Aav vector backbone optimization and use thereof

Abstract

The invention provides a recombinant plasmid vector containing a target gene expression cassette and a filling sequence, the filling sequence is inserted outside two ITR sequences to enlarge the size of a plasmid skeleton, and the plasmid residual quantity in a product can be obviously reduced under the condition of not influencing the target gene expression. The invention also provides a cell and a pharmaceutical composition containing the recombinant plasmid vector.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202380101981.6 (22) Application Date 2023.09.07 (85) PCT International Application Entering National Phase Date 2026.03.05 (86) PCT International Application Application Data PCT / CN2023 / 117429 2023.09.07 (87) PCT International Application Publication Data WO2025 / 050347 EN 2025.03.13 (71) Applicant Beijing Zhongyin Technology Co., Ltd. Address 102206, Building 1, No. 27, Life Science Park Road, Changping District, Beijing (72) Inventors Zhong Xiancheng, Chen Shaohong, Shi Tianyong (74) Patent Agency Beijing Qingmai Intellectual Property Agency (General Partnership) 16371 Patent Attorney Wu Xiaoming (51) Int.Cl. C12N 15 / 00 (2006.01) C12N 15 / 113 (2010.01) C12N 15 / 86 (2006.01) C12N 15 / 53 (2006.01) A61K 48 / 00 (2006.01) A61K 35 / 761 (2015.01) (54) Invention Title: AAV Vector Backbone Optimization and Application (57) Abstract: This invention provides a recombinant plasmid vector comprising a target gene expression cassette and a filler sequence, wherein by inserting a filler sequence outside the two ITR sequences to expand the size of the plasmid backbone, the amount of plasmid residue in the product can be significantly reduced without affecting the expression of the target gene. This invention also provides cell and pharmaceutical compositions comprising the recombinant plasmid vector. Claims (2 pages), Description (34 pages), Sequence Listing (electronic publication), Drawings (4 pages) CN 121794376 A 2026.04.03 CN 1 21 79 43 76 A 1. A recombinant plasmid vector comprising a target gene expression cassette and a filler sequence, wherein the target gene expression cassette is located between two inverted terminal repeat (ITR) sequences of adeno-associated virus (AAV), and the filler sequence is located outside the two ITR sequences, wherein the recombinant plasmid vector fragment comprising the target gene expression cassette (including the two ITR sequences) between the two ITR sequences is referred to as a first fragment, and the remaining portion of the recombinant plasmid vector including the filler sequence other than the first fragment is referred to as a second fragment, wherein the length of the second fragment is greater than or equal to 5.2 kb. 2. The recombinant plasmid vector according to claim 1, wherein the length of the first fragment is less than or equal to 5.2 kb and preferably greater than or equal to 3.0 kb. 3. The recombinant plasmid vector according to claim 1 or 2, wherein the length of the filler sequence is greater than 3.0 kb.More preferably greater than 4.0 kb, even more preferably greater than 6.0 kb, for example 5.0 kb to 10.0 kb; preferably the filling sequence is selected from introns, non-coding gene sequences or housekeeping gene sequences related to or unrelated to the target gene, more preferably selected from HPRT introns (SEQ ID NO: 1, 2, 21 or 22), EF1a introns (SEQ ID NO: 3) and CYP4V2 introns (SEQ ID NO: 4), even more preferably HPRT introns (SEQ ID NO: 1 or 2). 4. The recombinant plasmid vector according to any one of claims 1 to 3, wherein the target gene encodes a therapeutic protein, preferably CYP4V2 or Cas9. 5. The recombinant plasmid vector according to any one of claims 1 to 4, wherein the recombinant plasmid vector comprises an adeno-associated virus (AAV) vector. 6. The recombinant plasmid vector according to claim 5, wherein the filling sequence is selected from HPRT introns (SEQ ID NO: 1 or 2), and the target gene encodes a protein CYP4V2 (SEQ ID NO: 5), preferably the nucleotide sequence of the target gene is as shown in SEQ ID NO: 6. 7. The recombinant plasmid vector according to any one of claims 1 to 6, wherein the target gene expression cassette includes a promoter, enhancer, Kozak sequence, regulatory element, and / or polyadenylate signaling site for expressing the target gene. 8. The recombinant plasmid vector according to any one of claims 1 to 7, wherein the recombinant plasmid vector further includes a selective marker and / or a replication site in the second fragment, preferably the selective marker is an antibiotic resistance gene, such as a kanamycin resistance gene. 9. The recombinant plasmid vector according to any one of claims 1 to 8, comprising a sequence selected from the following (5'→ 3'): ITR-F1-KanR-Ori-HPRT-ITR (SEQ ID NO:7); ITR-F1-KanR-Ori-HPRT 5.8K-ITR (SEQ ID NO:8); ITR-F1-KanR-Ori-HPRT-ITR (11bp repaired) (SEQ ID NO:9); and ITR-F1-KanR-Ori-HPRT 5.8K-ITR (11bp repaired) (SEQ ID NO:10). 10. The recombinant plasmid vector according to any one of claims 1 to 9, wherein the recombinant plasmid vector is pAV-CAG-CYP4V2-HPRT-ITRres (ITR 11bp repaired) (SEQ ID NO: 11) or pAV-CAG-CYP4V2-HPRT 5.8K-ITRres (ITR 11bp repaired) (SEQ ID NO: 12).11. A cell comprising a recombinant plasmid vector according to any one of claims 1 to 10. 12. The cell according to claim 11, wherein the cell provides AAV Rep and / or Cap proteins. 13. Viral particles produced by culturing the cell according to claim 11 or 12, wherein the AAV is selected from AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV2 / 5, AAV2 / 8, AAV2 / 1, AAV2 / 9, AAV2 / 6, AAV2 / 4, AAV2 / 6, AAV5 / 2, AAV8 / 1, AAV8 / 2, AAV2 / 7, AAV2 / 12, and AAV2 / 10, preferably AAV2 and AAV8, more preferably AAV2 / 8. Claims 1 / 2 Page 2 CN 121794376 A 14. A pharmaceutical composition comprising: a) a recombinant plasmid vector according to any one of claims 1 to 10, a cell according to claim 11 or 12, or a virus particle according to claim 13; and b) a pharmaceutically acceptable adjuvant. 15. The pharmaceutical composition according to claim 14, wherein the adjuvant comprises a stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier, preservative, or any combination thereof. Claims 2 / 2 Page 3 CN 121794376 A AAV Vector Backbone Optimization and Application Technology

