CN117701638A - Method for inhibiting transcriptional activity of residual AAV vector plasmid and plasmid skeleton - Google Patents

Abstract

The present invention provides a method for inhibiting residual AAV vector plasmids and plasmid backbone transcriptional activity. In particular, the invention provides a nucleic acid construct comprising a transcription repressing element for inhibiting AAV vector plasmid residues and transcription products resulting from reverse packaging of the plasmid backbone by inhibiting the transcriptional activity of ITRs in the AAV vector plasmid. Recombinant AAV vector plasmids comprising the nucleic acid constructs, and AAV products prepared using the recombinant AAV vector plasmids, are also provided. The invention fundamentally solves the safety risks caused by non-specific transcription caused by plasmid residue and plasmid reverse package in AAV products and translation of unknown transcription products.

Description

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

Technical Field

The invention relates to the field of biotechnology and biological medicine, in particular to a design method of AAV vector plasmid carrying target genes, which is used for reducing the transcriptional activity of ITR in AAV vector plasmid on residual plasmid and plasmid skeleton and improving the safety of AAV products.

Background

In recent years, gene therapy has entered a rapid development stage, and more gene therapy products have entered a clinical research stage or marketed in batches, involving genetic diseases, malignant tumors, cardiovascular diseases, infectious diseases, and the like. Viral vectors and nonviral vectors can be classified according to the type of product, and commonly used viral vectors include adeno-associated virus (Adeno associated Virus, AAV), adenovirus (AdV), herpes simplex virus (Herpes Simplex Virus, HSV), and the like. Among them, AAV has good safety and high transduction efficiency, and is widely accepted. By the end of 2022, related studies in the field of gene therapy have shown a continuing trend towards the market of many cell therapies and gene therapy products. Taking the example of gene therapy preparations of recombinant adeno-Associated Virus (rAAV) vectors, there are 6 rAAV preparations commercially available worldwide, including Glybera (EMA, 2012), luxura (FDA, 2017), zolgensma (FDA, 2019), upstaza (EMA, 2022), roctavalian (EMA, 2022), and hemsenix (FDA, 2022). The total 300 AAV gene therapeutic drugs in clinical research stage are available in the global total, wherein 14 AAV gene therapeutic drugs in clinical stage 3 and above mainly surround the fields of rare diseases, hemophilia, ophthalmic diseases and the like. Meanwhile, the domestic AAV gene therapy also shows an explosion, 13 AAV gene therapies are approved by CDE in 2022 to enter clinic, and the domestic enterprises self-research 12 AAV gene therapies, which also indicate that the AAV therapies enter clinic acceleration progress.

Adeno-associated viruses are small replication-defective non-enveloped viruses that infect humans and some primates, with a virion size of about 20-25nm, a member of the parvoviral family. AAV relies on replication with co-infection with other viruses (mainly adenoviruses), originally found in contaminants of adenovirus preparations, and thus is known. AAV has 9 common serotypes (AAV 1-9), and serotypes such as AAV10, AAV-DJ/8, and the like also exist. More and more researches show that AAV gene therapy products have the advantages of weak immunogenicity, wide host range, stable physicochemical properties, long-term expression of exogenous genes and the like. The modified gene has been used as a high-efficiency and safe delivery carrier for in-vivo gene therapy, and is widely applied to the aspects of gene therapy, nerve loop marking, living body imaging, gene editing, and preparation of animal models of nervous system diseases and development of related medicaments such as rare diseases, ophthalmology, hemophilia and the like.

AAV genomes are composed of single stranded DNA (ssDNA) molecules of 4.7kb in length, between the ITR sequences (Inverted terminal repeat, ITR) at both ends, 145 nucleotides in length, with interrupted palindromic sequences that can fold into hairpin structures that act as primers during initiation of DNA replication. Between ITR sequences is a viral coding region containing two open reading frames, currently thought to express 4 Rep proteins, 3 Cap proteins and one AAP protein. ITR is the origin of DNA self-replication of AAV and a signal triggering viral packaging, plays a key role in viral replication and packaging, and is involved in the integration and escape process of viral genome on host genome. In addition, earley et al [1] have shown that ITR itself has strong promoter activity and can transcribe the carried gene sequence. Keiser et al [2] found that there was a strong transcription signal upstream and downstream of the 3'ITR and upstream of the 5' ITR, and that the transcribed fragment was a plasmid backbone fragment containing the ITR by RNA sequencing, and subsequent in vivo experiments in cynomolgus monkeys found that significant increases in gene expression associated with in vivo immunity were observed, consistent with the observed neuropathological results, which fully indicated that in AAV preparations, the plasmid backbone fragment containing the ITR remained transcribed in AAV preparations, resulting in toxicity in the body. The plasmid backbone of AAV vectors typically comprises elements such as antibiotic genes and bacterial replicons, and transcription of residual plasmid DNA occurs under the promoter activity of ITRs, possibly translating to unknown proteins, and both the transcription products of the residual plasmid DNA and the unknown proteins may elicit an immune response in the body. Furthermore, since the transcription initiation site in ITR is difficult to confirm, the transcribed protein is also difficult to identify.

