 and Y4 are forward transcriptional repressor elements, and Y1 and Y3 are reverse transcriptional repressor elements, and the reverse transcriptional repressor element is the reverse complementary sequence of the forward transcriptional repressor element;

[0134] Z1 is a target gene (GOI) expression cassette, which includes one or more promoter sequences and one or more target gene coding sequences;

[0135] "-" are each independently a linker sequence, wherein the length of the linker sequence in the "Y1-5'ITR-Y2" and "Y3-3'ITR-Y4" segments is 0-150 nt, preferably 0-100 nt; wherein the transcription repression element is as described above.

[0136] In one embodiment of the present invention, Y1 and Y4 in formula (I) are absent, that is, the recombinant AAV vector plasmid contains the following nucleic acid structure (5' end to 3' end):

[0137] 5'ITR-Y2-Z1-Y3-3'ITR (II)

[0138] Wherein, Y2 and Y3 are each independently None or a transcriptional repressor element, and Y2 and Y3 are not both None;

[0139] Furthermore, the transcriptional repressor element inhibits the transcriptional activity of ITR, resulting in decreased transcription of Z1, i.e., GOI.

[0140] An exemplary map of a recombinant AAV vector plasmid containing the nucleic acid structure represented by formula (II) is shown in BD in FIG1 .

[0141] In another embodiment of the present invention, Y2 and Y3 in formula (I) are absent, that is, the recombinant AAV vector plasmid contains the following nucleic acid structure (5' end to 3' end):

[0142] Y1-5'ITR-Z1-3'ITR-Y4 (III)

[0143] Wherein, Y1 and Y4 are each independently None or a transcriptional repressor element, and Y1 and Y4 are not both None;

[0144] In addition, the transcription repression element inhibits the transcription activity of ITR, resulting in decreased transcription of the AAV vector plasmid backbone region and inhibiting the reverse packaging of the plasmid backbone to produce transcription products.

[0145] An exemplary map of the recombinant AAV vector plasmid containing the nucleic acid structure represented by formula (III) is shown in BD in FIG3 .

[0146] The beneficial effects of the present invention include:

[0147] The present invention is based on the packaging mechanism of AAV products prepared by transient transfection of three plasmids. From the perspective of AAV plasmid design, 3×Stop codon-polyA signal sequences that inhibit ITR transcription and translation are added near the ITR of the AAV vector plasmid. This can effectively inhibit the residual plasmid DNA during the AAV production and preparation process and the production of random transcription products caused by the ITR promoter activity of the plasmid backbone DNA sequence of AAV packaging. The use of the 3×Stop codon sequence can also terminate the protein translation of unknown transcription products, thereby reducing the potential safety and genotoxicity of AAV products.

[0148] The present invention is based on the packaging mechanism of AAV products prepared by transient transfection of three plasmids. From the plasmid design level, 3×Stop codon-polyA signal sequences that inhibit ITR transcription are added downstream of the 3'ITR and upstream of the 5'ITR on the plasmid backbone of the target gene, respectively. This can effectively reduce the transcriptional activity of residual plasmid DNA and AAV reverse-packaged plasmid backbone DNA during the AAV production and preparation process, as well as the protein translation of unknown transcription products, thereby reducing the potential safety and genotoxicity of AAV products.

[0149] The present invention effectively reduces the generation of unknown transcription products and unknown proteins caused by residual plasmid DNA outside the AAV capsid and the plasmid backbone DNA sequence of the AAV package, thereby improving the safety of AAV products.

[0150] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0151] Unless otherwise specified, the materials, reagents, instruments, etc. used in the examples can be obtained from commercial sources.