[0001] This invention relates to the field of genetic engineering, and more specifically to a recombinant plasmid vector comprising a target gene expression cassette and a filler sequence, and a cell and pharmaceutical composition comprising the recombinant plasmid vector. Background Art

[0002] Adeno-associated virus (AAV) is a member of the parvovirus family, a non-autoreplicating, non-enveloped, icosahedral virus with a diameter of approximately 20-26 nm. Its genome is a 4.7kb single-stranded DNA, encoding Rep and Cap proteins. Recombinant adeno-associated virus (rAAV) is an engineered AAV vector that removes all Rep or Cap gene sequences encoded by the AAV genome, retaining only inverted repeat sequences (ITRs) at both ends as packaging signals. Due to the diversity of AAV serotypes, its ability to infect multiple cell types, low immunogenicity, high safety, and long in vivo expression time, it is widely used in scientific research and clinical practice.

[0003] Impurities in AAV products mainly include DNA residues, RNA residues, and protein residues. The amount of impurities on the outside of the viral capsid can be effectively reduced by column purification, ultracentrifugation, and nuclease digestion; however, the amount of impurities packaged in the virus capsid can be reduced.Impurities remaining inside the capsid are difficult to remove effectively during purification. The DNA impurities remaining inside the viral capsid mainly originate from the mispackaging of the plasmid's back backbone sequence (Bernd Hauck et al., Undetectable Transcription of cap in a Clinical AAV Vector: Implications for Preformed Capsid in Immune Responses, www.moleculartherapy.org, vol. 17 no. 1, 144-152, Jan. 2009). Therefore, by rationally designing the vector, reducing the residual plasmid backbone sequence is an effective and economical method to reduce the amount of plasmid mispackaging residue in viral products.