The main modes of AAV product production and preparation at present are three plasmid transient method (Helper-free AAV packaging system), stable rotation production cell method and baculovirus infection insect cell method. The AAV product produced by the three-plasmid transient method mainly uses plasmids as raw materials for production, is easier to prepare and control quality compared with viruses, does not need to introduce helper viruses, and reduces the safety risk. For AAV production without helper virus, the requirements for production environment facilities are relatively low, while AAV production with helper virus is relatively high, as should be produced in BSL2 laboratory. The AAV product prepared by the three-plasmid transient suspension HEK293 cell overcomes the limitation that the traditional adherent cell cannot be produced in large scale and the final product is difficult to reach higher titer, and is widely applied to rAAV mass production and preparation. The three-plasmid transient system is mainly obtained by transfecting HEK293 cells for a period of time through a target gene vector plasmid, 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, and collecting cell lysates, and performing ultracentrifugation and purification to obtain the high-titer viruses. In the AAV production process, plasmids are used as key raw materials for rAAV production and preparation, and residual plasmid DNA and AAV packaged plasmid skeleton DNA can be used as impurities to be accompanied with the whole AAV production and preparation process. In AAV preparation, residual plasmid DNA is derived from plasmid added during plasmid transient and plasmid DNA fragments reversely packed in AAV packaging process. Plasmid DNA remaining in the AAV harvest can be removed by adding a concentration of Benzonase and DNase I endonucleases. Plasmid backbone DNA that is entrapped within the AAV capsid, however, is not removed by nuclease means due to protection by the AAV capsid, and is always present in the AAV preparation. Since the vector plasmid containing the target gene contains 2 ITRs, and the ITRs have transcriptional activity in cells, the plasmid skeleton sequence containing the ITRs can be transcribed in vivo, so that unknown proteins are translated, the body can generate immune response and potential genetic toxicity, and the safety of AAV products is seriously influenced. The research of Chadeuf et al [3] by adopting different AAV packaging systems shows that plasmid impurities in AAV products are derived from a plasmid skeleton of GOI and AAV packaging plasmids, wherein GOI plasmid skeleton residues are a main source of the plasmid impurities in AAV products and account for about 1.3-6.1% of the AAV products. Furthermore, the residual plasmid is more stable in vivo than AAV DNA, and can express genes on the plasmid, including resistance genes, for a long period of time. For some high dose AAV products, doses up to 1E15vg per human, such as zolgensma, mean that there is 1E13vg of plasmid remaining into the human body, and potential plasmid transcription presents a significant safety concern. In this regard, regulatory authorities place greater demands on quality research and quality control of AAV products. The guidelines issued by CDE in 5 months 2022 for pharmaceutical research and evaluation of in vivo gene therapy products clearly indicate that AAV and other viral vectors which are easy to package non-vector DNA into viral particles should consider the potential risk of packaging relevant exogenous DNA into viral particles when selecting packaging plasmids, and the raw materials of plasmids in the production process of AAV need to be checked and controlled.

At present, aiming at the problem of residual plasmid of AAV vectors, the residual plasmid can only be reduced as far as possible from the design end because the residual plasmid cannot be effectively removed through a downstream process, or the transcription and translation of the plasmid can be reduced under the condition that the residual plasmid cannot be reduced, so that the possible side effect caused by the residual plasmid can be further reduced. The current methods for reducing AAV plasmid residues mainly comprise the following steps: 1) The backbone, the resistance and other unnecessary sequences in the AAV vector are removed, and only the microring plasmid [4] containing the target gene fragment is used, but the preparation of the microring plasmid is complex, the operation is complex, the technical requirement is high, the price is high, and the promotion is not widely achieved; 2) Optimizing the transfection system, and reducing the total amount of transfected plasmid, thereby reducing the residue of plasmid in AAV product. However, the total amount of transfected plasmid is often directly related to AAV production, and low total plasmid transfection often results in low total AAV production; 3) Under the condition that the plasmid skeleton is not more than 4.7kbp, reverse packaging is easy to occur on the plasmid skeleton connected with ITR, and the AAV vector plasmid skeleton part is used as GOI for packaging, so that the packaged AAV has no function, an unknown sequence can be transcribed by taking the ITR as a promoter, even the unknown protein is translated, and the uncertain safety risk [4] is brought; therefore, researchers have added a stuffer sequence to the AAV plasmid backbone, so that the plasmid backbone length is much longer than 4.7kbp, effectively reducing the efficiency of reverse packaging of AAV [5]. Hauck et al [6] showed that the residue of plasmid DNA of AAV products was mainly derived from the ITR-containing plasmid backbone, and that the plasmid residue in AAV showed a decreasing trend as the size of the plasmid backbone increased. Although these methods reduce the residual plasmid in AAV products, they still do not prevent transcription of the residual plasmid and the possible safety risks. Thus, in cases where plasmid residues in AAV products cannot be removed efficiently at all, how to reduce the nonspecific transcription of residual plasmids in AAV products and the translation of unknown transcription products would be an important factor affecting the safety of AAV gene therapy products.

In summary, since plasmid residues in AAV products or the plasmid backbone of AAV packaging may bring about abnormal unknown protein expression, not only does the effectiveness of the product be affected, but also potential safety risks are brought. Therefore, there is a need in the art to develop a method that can effectively solve the problem of plasmid residue in AAV products.

Disclosure of Invention

The present invention aims at providing a method for inhibiting the reverse package of plasmid skeleton and unknown transcription product and translation product generated by residual plasmid DNA in AAV product and plasmid DNA in AAV capsid.

In a first aspect of the invention, there is provided a nucleic acid construct comprising a transcription repressing element for inhibiting AAV vector plasmid residue and transcription products resulting from reverse packaging of the plasmid backbone by inhibiting the transcriptional activity of ITRs in the AAV vector plasmid;

the nucleic acid construct has a structure (5 'to 3' end) as shown in formula I below:

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

in the method, in the process of the invention,

y1, Y2, Y3, Y4 are each independently absent or transcription repressing elements, and Y1, Y2, Y3, Y4 are not absent at the same time; wherein Y2 and Y4 are forward transcription repressing elements, Y1 and Y3 are reverse transcription repressing elements, and the reverse transcription repressing elements are reverse complements of the forward transcription repressing elements;

Z1 is a gene of interest (GOI) expression cassette comprising one or more promoter sequences and one or more gene of interest 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 0-150nt, preferably 0-100nt;

wherein the transcription repressing element is selected from the group consisting of: (a) a hairpin-containing sequence; (b) a polyA signal tailing signal sequence; (c) A polyA signal tailing signal sequence containing a stop codon; (d) a polyadenylation sequence, i.e., a PolyA sequence; (e) a polyadenylation sequence containing a stop codon; or a combination thereof.

In another preferred example, the "hairpin-containing sequence" includes a hairpin structure formed by an inverted repeat sequence (e.g., shRNA) or a RNA hairpin structure formed by a bulk, or other forms of hairpin structure.

In another preferred embodiment, the transcription repressing element is (c) a polyA tailing signal sequence containing a stop codon.

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

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

In another preferred embodiment, the stop codon is selected from the group consisting of: TAA, TAG, TGA, or a combination thereof.

In another preferred embodiment, the polyA signal tailing signal sequence is derived from the 3' UTR sequence of a prokaryotic or eukaryotic gene, or a sequence which is artificially designed and synthesized to function as a polyA signal sequence.

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

In another preferred embodiment, the polyA signal tailing signal sequence is derived from the 3' utr sequence or an artificially designed synthetic sequence of a gene selected from the group consisting of seq id nos: SV40, bGH, hGH (artificial design), or combinations thereof.

In another preferred embodiment, the polyA signal tailing signal sequence has a sequence common to AATAAA, and a GT-rich sequence.