[0152] Example 1 PolyA signal effectively inhibits the transcriptional activity of the target gene in the AAV vector

[0153] The present invention is based on the AAV9 plasmid AAV9-Vector (A in Figure 1), and a 3×Stop codon-polyA signal sequence is added near the ITR to construct AAV9-Vector-5Pn3, AAV9-Vector-5nrP3 and AAV9-Vector-5PnrP3, respectively. In this example, bGH polyA signal is used as an example, and the vector construction is described as follows: based on AAV9-Vector, in order to inhibit the transcriptional activity of the 5'ITR of the AAV vector, a 3×Stop codon-bGH polyA signal sequence is added downstream of the 5'ITR, and the constructed vector is named AAV9-Vector-5Pn3; based on AAV9-Vector, in order to inhibit the transcriptional activity of the 3'ITR of the AAV vector, a reverse complementary 3×Stop codon-bGH polyA signal sequence is added upstream of the 3'ITR, and the constructed vector is named AAV9-Vector-5nrP3; in order to inhibit the transcriptional activity of the 3' and 5'ITRs, reverse complementary and forward 3×Stop codon-bGH polyA signal sequences are added upstream of the 3'ITR and downstream of the 5'ITR, respectively, and the constructed vector is named AAV9-Vector-5PnrP3; the forward nucleotide sequence of 3×Stop codon-bGH polyA signal is shown in SEQ ID NO: 1 (the italics are the PolyA signal sequence, and the underlined are the stop codon sequence, bold is the AATAAA termination signal sequence), the reverse complementary nucleotide sequence is shown in SEQ ID NO: 2 (italic is the PolyA signal sequence, underlined is the stop codon sequence, bold is the AATAAA termination signal sequence), and the constructed sequence map is shown in BD in Figure 1.

[0154] 1.1 Passaging and culture of HEK293T cells

[0155] Take HEK293T cells cultured in a 100mm dish as an example. Discard the culture medium and slowly add 3mL of D-PBS along the side of the dish, gently shaking to mix. Discard the D-PBS and add 1mL of trypsin containing EDTA. Digest the cells at room temperature for 3 minutes. Terminate the reaction with complete DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin. Harvest the cells, centrifuge at 1500 rpm for 3 minutes, discard the supernatant, and resuspend in 4mL of complete culture medium. Count the cells under a microscope.

[0156] 1x10 6HEK293T cells were plated in a 100 mm dish containing 10 mL of complete culture medium and cultured in a 37°C cell culture incubator for 2 to 3 days. The next passage was performed when the cell confluence reached 80% to 90%. The HEK293T cells used in the following examples were all between passages P8 and P15.

[0157] 1.2 AAV vector plasmid transfection of HEK293T cells

[0158] 1) One day before transfection, HEK293T cells were passaged once normally and 1x10 6 Cells were added to 2 mL of culture medium and cultured in an incubator;

[0159] 2) After approximately 24 hours, when the cell density reaches about 80%, plasmid transfection is performed using PEI;

[0160] 3) Dilute 2 μg of target plasmid in 125 μL of serum-free Opti-MEM medium and mix thoroughly. pcDNA3.1-EGFP is used as a negative control.

[0161] 4) Dilute 6 μL of PEI (1 mg / mL) in 125 μL of serum-free Opti-MEM medium and mix thoroughly.

[0162] 5) Immediately add the mixture (4) to the mixture (3) and mix thoroughly, then incubate at room temperature for 15 minutes;

[0163] 6) Add the plasmid-PEI complex from (5) to HEK293T cells;

[0164] 7) 8 hours after transfection, discard the culture medium and add 3 mL of DMEM complete medium;

[0165] 8) 48 hours after transfection, cells were harvested for analysis.

[0166] 1.3 Detection and analysis of transcriptional activity of the target gene region of AAV vector

[0167] 1) Extract total RNA from cells according to the instructions of the EZNAHP Total RNA Kit (Omega bio-tek, R6812). Proceed to the next step after passing the test.

[0168] 2) According to PrimeScript TM Reverse transcription PCR was performed according to the instructions of the RT reagent Kit with gDNA Eraser (TAKARA, RR047A). The reaction system is shown in Table 1 below:

[0169] Table 1 Total RNA reverse transcription reaction system

[0170] 3) Follow the Premix Ex Taq TM Probe qPCR (TAKARA, RR390A) kit instructions, using specific probes and primers to detect the transcriptional activity of ITRs in AAV vectors, with γ-actin as a normalized internal reference. The primers and probes used were all selected from the target gene region, with nucleotide sequences shown in SEQ ID NO: 3-SEQ ID NO: 17. The qPCR reaction system is shown in Table 2. Each experiment was repeated three times, and the results are shown in Figure 2.