[0004] Studies have found that the length of the packaged AAV vector genome cannot exceed 5.2 kb, regardless of the size of the plasmid vector or the type of capsid protein (see Zhijian Wu et al., Effect of Genome Size on AAV Vector Packaging, www.moleculartherapy.org, vol. 18 no. 1, 80-86, Jan. 2010).

[0005] To address the above-mentioned problems, the inventors have modified a recombinant plasmid vector (AAV vector) containing a target gene expression cassette. On one hand, by increasing the capacity of the genome including the target gene expression cassette, making it as close as possible to the 5.2 kb packaging limit, the AAV capsid is free of excess space to accommodate impurities such as genome or plasmid DNA. On the other hand, by increasing the plasmid backbone of the vector other than the genome, it exceeds the 5.2 kb packaging limit and cannot be packaged into the capsid as an AAV genome. In this invention, the above-mentioned expansion is achieved by inserting filler sequences.

[0006] The inventors have discovered that by increasing the size of the plasmid vector by inserting a filler sequence into a plasmid vector fragment (first fragment) comprising a genome including a target gene expression cassette, or by inserting a filler sequence into the remainder of the plasmid vector other than the first fragment (second fragment), it is possible to reduce the amount of plasmid residue in the packaged viral product. In particular, compared with inserting into the first fragment, the reduction in plasmid impurities is particularly significant when the filler sequence is inserted into the second fragment, and unexpectedly, the protein expression level of the target gene is significantly better (although the mRNA expression level of the target gene is similar in both cases).

[0007] Therefore, a first aspect of the present invention relates to a recombinant plasmid vector comprising a target gene expression cassette and a filler sequence. (Specification 1 / 34 pages 4 CN 121794376 A)The recombinant plasmid vector containing the target gene expression cassette is located between two inverted terminal repeat (ITR) sequences of adeno-associated virus (AAV), and the filler sequence is located outside the two ITR sequences. The portion of the recombinant plasmid vector containing the target gene expression cassette (including the ITR sequences at both ends) between the two ITR sequences is referred to as the first fragment, and the remaining portion of the recombinant plasmid vector containing the filler sequence, excluding the first fragment, is referred to as the second fragment. The length of the second fragment is greater than or equal to 5.2 kb. In some embodiments, due to the insertion of the filler sequence, the length of the second fragment is greater than or equal to 5.2 kb, 5.5 kb, 6.0 kb, 6.5 kb, 7.0 kb, 7.5 kb, 8.0 kb, 8.5 kb, 9.0 kb, 9.5 kb, 10.0 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 20 kb, or more.

[0008] In some embodiments, the length of the first segment is less than or equal to 5.2 kb and preferably greater than or equal to 3.0 kb, for example 1.0 kb, 1.5 kb, 2.0 kb, 2.5 kb, 3.0 kb, 3.5 kb, 4.0 kb, 4.5 kb, 5.0 kb, 5.2 kb or less.

[0009] In some embodiments, the filler sequence is selected from introns, non-coding gene sequences, or housekeeping gene sequences that are related to or unrelated to the target gene, preferably selected from HPRT introns (SEQ ID NO: 1, 2, 21, or 22), EF1a introns (SEQ ID NO: 3), and CYP4V2 introns (SEQ ID NO: 4), more preferably HPRT introns (SEQ ID NO: 1 or 2); preferably, the length of the filler sequence is greater than 3.0 kb, more preferably greater than 4.0 kb, and even more preferably greater than 6.0 kb, for example, 5.0 kb to 10.0 kb.

[0010] In some embodiments, the target gene encodes a therapeutic protein, preferably CYP4V2 or Cas9.

[0011] In some embodiments, the recombinant plasmid vector comprises an adeno-associated virus (AAV) vector.

[0012] In some embodiments, the filling sequence is selected from HPRT introns (SEQ ID NO: 1), and the target gene encodes the protein CYP4V2 (SEQ ID NO: 5), preferably the nucleotide sequence of the target gene is shown in SEQ ID NO: 6.