In another preferred embodiment, 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:

S-P(A)

wherein S is the stop codon sequence; p is the polyA signal sequence and "-" is a phosphodiester bond.

In another preferred embodiment, the forward element (c) has the nucleotide sequence shown in SEQ ID NO. 1.

In another preferred embodiment, the inverted element (c) has the nucleotide sequence shown as SEQ ID NO. 2 or 18.

In another preferred embodiment, the transcription repressing element performs the following functions: (1) inhibiting transcription of AAV vector plasmid residues; (2) Inhibiting transcription of the AAV vector plasmid after it has been reverse-packaged into an AAV capsid; (3) inhibiting the transcriptional activity of an ITR in an AAV vector; (4) inhibiting transcription of the backbone region in the AAV vector plasmid.

In another preferred embodiment, the AAV vector plasmid is an AAV vector plasmid loaded with an exogenous gene of interest in an AAV three-plasmid packaging system.

In another preferred embodiment, the AAV vector plasmid comprises: AAV9, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV10, AAV-DJ/8, or other coat mutated AAV vector plasmids.

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

In another preferred example, in the nucleic acid construct of formula (I), Y1 and Y4 are absent, and the nucleic acid construct has a structure (5 '-end to 3' -end) as shown in formula II below:

5'ITR-Y2-Z1-Y3-3'ITR(II)

wherein Y2, Y3 are each independently absent or transcription repressing element, and Y2 and Y3 are not absent at the same time;

Furthermore, the transcription repressing element therein represses the transcriptional activity of ITR, leading to a decrease in Z1, GOI transcription.

In another preferred embodiment, in formula (II), Y2 is a forward transcription repressing element and Y3 is a reverse transcription repressing element; preferably, wherein Y2 has the nucleotide sequence shown as SEQ ID NO. 1 and Y3 has the nucleotide sequence shown as SEQ ID NO. 2.

In another preferred embodiment, in formula (II), Y2 is a forward transcription repressing element and Y3 is absent; preferably, wherein Y2 has a nucleotide sequence as shown in SEQ ID NO. 1.

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

In another preferred example, in the nucleic acid construct of 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:

Y1-5'ITR-Z1-3'ITR-Y4(III)

wherein Y1, Y4 are each independently absent or transcription repressing element, and Y1 and Y4 are not absent at the same time;

in addition, the transcription inhibition element inhibits the transcriptional activity of ITR, which results in the transcriptional reduction of AAV vector plasmid skeleton region and inhibits the reverse packaging of plasmid skeleton to produce transcription product.

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

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

In another preferred embodiment, in formula (III), Y1 is absent and Y4 is a forward transcription repressing element; preferably, wherein said Y1 has the nucleotide sequence as shown in SEQ ID NO. 18.

In a second aspect of the invention, there is provided a recombinant AAV vector plasmid comprising a nucleic acid construct according to the first aspect of the invention.

In another preferred embodiment, the AAV vector plasmid is an AAV vector plasmid loaded with an exogenous gene of interest in an AAV three-plasmid packaging system.

In another preferred embodiment, the AAV vector plasmid comprises: AAV9, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV10, AAV-DJ/8, or other coat mutated AAV vector plasmids.

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

In a third aspect of the invention, there is provided a method of 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 AAV vector plasmid of interest, or constructing a nucleic acid construct according to the first aspect of the invention in an AAV vector plasmid of interest;

wherein the transcription repressing element is selected from the group consisting of: (a) a hairpin-containing sequence; (b) a polyA signal tailing signal sequence; (c) A polyA signal tailing signal sequence containing a stop codon; (d) a polyadenylation sequence, i.e., a PolyA sequence; (e) a polyadenylation sequence containing a stop codon; or a combination thereof.

In another preferred embodiment, the method comprises adding the transcription repressing element downstream of the 5'ITR and/or upstream of the 3' ITR of the AAV vector plasmid of interest.

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;

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 (b)

Upstream of the 3' ITR a reverse element (c) is added.

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

In another preferred embodiment, the method comprises adding the nucleotide sequence shown as SEQ ID NO. 2 upstream of the 3' ITR.

In another preferred embodiment, the method comprises adding the transcription repressing element upstream of the 5'ITR and/or downstream of the 3' ITR of the AAV vector plasmid of interest.

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;

preferably, a reverse element (c) is added upstream of the 5'ITR and a forward element (c) is added downstream of the 3' ITR.

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

In another preferred embodiment, the AAV vector plasmid is an AAV vector plasmid loaded with an exogenous gene of interest in an AAV three-plasmid packaging system.

In another preferred embodiment, the AAV vector plasmid comprises: AAV9, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV10, AAV-DJ/8, or other coat mutated AAV vector plasmids.

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

In a fourth aspect of the invention, there is provided a method of preparing an AAV product, the method comprising: AAV packaging is performed using the recombinant AAV vector plasmid according to the second aspect of the invention, and the packaged AAV is harvested for use in the preparation of AAV products.

In another preferred embodiment, the method comprises the steps of:

(i) Constructing a recombinant AAV vector plasmid according to the second aspect of the invention;

(ii) Transfecting a host cell with the recombinant AAV vector plasmid constructed in step (i) using a three plasmid packaging system;

(iii) Culturing host cells for 48-72 hr after transfection, collecting cell culture solution (virus solution for short) containing virus, and purifying to prepare AAV product.

In another preferred embodiment, the host cell is selected from the group consisting of: HEK293T cells, suspension HEK293, hela cells.

In another preferred embodiment, the AAV vector plasmid is an AAV vector plasmid carrying an exogenous gene of interest in an AAV three-plasmid packaging system.

In another preferred embodiment, the AAV vector plasmid comprises: AAV9, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV10, AAV-DJ/8, or other coat mutated AAV vector plasmids.

In another preferred embodiment, the AAV vector plasmid is an AAV9 vector plasmid, and AAV having a serotype of AAV9 is prepared.

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

wherein the helper plasmid comprises: an adenovirus helper plasmid comprising a helper Rep/Cap protein expression gene and an adeno-associated virus replication plasmid comprising Rep and Cap protein expression genes.

In a fifth aspect of the invention, there is provided an AAV product prepared using the method according to the fourth aspect of the invention.

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

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

In another preferred embodiment, the AAV vector plasmid residues and the transcripts produced by inverse packaging of the AAV vector plasmid backbone in the AAV product are significantly reduced.

In a sixth aspect of the invention, there is provided the use of a recombinant AAV vector plasmid according to the second aspect of the invention for the preparation of an AAV product.