[0171] Table 2 qPCR reaction system

[0172] Reaction procedure:

[0173] Results: Specific primers and probes for different segments were used to detect and analyze the transcriptional activity of the ITRs of AAV vectors. γ-actin was used as a normalized internal control. The results showed that AAV9-vectors without the 3×Stop codon-bGH polyA signal sequence displayed high ITR transcriptional activity in HEK293T cells. In contrast, the addition of the 3×Stop codon-bGH polyA signal sequence near the ITR significantly reduced the transcriptional activity of the ITRs in AAV vectors. Specifically, AAV9-Vector-5nrP3 (which only added the reverse-complementary 3×Stop codon-bGH polyA signal sequence upstream of the 3'ITR) and AAV9-Vector-5PnrP3 (which added both forward and reverse 3×Stop codon-bGH polyA signal sequences near the ITR) exhibited more significant inhibitory effects on ITR transcriptional activity than AAV9-Vector-5Pn3 (which only added the 3×Stop codon-bGH polyA signal sequence downstream of the 5'ITR).

[0174] Conclusion: Adding PolyA signal downstream of 5'ITR and upstream of 3'ITR can effectively inhibit the transcriptional activity of ITR in AAV vector and significantly reduce the transcription level of target gene.

[0175] Example 2 PolyA signal effectively inhibits the transcriptional activity of the AAV vector backbone

[0176] Due to the packaging limitations of the AAV virus, the maximum target gene that can be packaged is 4.7kb. To further expand the target gene capacity, 3×Stop codon-polyA signal sequences are added to the backbone sequence of the AAV vector. Based on the AAV9-Vector plasmid (the plasmid map is shown in Figure 1A), the reverse complementary and forward 3×Stop codon-polyA signal sequences are added upstream of the 5'ITR and downstream of the 3'ITR, respectively, to inhibit the transcriptional activity of residual AAV vector DNA and the AAV-packaged plasmid backbone DNA. Vector construction is described as follows: Based on AAV9-Vector, to inhibit the transcriptional activity of the ITRs in the AAV vector on the plasmid backbone, a reverse-complementary 3×Stop codon-SV40 polyA signal was added upstream of the 5'ITR. The resulting vector was named AAV9-Vector-5'PolyA. Based on AAV9-Vector, a forward-directed 3×Stop codon-bGH polyA signal sequence was added downstream of the 3'ITR. The resulting vector was named AAV9-Vector-3'PolyA. Reverse-complementary 3×Stop codon-SV40 polyA signal and forward-directed 3×Stop codon-bGH polyA signal sequences were added upstream of the 5'ITR and downstream of the 3'ITR, respectively. The resulting vector was named AAV9-Vector-bPolyA. The nucleotide sequences of the reverse-complementary 3×Stop codon-SV40 polyA signal are shown in SEQ ID NO:18, and the nucleotide sequences of the 3×Stop codon-bGH polyA signal are shown in SEQ ID NO:1. The constructed AAV plasmids have the same target gene (GOI) sequence but different backbone designs. The sequence maps are shown in AD in Figure 3.

[0177] The extracted plasmid was tested and then transfected into HEK293T cells. The cell passage and culture and plasmid transfection were the same as described in Example 1. The total RNA extraction and reverse transcription system are shown in Table 1, and the qPCR system and reaction conditions are shown in Table 2. Specific primers and probes were designed for the AAV plasmid backbone, and the nucleotide sequences of the primers and probes used are shown in SEQ ID NO: 19-SEQ ID NO: 21. γ-actin was used as a normalized internal reference, and the nucleotide sequences of the primers and probes used are shown in SEQ ID NO: 15-SEQ ID NO: 17. Each experiment was repeated three times, and the results are shown in Figure 4.

[0178] Results: Specific primers and probe sequences for the AAV vector plasmid backbone region were selected for transcriptional activity analysis. Compared with the AAV9-Vector without 3×Stop codon-PolyA signal near the ITR, the AAV vectors with 3×Stop codon-bGH polyA signal and reverse-complementary 3×Stop codon-SV40 polyA signal sequences added only downstream of the 3'ITR or upstream of the 5'ITR, respectively, inhibited the transcriptional activity of the ITR on the plasmid backbone, and the transcriptional activity decreased by 18.39% and 37.53% compared with the control, respectively; while the AAV vectors with 3×Stop codon-bGH polyA signal and reverse-complementary 3×Stop codon-SV40 PolyA signal sequences added downstream of the 3'ITR and upstream of the 5'ITR, respectively, significantly inhibited the transcriptional activity of the ITR plasmid backbone, and the transcriptional activity decreased by 86.70% compared with the control.