[0013] In some embodiments, the target gene expression cassette includes a promoter, enhancer, Kozak sequence, regulatory element, and / or polyadenylate signaling site for expressing the target gene.

[0014] In some embodiments, the recombinant plasmid vector further includes a selective marker and / or a replication site in the second fragment, preferably the selective marker being an antibiotic resistance gene, such as a kanamycin resistance gene.

[0015] In some embodiments, the recombinant plasmid vector includes sequences selected from the following (5'→3'): ITR-F1-KanR-Ori-HPRT-ITR (SEQ ID NO:7); ITR-F1-KanR-Ori-HPRT 5.8K-ITR (SEQ ID NO:8); ITR-F1-KanR-Ori-HPRT-ITR (11bp repaired) (SEQ ID NO:9); and ITR-F1-KanR-Ori-HPRT 5.8K-ITR (11bp repaired) (SEQ ID NO:10).

[0016] In some embodiments, the recombinant plasmid vector is pAV-CAG-CYP4V2-HPRT-ITRres (SEQ ID NO: 11) or pAV-CAG-CYP4V2-HPRT 5.8K-ITRres (SEQ ID NO: 12).

[0017] In a second aspect, the present invention relates to a cell comprising the above-described recombinant plasmid vector.

[0018] In some embodiments, the cell provides AAV Rep and / or Cap protein.

[0019] The present invention also relates to viral particles produced by culturing the cells described above, wherein the AAV is selected from AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV2 / 5, AAV2 / 8, AAV2 / 1, AAV2 / 9, AAV2 / 6, AAV2 / 4, AAV2 / 6, AAV5 / 2, AAV8 / 1, AAV8 / 2, AAV2 / 7, AAV2 / 12, and AAV2 / 10, preferably AAV2 and AAV8, more preferably AAV2 / 8.

[0020] The present invention also relates to pharmaceutical compositions comprising: a) the recombinant plasmid vector, cells, or viral particles described above; and b) a pharmaceutically acceptable adjuvant. Instructions for Use, Page 2 / 34, CN 121794376 A

[0021] In some embodiments, the excipients include stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, preservatives, or any combination thereof.

[0022] By designing a backbone sequence for an AAV master plasmid, the inventors have expanded the master plasmid backbone, for example, from 2.5 kb to 6.8 kb, resulting in a plasmid residue in the finished AAV product as low as 4.7~8.9 pg / 109 vg. Under the same packaging and purification conditions, compared with...Compared to before plasmid expansion, the residual plasmid content can be reduced by 6-40 times, which can effectively reduce plasmid mispackaging in AAV products. Moreover, it does not affect product packaging yield or other key attributes, and can be widely applied to the plasmid packaging construction of other recombinant AAV products.

[0023] Other aspects and advantages of this application will be readily apparent to those skilled in the art from the following detailed description. The following detailed description shows and describes only exemplary embodiments of this application. As those skilled in the art will recognize, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention involved in this application. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive.

[0024] The above-mentioned features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which: Figure 1 is a conceptual diagram of AAV master plasmid modification; Figure 2 is an expansion of the front (first fragment) of the AAV master plasmid; Figure 3 is an expansion of the back backbone (second fragment) of the AAV master plasmid; Figure 4 is a comparison of plasmid expression before and after vector modification, where CYP represents the original unmodified plasmid, HPRT represents the insertion of a 4.1 kb HPRT intron sequence into the back backbone, 5.8 k represents the insertion of a 5.8 kb HPRT intron sequence into the back backbone, inA represents the insertion of an EF1a intron-A (EF1a-intron A) sequence between the CAG promoter and CYP4V2 CDS, and in2 represents the insertion of the second intron of CYP4V2 between the original exon 2 and the original exon 3 inside the CYP4V2 CDS region; Figure 5 is a comparison of the expression of AAV8-HPRT and AAV8-inA viruses; Figure 6 is a schematic diagram of AAV master plasmid ITR repair modification; and Figure 7 is a comparison of expression levels after AAV master plasmid ITR repair. Detailed Implementation

[0025] Unless otherwise indicated, the terms used herein have their general technical meanings as understood by those skilled in the art. For definitions and terms in this art, those skilled in the art are particularly recommended to refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0026] In this application, the term “AAV” is the standard abbreviation for adeno-associated virus. Adeno-associated viruses are viruses that grow only in cells.A single-stranded DNA parvovirus, in which some functions are provided by co-infected helper viruses. General information and reviews on AAVs can be found, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169–228, and Berns, 1990, Virology, pp. 1743–1764, Raven Press, (New York).