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

In a seventh aspect of the invention, there is provided an AAV packaging system comprising a recombinant AAV vector plasmid according to the second aspect of the invention.

In another preferred embodiment, the packaging system further comprises: a helper plasmid for assisting the recombinant AAV vector plasmid in AAV packaging.

In another preferred embodiment, the AAV packaging system is a three plasmid packaging system.

In another preferred embodiment, the three plasmid packaging system comprises: the recombinant AAV vector plasmid and helper plasmid of the second aspect of the invention, the helper plasmid comprising: an adenovirus helper plasmid comprising a helper Rep/Cap protein expression gene and an adeno-associated virus replication plasmid comprising Rep and Cap protein expression genes.

In an eighth aspect of the invention, there is provided a kit comprising:

(C1) A recombinant AAV vector plasmid according to the second aspect of the invention;

(C2) A helper plasmid for assisting the recombinant AAV vector plasmid in AAV packaging;

and, (C3) instructions or tags, the instructions or tags stating that the kit is for AAV viral packaging.

In another preferred embodiment, the helper plasmid comprises: an adenovirus helper plasmid comprising a helper Rep/Cap protein expression gene and an adeno-associated virus replication plasmid comprising Rep and Cap protein expression genes.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 is a schematic diagram of AAV9 vector comprising a 3 XStop codon-bGH polyA signal sequence. (a) AAV9 plasmid AAV9-Vector structural schematic; (B) AAV9-Vector-5Pn3 structural schematic; (C) AAV9-Vector-5nrP3 schematic structure; (D) AAV9-Vector-5PnrP3 structural schematic.

FIG. 2 assay of the ITR transcriptional activity of AAV vectors in HEK293T cells.

FIG. 3 is a schematic diagram of a vector constructed by adding a 3 XStop codon-polyA signal sequence to the backbone region of an AAV9 vector. (A) AAV9-Vector structure schematic (3 XStop codon-polyA signal sequence is not added); (B) schematic AAV9-Vector-5' PolyA structure; (C) AAV9-Vector-3' PolyA schematic structural representation; (D) schematic structure of AAV 9-Vector-bPolyA.

FIG. 4. Analysis of transcriptional activity of the backbone region of the constructed AAV vector.

FIG. 5 assay for transcriptional activity of ITR in AAV products in mice.

Detailed Description

The present inventors have made extensive and intensive studies and have unexpectedly found for the first time that the introduction of a transcription repressing element upstream and downstream of the ITR of an AAV vector plasmid can efficiently repress the transcriptional activity of the ITR. The inventor realizes effective inhibition of ITR transcription effect by adding a PolyA signal tailing signal sequence with a hairpin structure or a combination of the PolyA signal tailing signal sequence and a termination codon sequence into an AAV plasmid skeleton, and remarkably reduces the generation of unknown transcription products of residual plasmid DNA and/or AAV packaged plasmid skeleton DNA. On the other hand, the use of the stop codon sequence can effectively inhibit the protein translation of unknown transcription products, thereby being beneficial to further improving the safety of AAV products.

On this basis, the present invention has been completed.

Terminology

In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meanings given below, unless expressly specified otherwise herein. Other definitions are set forth throughout the application.

The term "about" may refer to a value or composition that is within an acceptable error of 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 measured. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the range as well as fractional values thereof (e.g., one tenth and one hundredth of an integer) as appropriate.

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.

As used herein, a "therapeutically effective amount" refers to a dose that produces an effect upon administration to a subject. The exact dosage will depend on the purpose of the treatment and can be determined by one skilled in the art using known techniques.

As used herein, "store" or "preservation" refers to not immediately administering to a subject after preparation of a formulation, but rather, to a period of time under specific conditions (e.g., specific temperature, etc.) prior to use.

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

Transcription repressing element

The present invention provides a nucleic acid construct comprising a transcription repressing element for inhibiting AAV vector plasmid residues in AAV preparations and transcription products generated by reverse packaging of the plasmid backbone by inhibiting the transcriptional activity of ITRs in AAV vectors.

As used herein, the term "transcription repressing element" refers to a particular nucleotide sequence that has a transcription termination effect that is added upstream and/or downstream of the AAV vector plasmid ITR, thereby repressing transcription that occurs under the promoter activity of the ITR. Transcription repressing elements include naturally occurring nucleotide sequences and artificially synthesized nucleotide sequences, and can be used as transcription repressing elements as long as the nucleotide sequences can exert transcription termination effects.

In an embodiment of the invention, the transcription repressing element is selected from the group consisting of: (a) a hairpin-containing sequence; (b) a polyA signal tailing signal sequence; (c) A polyA signal tailing signal sequence containing a stop codon; (d) a polyadenylation sequence, i.e., a PolyA sequence; (e) a polyadenylation sequence containing a stop codon; or a combination thereof. These transcription repressing elements include naturally occurring and synthetic ones. For example, naturally occurring polyA signal sequences derived from the 3'UTR sequence of a prokaryotic or eukaryotic gene, including, but not limited to, polyA signal sequences derived from the 3' UTR sequence of the SV40, bGH gene; and artificially synthesized sequences with the function of polyA signal sequences. Furthermore, a sequence containing a stop codon may be formed by concatenating a polyA signal sequence with a polyA signal sequence to form a polyA signal tailing signal sequence containing a stop codon, and the polyA signal tailing signal sequence may be used as a transcription repressing element.

In some embodiments of the invention, a polyA signal tailing signal sequence or a polyA signal tailing signal sequence containing a stop codon is used as a transcription repressing element, added upstream and/or downstream of the AAV vector plasmid ITR, and the polyA tail is added at the time of transcription using the polyA signal tailing signal sequence, thereby terminating transcription.

In other embodiments of the invention, the transcription repressing element is a polyadenylation sequence directly added upstream and/or downstream of the AAV vector plasmid ITR or a polyadenylation sequence containing a stop codon, which likewise serves as a transcription termination.

As used herein, the term "hairpin-containing sequence" refers to a sequence that forms an RNA hairpin structure (e.g., shRNA) by an inverted repeat sequence, or an RNA hairpin structure by a bulge (bulge). There are two modes of hairpin formation: 1. consisting of three or more nucleotide sequences, a multi-layered structure is formed by interactions between the sequences, thereby generating an RNA hairpin structure. 2. By the generation of a bulge, the bulge can interact with the U-rich sequence in the corresponding mRNA molecule, forming a hairpin structure.