[0179] Conclusion: Adding 3×Stop codon-PolyA signal (AAV9-Vector-bPolyA) upstream of 5'ITR and downstream of 3'ITR is the optimal combination, which can significantly inhibit the transcriptional activity of ITR on the plasmid backbone and significantly reduce the transcription level of AAV vector plasmid backbone.

[0180] Example 3 PolyA signal effectively inhibits the transcriptional activity of AAV virus ITR

[0181] 3.1 Virus packaging

[0182] The optimal combination AAV9-Vector-bPolyA (i.e., adding 3×Stop codon-PolyA signals upstream of the 5'ITR and downstream of the 3'ITR, respectively) that was verified to significantly inhibit the transcriptional activity of the AAV vector plasmid backbone in cells and the unchanged plasmid AAV9-Vector were used for viral packaging. AAV9-Vector-bPolyA and AAV9-Vector have the same target gene sequence. The difference between the two is that AAV9-Vector-bPolyA has a 3×Stop codon-PolyA signal added near the ITR, while AAV9-Vector has no sequence elements added. AAV packaging uses a three-plasmid transient transfection HEK293 system. 72 hours after transfection, the virus liquid is harvested for downstream purification. The specific operation process can be found in the literature [Grieger, J., Choi, V. & Samulski, R. Production and characterization of adeno-associated viral s. Nat Protoc 1, 1412–1428 (2006).], and the final titer is 1×10 13 vg / mL of AAV virus with serotype AAV9 was used for in vivo experiments in mice.

[0183] 3.2 Drug administration and detection of residual plasmid transcriptional activity

[0184] Dosing: Six-week-old C57BL / 6 mice were purchased from Jicui Yaokang. Packaged virus was injected intramuscularly into the left and right legs at a low dose of 3E10 vg / leg and a high dose of 1.5E11 vg / leg, respectively. The virus was mixed with 5% (v / v%) ink during the injection. Control mice were also given an equal volume of sterile PBS. Mice were sacrificed 10 days after dosing, and the ink-soaked muscle tissue was recovered.

[0185] Detection: RNA from muscle tissue was extracted using the TRIZOL method. Specifically, 50 mg of tissue was weighed, 1 mL of TRIZOL was added, and the tissue was ground using a grinder. After incubation at room temperature for 10 minutes, 0.2 mL of chloroform was added and incubated at room temperature for 5 minutes. The supernatant was transferred to a new 1.5 mL centrifuge tube at 12,000 rpm in a pre-cooled 4°C centrifuge for 20 minutes. The tube was then inverted to mix thoroughly, incubated at room temperature for 10 minutes, and centrifuged at 12,000 rpm at 4°C for 30 minutes. The supernatant was discarded, and the pellet was washed twice with 75% ethanol and finally dissolved in DEPC water for subsequent steps. Reverse transcription-PCR and qPCR detection were performed using the reaction systems shown in Tables 1 and 2. The probes and primers used are shown in SEQ ID NOs: 19 to 24. To minimize variability in the amount of virus entering cells, a GOI was used as a normalization control. The specific probe and primer sequences for the GOI are shown in SEQ ID NOs: 25 to 27. The results are shown in Figure 5.

[0186] Results: Primers and probes specific to the AAV vector plasmid backbone were used to detect plasmid backbone transcription levels. To minimize potential differences in transfection efficiency, the GOI was used as a normalized internal control. The results showed that, compared with AAV9-Vector, both low-dose (3E10 vg / leg) and high-dose (1.5E11 vg / leg) intramuscular injections of AAV virus significantly reduced the transcription of residual plasmid DNA or reverse-packaged plasmid backbone DNA in mice. Compared to the control, plasmid backbone transcription levels decreased by over 99% at both low and high doses.