As used herein, the term "AAV vector plasmid backbone reverse packaging" refers to packaging of sequences of AAV vector plasmid backbone portions other than GOI in AAV packaging; wherein the plasmid backbone portion generally comprises elements such as antibiotic genes and bacterial replicons.

PolyA signal tailing signal sequence

DNA is a carrier of genetic information that is transferred from DNA to RNA by transcription and then translated into protein. Transcription of DNA requires the RNA polymerase to recognize the start (promoter) and stop (terminator) sequences of the gene in a stringent manner. You et al show that RNA hairpin structure transcribed in bacteria can enter the interior of RNA polymerase through folding, induce its structure change, release RNA and terminate transcription [7]. There are 3 RNA polymerases in eukaryotes, and the transcription termination patterns vary. Transcription termination by Pol I requires that the transcription termination factor TTF-1 bind to a terminator downstream of the rRNA gene, resulting in a pause in the polymerase. The Pol II transcribed genes are smaller, a section of ol igo (dA) (poly A) is arranged in a template chain, and after poly U is transcribed, the combination of the two genes is weaker, so that the complex is unstable. The most studied transcription termination of Pol ii is a polyA-dependent transcription termination. More than 90% of the modification of the 3' -end of eukaryotic mRNA is achieved by endonuclease cleavage and addition of polyA. In mammalian cells, this modification depends on the AAUAAA sequence upstream of the cleavage site, the AAUAAA hexamer being the polyadenylation signal of the earliest recognized mRNA found, the GU or U rich sequence downstream of the cleavage site and the stimulatory sequence upstream of the AAUAAA sequence. Endonuclease cleavage occurs between the AAUAAA sequence and the downstream U-rich sequence, thereby producing an upstream sequence containing a 3'OH at one end and a downstream sequence containing a 5' phosphate gene at one end. Polyadenylation occurs at the end of the upstream sequence and the downstream sequence is degraded by addition of the poly A tail. Thus, the poly A tail is not present in the gene, but is the end product of the polyadenylation reaction after internal cleavage of the 3' -end of the pre-mRNA. In eukaryotic cells, tail length can range from 90 adenylates in yeast to approximately 250 adenylates in mammals [8]. The main function of the polyA tail is to maintain mRNA activity as a translation template and to increase its mRNA stability, which is commonly used in plasmid construction for gene and protein expression and the like. It was found by analysis that polyA signal consisted of a hairpin structure and a specific 5'-AAUAAA-3', presumably recognized by the RNA polymerase transcription complex, redirecting the RNA polymerase structure under the action of the hairpin structure, thus stopping transcription.

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

polyA tailing signal sequence containing stop codon

As used herein, the terms "Stop codon-containing polyA signal tailing signal sequence", "Stop codon-polyA signal sequence" are used interchangeably and refer to a nucleotide sequence comprising a Stop codon sequence and a polyA signal tailing signal sequence.

The Stop codon-polyA signal sequence has a structure shown in the following formula A from a 5 'end to a 3' end:

S-P(A)

wherein S is the stop codon sequence; p is the polyA signal sequence. The stop codon sequence contains 1 to 10 stop codons, preferably 3 to 5, more preferably 3. Suitable codons are selected from the group consisting of:

in some embodiments of the invention, a sequence containing 3 Stop codons is concatenated with 1 polyA signal sequence to form a "3 XStop codon-polyA signal sequence". In one embodiment of the present invention, 3 sequences containing a Stop codon are concatenated with a polyA signal sequence derived from the bGH gene to constitute a 3 XSTOP-bGH polyA signal sequence having a nucleotide sequence as shown in SEQ ID NO. 1; the reverse complement of the sequence has a nucleotide sequence shown as SEQ ID NO. 2. In another embodiment of the present invention, 3 sequences containing a Stop codon are concatenated with a polyA signal sequence derived from the SV40 gene, thereby constituting a 3 XSTOC code-SV 40 polyA signal sequence.

In the embodiment of the invention, the polyA tailing signal sequence containing the termination codon is added to the upstream and/or downstream of the ITR of the AAV vector plasmid as a transcription inhibition element, wherein the polyA tailing signal sequence plays a role in transcription termination, and further, the termination codon plays a role in translation termination, so that the transcription and the expression of unknown proteins by using the ITR as a promoter of the residual plasmid can be effectively reduced, and the potential safety risk is reduced.

Recombinant AAV vector plasmids of the invention

The nucleic acid construct of the present invention is used to construct a recombinant AAV vector plasmid, and thus, the present invention provides a recombinant AAV vector plasmid comprising the nucleic acid structure (5 'to 3' ends) as shown below:

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

in the method, in the process of the invention,

y1, Y2, Y3, Y4 are each independently absent or transcription repressing elements, and Y1, Y2, Y3, Y4 are not absent at the same time; wherein Y2 and Y4 are forward transcription repressing elements, Y1 and Y3 are reverse transcription repressing elements, and the reverse transcription repressing elements are reverse complements of the forward transcription repressing elements;

z1 is a gene of interest (GOI) expression cassette comprising one or more promoter sequences and one or more gene of interest 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 0-150nt, preferably 0-100nt; wherein the transcription repressing element is as described above.

In one embodiment of the invention, Y1 and Y4 in formula (I) are absent, i.e., the recombinant AAV vector plasmid contains the nucleic acid structure (5 'to 3' end) as shown below:

5'ITR-Y2-Z1-Y3-3'ITR(II)

wherein Y2, Y3 are each independently absent or transcription repressing element, and Y2 and Y3 are not absent at the same time;

furthermore, the transcription repressing element therein represses the transcriptional activity of ITR, leading to a decrease in Z1, GOI transcription.

An exemplary map of a recombinant AAV vector plasmid comprising the nucleic acid structure of formula (II) is shown in FIGS. 1B-D.

In another embodiment of the invention, Y2 and Y3 in formula (I) are absent, i.e., the recombinant AAV vector plasmid contains the nucleic acid structure (5 'to 3') as shown below:

Y1-5'ITR-Z1-3'ITR-Y4(III)

wherein Y1, Y4 are each independently absent or transcription repressing element, and Y1 and Y4 are not absent at the same time;

in addition, the transcription inhibition element inhibits the transcriptional activity of ITR, which results in the transcriptional reduction of AAV vector plasmid skeleton region and inhibits the reverse packaging of plasmid skeleton to produce transcription product.

An exemplary map of a recombinant AAV vector plasmid comprising the nucleic acid structure of formula (III) is shown in FIGS. 3B-D.

The beneficial effects of the invention include:

the invention is based on the packaging mechanism of three plasmid transient preparation AAV products, from the AAV plasmid design level, 3 x Stop codon-polyA signal sequences for inhibiting ITR transcription and translation are respectively added near ITR of AAV vector plasmid, thus being capable of effectively inhibiting plasmid DNA residues and random transcription living products generated by the plasmid skeleton DNA sequences of AAV packaging in the AAV production preparation process due to ITR promoter activity, and the use of 3 x Stop codon sequences can also terminate protein translation of unknown transcription products, thereby reducing potential safety and genetic toxicity of AAV products.

The invention is based on the packaging mechanism of preparing AAV products by three plasmid transient transformation, from the plasmid design level, 3 xStop codon-polyA signal sequences for inhibiting ITR transcription are respectively added at the downstream of 3'ITR and the upstream of 5' ITR on the target gene plasmid skeleton, thereby being capable of effectively reducing the transcriptional activity of residual plasmid DNA and AAV reversely packaged plasmid skeleton DNA and the protein translation of unknown transcription products in the AAV production and preparation process, and reducing the potential safety and genetic toxicity of AAV products.

The invention can radically reduce the generation of unknown transcription products and unknown proteins caused by residual plasmid DNA outside AAV capsid and plasmid skeleton DNA sequence of AAV package, and improve the safety of AAV products.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Materials, reagents, instruments and the like used in the examples are commercially available unless otherwise specified.

EXAMPLE 1PolyA Signal is effective in inhibiting transcriptional activity of a target gene in AAV vectors

The invention is based on AAV9 plasmid AAV9-Vector (A in figure 1), and 3 XStop codon-polyA signal sequence is added near ITR to respectively construct AAV9-Vector-5Pn3, AAV9-Vector-5nrP3 and AAV9-Vector-5PnrP3. Taking bGH polyA signal as an example, the vector construction is described as follows: based on AAV9-Vector, 3 Xstop codon-bGH polyA signal sequence is added downstream of 5'ITR in order to inhibit the transcriptional activity of AAV Vector 5' ITR, and the constructed Vector is named AAV9-Vector-5Pn3; based on AAV9-Vector, in order to inhibit the transcriptional activity of 3'ITR of AAV Vector, adding reverse complementary 3×stop codon-bGH polyA signal sequence upstream of 3' ITR, and the constructed Vector is named AAV9-Vector-5nrP3; in order to inhibit the transcriptional activity of 3 'and 5' ITRs, a reverse complementary and forward 3×stop codon-bGH polyA signal sequence is added at the upstream of the 3'ITR and the downstream of the 5' ITR, and the constructed Vector is named AAV9-Vector-5PnrP3; the 3×stop codon-bGH polyA signal forward nucleotide sequence is shown in SEQ ID NO 1 (italic PolyA signal sequence, underlined Stop codon sequence, bold AATAAA termination signal sequence), the reverse complementary nucleotide sequence is shown in SEQ ID NO 2 (italic PolyA signal sequence, underlined Stop codon sequence, bold AATAAA termination signal sequence), and the constructed sequence diagram is shown in B-D in FIG. 1.

Passage and culture of 1.1HEK293T

HEK293T cells were cultured in 100mm dishes as an example. The medium was discarded, 3mL of D-PBS was slowly added along the walls of the dish and gently shaken. D-PBS was discarded and 1mL of trypsin containing EDTA was added and digested at room temperature for 3 minutes. The reaction was stopped with DMEM complete medium containing 10% FBS,1% Penicillin-Streptomycin, cells were collected, centrifuged at 1500rpm for 3 min, the supernatant was discarded and resuspended with 4mL complete medium and counted under a microscope;

will be 1x10 ⁶ Each HEK293T cell was plated on a 100mm dish containing 10mL of complete medium, cultured in a cell incubator at 37℃for 2 to 3 days, and passaged next time at a cell confluency of 80% -90%. HEK293T cells used in the following examples were all cells with passage numbers of P8 to P15.

AAV vector plasmid transfection of 1.2HEK293T cells

1) HEK293T cells were passaged normally once the day before transfection, plated 1X10 in each well of a 6-well plate ⁶ Cells per 2mL of medium are placed into an incubator for culture;

2) When the cell density reaches about 80% after about 24 hours, PEI is used for plasmid transfection;

3) Mu.g of the plasmid of interest was diluted with 125. Mu.L of serum-free medium Opti-MEM and thoroughly mixed, with pcDNA3.1-EGFP as negative control;

4) mu.L PEI (1 mg/mL) was diluted with 125. Mu.L of serum-free medium Opti-MEM and thoroughly mixed;

5) Immediately adding the mixed solution (4) into the mixed solution (3), fully and uniformly mixing, and incubating for 15 minutes at room temperature;

6) Adding the (5) plasmid PEI complex to HEK293T cells;

7) After 8 hours of transfection, the medium was discarded and 3mL DMEM complete medium was added;

8) Cells were harvested 48 hours after transfection for analysis.

1.3 analysis of transcriptional Activity of target Gene region of AAV vector

1) Extracting Total RNA of cells according to the instruction of an E.Z.N.A.HP Total RNA Kit (Omega bio-tek, R6812) Kit, and performing the next operation after the Total RNA of the cells is qualified by inspection;

2) According to PrimeScript ^TM RT reagent Kit with gDNA Eraser (TAKARA, RR 047A) kit instruction for reverse transcription PCR, reaction systemSee table 1 below:

TABLE 1 Total RNA reverse transcription reaction System

3) According to Premix Ex Taq ^TM The specification of a Probe qPCR (TAKARA, RR 390A) kit respectively uses a specific Probe and a primer to detect the transcriptional activity of ITR in an AAV vector, wherein gamma-actin is used as a standardized internal reference, the primer and the Probe are selected from target gene regions, the nucleotide sequence is shown as SEQ ID NO. 3-SEQ ID NO. 17, a qPCR reaction system is shown in Table 2, and each experiment is repeated three times, and the result is shown in figure 2.