[0187] Conclusion: Low and high doses of AAV plasmids were injected intramuscularly. The results showed that the design of adding 3×Stop codon-polyA signal upstream of 5'ITR and downstream of 3'ITR can effectively inhibit the transcriptional activity of ITR and significantly reduce the transcription level of residual plasmid backbone in AAV virus products in vivo.

[0188] Sequence information

[0189] References

[0190] 1.Earley LF,Conatser LM,Lue VM,Dobbins AL,Li C,Hirsch ML,Samulski RJ.Adeno-Associated Virus Serotype-Specific Inverted Terminal Repeat Sequence Role in Vector Transgene Expression.Hum Gene Ther.2020Feb;31(3-4):151-162.doi:10.1089 / hum.2019.274.PMID:31914802;PMCID:PMC7047122.

[0191] 2.Keiser MS,Ranum PT,Yrigollen CM,Carrell EM,Smith GR,Muehlmatt AL,Chen YH,Stein JM,Wolf RL,Radaelli E,Lucas TJ 2nd,Gonzalez-Alegre P,Davidson BL.Toxicity after AAV delivery of RNAi expression constructs into nonhuman primate brain.Nat Med.2021 Nov;27(11):1982-1989.doi:10.1038 / s41591-021-01522-3.Epub 2021Oct 18.PMID:34663988;PMCID:PMC8605996.

[0192] 3.Chadeuf G,Ciron C,Moullier P,et al.Evidence for encapsidation of prokaryotic sequences during recombinant adeno-associated virus production and their in vivo persistence after vector delivery.Mol Ther,2005,12(4):744-753.

[0193] 4. M,Schmeer M,Kracher B,Krüsemann C,Espinosa LE,Grünert A,Fuchsluger T,Rischmüller A,Schleef M,Büning H.DNA Minicircle Technology Improves Purity of Adeno-associated Viral Vector Preparations.Mol Ther Nucleic Acids.2016;5(8):e355.

[0194] 5.Wright JF.Product-Related Impurities in Clinical-Grade Recombinant AAV Vectors:Characterization and Risk Assessment.Biomedicines.2014;2(1):80-97.

[0195] 6.Hauck B,Murphy SL,Smith PH,Qu G,Liu X,Zelenaia O,Mingozzi F,Sommer JM,High KA,Wright JF.Undetectable transcription of cap in a clinical AAV vector:implications for preformed capsid in immune responses.Mol Ther.2009Jan;17(1):144-52.doi:10.1038 / mt.2008.227.Epub 2008Oct 21.PMID:18941440;PMCID:PMC2834997.

[0196] 7.You L,Omollo EO,Yu C,et al.Structural basis for intrinsic transcription termination.Nature.2023;613(7945):783-789.

[0197] 8.WAHLE E,R EGSEGGER U.3'-End processing of pre-mRNA in eukaryotes[J].FEMS microbiology reviews,1999,23(3):277-95.

[0198] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A nucleic acid construct, characterized in that The nucleic acid construct comprises a transcription inhibitory element, which is used to inhibit the transcriptional activity of ITR in the AAV vector plasmid to inhibit the transcription product generated by the AAV vector plasmid residue and the reverse packaging of the plasmid backbone; The nucleic acid construct has a structure as shown in the following formula I (5' end to 3' end): Y1-5'ITR-Y2-Z1-Y3-3'ITR-Y4 (I) In the formula, Y1, Y2, Y3, and Y4 are each independently none or a transcription inhibitory element, and Y1, Y2, Y3, and Y4 are not all none at the same time; wherein, Y2 and Y4 are forward transcription inhibitory elements, and Y1 and Y3 are reverse transcription inhibitory elements, and the reverse transcription inhibitory element is the reverse complementary sequence of the forward transcription inhibitory element; Z1 is a target gene (GOI) expression cassette, which includes one or more promoter sequences and one or more target gene coding sequences; "-" are each independently a linker sequence, wherein the length of the linker sequence in the "Y1-5'ITR-Y2" and "Y3-3'ITR-Y4" segments is 0-150 nt, preferably 0-100 nt; Wherein, the transcription inhibition element is selected from the following groups: (a) a sequence containing a hairpin structure; (b) a polyA signal tailing signal sequence; (c) a polyA signal tailing signal sequence containing a termination codon; (d) a polyadenylic acid sequence, i.e., a PolyA sequence; (e) a polyadenylic acid sequence containing a termination codon; or a combination thereof.