TABLE 2 qPCR reaction System

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The reaction procedure:

analysis of results: the specific primers and probes of different fragments are respectively selected to detect and analyze the transcriptional activity of the ITR of the AAV Vector, and gamma-actin is used as a standardized internal reference, and the result shows that AAV9-Vector without adding 3 x Stop codon-bGH polyA signal sequence shows very high ITR transcriptional activity in HEK293T cells, compared with the situation that 3 x Stop codon-bGH polyA signal sequence is added near the ITR to obviously reduce the transcriptional activity of the ITR in the AAV Vector. Among them, AAV9-Vector-5nrP3 (only the 3 x Stop codon-bGH polyA signal sequence complementary in reverse is added upstream of the 3 'ITR) and AAV9-Vector-5PnrP3 (only the 3 x Stop codon-bGH polyA signal sequence complementary in forward and reverse is added near the ITR) have more remarkable inhibitory effect on the transcriptional activity of ITR than AAV9-Vector-5Pn3 (only the 3 x Stop codon-bGH polyA signal sequence complementary in downstream of the 5' ITR).

Conclusion: polyA signal is added downstream of the 5'ITR and upstream of the 3' ITR respectively to effectively inhibit the transcriptional activity of ITR in AAV vectors, thereby significantly reducing the transcriptional level of the target gene.

Example 2PolyA Signal is effective in inhibiting transcriptional activity of AAV vector backbones

Due to packaging limitations of AAV viruses, a target gene of 4.7kb at maximum can be packaged, and in order to further expand the capacity of the target gene, 3 XStop codon-polyA signal sequences are added to the backbone sequence of AAV vectors, respectively. Based on AAV9-Vector plasmid, the plasmid map is shown in FIG. 1A, and reverse complementary and forward 3 XStop codon-polyA signal sequences are added respectively at the upstream of 5'ITR and downstream of 3' ITR, so as to inhibit the residual AAV Vector and the transcriptional activity of AAV packaged plasmid backbone DNA. The vector construction is described as follows: based on AAV9-Vector, in order to inhibit the transcriptional activity of ITR on plasmid skeleton in AAV Vector, adding reverse complementary 3×stop codon-SV40polyA signal upstream of 5'ITR, and the constructed Vector is named AAV9-Vector-5' polyA; based on AAV9-Vector, a forward 3 XStop codon-bGH polyA signal sequence is added at the downstream of 3'ITR, and the constructed Vector is named AAV9-Vector-3' polyA; the constructed Vector was named AAV9-Vector-bPolyA with the addition of reverse complementary 3 XStop codon-SV40polyA signal and forward 3 XStop codon-bGH polyA signal sequences upstream of the 5'ITR and downstream of the 3' ITR, respectively. The reverse complementary 3×stop codon-SV40polyA signal nucleotide sequence is shown as SEQ ID NO. 18, and the 3×stop codon-bGH polyA signal nucleotide sequence is shown as SEQ ID NO. 1. The constructed AAV plasmid has the same Gene of interest (GOI) sequence, different framework designs and sequence patterns shown as A-D in FIG. 3.

HEK293T cells were transfected after the extracted plasmids were detected as being acceptable, and the passage and culture of the cells and plasmid transfection were as described in example 1. The extraction and reverse transcription system of total RNA is shown in Table 1, the system and reaction conditions of qPCR are shown in Table 2, specific primers and probes are designed for AAV plasmid skeleton, the nucleotide sequences of the primers and probes are shown as SEQ ID NO. 19-SEQ ID NO. 21, gamma-actin is used as a standardized internal reference, the nucleotide sequences of the primers and probes are shown as SEQ ID NO. 15-SEQ ID NO. 17, and each experiment is repeated three times, and the result is shown as figure 4.

Analysis of results: selecting AAV Vector plasmid skeleton region specific primers and probe sequences for transcriptional activity analysis, wherein compared with AAV9-Vector without 3 x Stop codon-PolyA signal near ITR, AAV vectors with 3 x Stop codon-bGH polyA signal and reverse complementary 3 x Stop codon-SV40 polyA signal sequences respectively added only at the downstream of 3'ITR or at the upstream of 5' ITR inhibit the transcriptional activity of ITR on plasmid skeleton, and compared with control, the transcriptional activity is respectively reduced by 18.39% and 37.53%; whereas AAV vectors with 3 XSTOC-bGH polyA signal and reverse complementary 3 XSTOC-SV 40 polyA signal sequences added downstream of the 3'ITR and upstream of the 5' ITR, respectively, significantly inhibited the transcriptional activity of the plasmid backbone of the ITR, which was 86.70% lower than the control.

Conclusion: the 3 x Stop codon-PolyA signal (AAV 9-Vector-bPolyA) is added at the upstream of the 5'ITR and the downstream of the 3' ITR as the optimal combination, so that the transcriptional activity of the ITR on the plasmid skeleton can be obviously inhibited, and the transcriptional level of the AAV Vector plasmid skeleton can be obviously reduced.

Example 3PolyA Signal is effective in inhibiting transcriptional activity of AAV viral ITRs

3.1 viral packaging

The optimal combination of AAV9-Vector-bPolyA (i.e., 3 XStop codon-PolyA signal added upstream of the 5'ITR and downstream of the 3' ITR, respectively) and the unchanged plasmid AAV9-Vector, which were verified in the cell to significantly inhibit the transcriptional activity of the AAV Vector plasmid backbone, were virus packaged, with AAV9-Vector-bPolyA and AAV9-Vector having the same gene sequences of interest, with the difference that AAV9-Vector-bPolyA had 3 XStop codon-PolyA signal added near ITR, and AAV9-Vector was without any sequence elements added. AAV packaging employs a three plasmid transient HEK293 system, and virus liquid is harvested 72h after transfection for downstream purification, and specific operational procedures are described in the literature [ Grieger, J., choi, V.&Samulski,R.Production and characterization of adeno-associated viral s.Nat Protoc 1,1412-1428(2006).]Finally, the titer of 1X 10 is obtained ¹³ AAV disease with vg/mL serotype AAV9Toxicity was used in mice in vivo experiments.

3.2 administration and residual plasmid transcriptional Activity assay

Administration: experimental mice C57BL/6 at 6 weeks of age were purchased from Ji Cui Ji Xie kang, and the packaged virus was injected into the left and right legs of mice by intramuscular injection at 3E10 vg/leg and 1.5E11 vg/leg at high doses, respectively, with 5% (V/V) of ink mixed during injection, and the control mice were given equal volumes of sterile PBS simultaneously. Mice were sacrificed 10 days after dosing and ink-stained muscle tissue was recovered.