2. The nucleic acid construct according to claim 1, wherein In the structure represented by formula (I), Y1 and Y4 are absent, and the nucleic acid construct has a structure represented by formula II below (5' end to 3' end): 5'ITR-Y2-Z1-Y3-3'ITR (II) In the formula, Y2 and Y3 are each independently None or a transcriptional repressor element, and Y2 and Y3 are not both None; Furthermore, the transcriptional repressor element inhibits the transcriptional activity of ITR, resulting in decreased transcription of Z1, i.e., GOI.

3. The nucleic acid construct according to claim 1, wherein In the structure represented by formula (I), Y2 and Y3 are absent, and the nucleic acid construct has a structure represented by formula III below (5' end to 3' end): Y1-5'ITR-Z1-3'ITR-Y4 (III) In the formula, Y1 and Y4 are each independently None or a transcriptional repressor element, and Y1 and Y4 are not both None; In addition, the transcription repression element inhibits the transcription activity of ITR, resulting in decreased transcription of the AAV vector plasmid backbone region and inhibiting the reverse packaging of the plasmid backbone to produce transcription products.

4. The nucleic acid construct according to claim 1, wherein The transcription inhibition element is (c) a polyA signal tailing signal sequence containing a stop codon.

5. The nucleic acid construct according to claim 1, wherein The stop codon sequence contains 1-10 stop codons, preferably 3-5, more preferably 3.

6. The nucleic acid construct according to claim 1, wherein The polyA signal tailing signal sequence is derived from the 3'UTR sequence of a prokaryotic or eukaryotic gene, or an artificially designed and synthesized sequence having the function of a polyA signal sequence.

7. The nucleic acid construct according to claim 1, wherein The polyA signal tailing signal sequence (c) containing a stop codon has a structure shown in the following formula A from the 5' end to the 3' end: SP (A) Wherein, S is the stop codon sequence; P is the polyA signal sequence, and "-" is a phosphodiester bond.

8. The nucleic acid construct according to claim 1, wherein The transcription repression element plays the following roles: (1) inhibiting the transcription of the AAV vector plasmid residue; (2) inhibiting the transcription of the AAV vector plasmid after being reversely packaged into the AAV capsid; (3) inhibiting the transcription activity of the ITR in the AAV vector; and (4) inhibiting the transcription of the backbone region in the AAV vector plasmid.

9. A recombinant AAV vector plasmid, characterized in that: The plasmid contains the nucleic acid construct according to claim 1.

10. A method for inhibiting ITR transcription activity in an AAV vector plasmid, characterized in that: The method comprises adding a transcription inhibitory element upstream of the 5'ITR, downstream of the 5'ITR, upstream of the 3'ITR and / or downstream of the 3'ITR of the target AAV vector plasmid, or constructing the nucleic acid construct as claimed in claim 1 in the target AAV vector plasmid; Wherein, the transcription inhibition element is selected from the following groups: (a) a sequence containing a hairpin structure; (b) a polyA signal tailing signal sequence; (c) a polyA signal tailing signal sequence containing a termination codon; (d) a polyadenylic acid sequence, i.e., a PolyA sequence; (e) a polyadenylic acid sequence containing a termination codon; or a combination thereof.

11. A method for preparing an AAV product, the method comprising: AAV packaging is performed using the recombinant AAV vector plasmid as described in claim 9, and the packaged AAV is harvested for use in preparing AAV products.

12. An AAV product, characterized in that: The AAV virus product is prepared using the method as claimed in claim 10.

13. A use of the recombinant AAV vector plasmid according to claim 9, characterized in that: Used to prepare AAV products.

14. An AAV packaging system, characterized in that The packaging system comprises the recombinant AAV vector plasmid as described in claim 9.

15. A kit, characterized in that: The kit comprises: (C1) the recombinant AAV vector plasmid according to claim 9; (C2) an auxiliary plasmid, used to assist the recombinant AAV vector plasmid in AAV packaging; And, (C3) instructions or labels, wherein the instructions or labels indicate that the kit is used for AAV virus packaging.

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