And (3) detection: RNA of muscle tissue was extracted by TRIZOL method. Specifically, 50mg of tissue was weighed and 1mL of TRIZOL was added and broken with a mill, and after 10 minutes incubation at room temperature, 0.2mL of chloroform was added and incubated at room temperature for 5 minutes. Centrifuge at 12000rpm pre-chilled at 4℃for 20 minutes, aspirate the upper aqueous phase into a new 1.5mL centrifuge tube and add the same volume of isopropanol, incubate at room temperature for 10 minutes after inversion mixing, centrifuge at 12000rpm at 4℃for 30 minutes. The supernatant was discarded and the pellet was washed twice with 75% alcohol and finally dissolved in DEPC water for the subsequent step. Reverse transcription PCR and qPCR detection are shown in Table 1 and Table 2, probes and primers are shown in SEQ ID NO. 19-SEQ ID NO. 24, GOI is selected as a standardized internal reference, and specific probes and primers are shown in SEQ ID NO. 25-SEQ ID NO. 27, and the result is shown in FIG. 5.

Analysis of results: the specific primers and probes on the AAV vector plasmid backbone are selected to detect the transcription level of the plasmid backbone, and GOI is selected as a standardized internal reference in order to reduce the possible difference caused by different transfection efficiencies. The results show that AAV9-Vector-bPolyA showed a significant reduction in the level of transcription of residual plasmid DNA or AAV reverse packaged plasmid backbone DNA in AAV in mice, compared to controls, at either low (3E 10 vg/leg) or high (1.5E 11 vg/leg) doses of AAV virus, with a 99% reduction in the level of transcription of plasmid backbone in both low and high doses compared to controls.

Conclusion: the results of intramuscular injection of low-dose and high-dose AAV plasmids show that the design of adding 3 XStop codon-polyA signal upstream of 5'ITR and downstream of 3' ITR can effectively inhibit the transcriptional activity of ITR and obviously reduce the transcriptional level of residual plasmid skeleton in AAV virus products in vivo.

Sequence information

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Reference to the literature

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 VectorTransgene Expression.Hum Gene Ther.2020Feb；31(3-4):151-162.doi:10.1089/hum.2019.274.

PMID:31914802；PMCID:PMC7047122.

2.Keiser MS,Ranum PT,Yrigollen CM,Carrell EM,Smith GR,Muehlmatt AL,Chen YH,SteinJM,Wolf RL,Radaelli E,Lucas TJ 2nd,Gonzalez-Alegre P,Davidson BL.Toxicity after AAVdelivery of RNAi expression constructs into nonhuman primate brain.Nat Med.2021

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PMCID:PMC8605996.

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

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 ofAdeno-associated Viral Vector Preparations.Mol Ther Nucleic Acids.2016；5(8):e355.

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Characterization and Risk Assessment.Biomedicines.2014；2(1):80-97.

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 preformedcapsid in immune responses.Mol Ther.2009Jan；17(1):144-52.doi:10.1038/mt.2008.227.Epub

2008Oct 21.PMID:18941440；PMCID:PMC2834997.

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All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims (10)

1. A nucleic acid construct comprising a transcription repressing element for repressing transcription products produced by AAV vector plasmid residue and plasmid backbone reverse packaging by repressing transcription activity of ITRs in AAV vector plasmids;

the nucleic acid construct has a structure (5 'to 3' end) as shown in formula I below:

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

in the method, in the process of the invention,

y1, Y2, Y3, Y4 are each independently absent or transcription repressing elements, and Y1, Y2, Y3, Y4 are not absent at the same time; wherein Y2 and Y4 are forward transcription repressing elements, Y1 and Y3 are reverse transcription repressing elements, and the reverse transcription repressing elements are reverse complements of the forward transcription repressing elements;

Z1 is a gene of interest (GOI) expression cassette comprising one or more promoter sequences and one or more gene of interest 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 0-150nt, preferably 0-100nt;

wherein the transcription repressing element is selected from the group consisting of: (a) a hairpin-containing sequence; (b) a polyA signal tailing signal sequence; (c) A polyA signal tailing signal sequence containing a stop codon; (d) a polyadenylation sequence, i.e., a PolyA sequence; (e) a polyadenylation sequence containing a stop codon; or a combination thereof.

2. The nucleic acid construct of claim 1, wherein in the structure of formula (I), Y1 and Y4 are absent and the nucleic acid construct has the structure of formula II (5 'to 3') below:

5'ITR-Y2-Z1-Y3-3'ITR(II)

wherein Y2, Y3 are each independently absent or transcription repressing element, and Y2 and Y3 are not absent at the same time;

furthermore, the transcription repressing element therein represses the transcriptional activity of ITR, leading to a decrease in Z1, GOI transcription.

3. The nucleic acid construct of claim 1, wherein in the structure of formula (I), Y2 and Y3 are absent and the nucleic acid construct has the structure of formula III (5 'to 3') below:

Y1-5'ITR-Z1-3'ITR-Y4(III)

Wherein Y1, Y4 are each independently absent or transcription repressing element, and Y1 and Y4 are not absent at the same time;

in addition, the transcription inhibition element inhibits the transcriptional activity of ITR, which results in the transcriptional reduction of AAV vector plasmid skeleton region and inhibits the reverse packaging of plasmid skeleton to produce transcription product.

4. A recombinant AAV vector plasmid comprising the nucleic acid construct of claim 1.

5. A method of inhibiting ITR transcriptional activity in an AAV vector plasmid, 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 AAV vector plasmid of interest, or constructing the nucleic acid construct of claim 1 in the AAV vector plasmid of interest;

wherein the transcription repressing element is selected from the group consisting of: (a) a hairpin-containing sequence; (b) a polyA signal tailing signal sequence; (c) A polyA signal tailing signal sequence containing a stop codon; (d) a polyadenylation sequence, i.e., a PolyA sequence; (e) a polyadenylation sequence containing a stop codon; or a combination thereof.

6. A method of preparing an AAV product, the method comprising: AAV packaging using the recombinant AAV vector plasmid according to claim 4, and harvesting the packaged AAV for use in the production of AAV products.

7. An AAV product prepared using the method of claim 6.

8. Use of the recombinant AAV vector plasmid according to claim 4, for the preparation of an AAV product.

9. An AAV packaging system comprising the recombinant AAV vector plasmid according to claim 4.

10. A kit, comprising:

(C1) The recombinant AAV vector plasmid of claim 4;

(C2) A helper plasmid for assisting the recombinant AAV vector plasmid in AAV packaging;

and, (C3) instructions or tags, the instructions or tags stating that the kit is for AAV viral packaging.