[0027] In this application, the term “AAV vector” generally refers to a vector containing one or more polynucleotides (or transgenes) of interest side-attached by an AAV terminal repeat sequence (ITR). When present in host cells that have been transfected with a vector encoding and expressing the rep and cap gene products, such AAV vectors can be replicated and packaged into infectious viral particles. The terms “recombinant AAV virus” or “recombinant AAV virus particle” or “AAV vector particle” refer to a viral particle composed of at least one AAV capsid protein and a capsidated polynucleotide AAV vector. If the particle contains heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, such as transgenes to be delivered into mammalian cells), then it is generally referred to as an “AAV vector particle” or simply “AAV vector”. Therefore, the production of AAV vector particles necessarily includes the production of AAV vectors such that the vector is contained within the AAV vector particle.

[0028] In this application, the term “target gene” refers to the target gene intended to be expressed by recombinant AAV (rAAV), which, in the recombinant plasmid vector of the present invention, typically replaces the entire Rep or Cap gene sequence encoded by the wild-type AAV genome, retaining only inverted repeat sequences (i.e., ITRs) at both ends as packaging signals. In a preferred embodiment of the present invention, the target gene encodes a therapeutic protein, such as a functional protein for treating various genetic diseases, preferably CYP4V2 or Cas9.

[0029] In this application, the term "filler sequence" refers to any nucleotide sequence that can be used to augment the backbone of the plasmid vector of the present invention, as long as it does not affect the expression of the target gene of the present invention. Preferably, the filler sequence of the present invention and the target gene expression cassette of the present invention are located in different segments of the recombinant plasmid vector with separate ITR sequences, that is, the filler sequence of the present invention and the target gene expression cassette of the present invention do not coexist in the same segment of the recombinant plasmid vector with separate ITR sequences. In the present invention, the recombinant plasmid vector segment (including the ITR sequences at both ends) between the two ITR sequences is referred to as the first segment, while the remaining segments of the recombinant plasmid vector including the filler sequence are referred to as the first segment.This part is referred to as the second segment. There is no particular limitation on the length of the padding sequence in this invention, as long as it makes the length of the second segment greater than or equal to 5.2 kb. In one embodiment of the present invention, the length of the padding sequence is 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, 1,000-1,500, 1,500-2,000, 2,000-2,500, 2,500-3,000, 3,000-3,500, 3,500-4,000, 4,000-4,500, 4,500-5,000, 5,500-6,000, 6,000- 7,000, 7,000-8,000 or 8,000-9,000 bp, 9,000-10,000, 10,000-11,000, 11,000-12,000, 12,000-13,000, 13,000-14,000, 14,000-15,000, 15,000-16,000, 16,000-17,000, 17,000-18,000, 18,000-19,000, 19,000-20,000 bp or greater. In a preferred embodiment of the invention, the length of the padding sequence is greater than 3.0 kb, more preferably greater than 4.0 kb, and even more preferably greater than 6.0 kb, for example, 5.0 kb to 10.0 kb. There are no particular restrictions on the type of filler sequence in this invention, as long as it does not affect the expression of the target gene. In a preferred embodiment of this invention, the filler sequence is selected from introns, non-coding gene sequences or housekeeping gene sequences that are related to or unrelated to the target gene, more preferably from HPRT introns (SEQ ID NO: 1, 2, 21 or 22), EF1a introns (SEQ ID NO: 3) and CYP4V2 introns (SEQ ID NO: 4), and even more preferably from HPRT introns (SEQ ID NO: 1 or 2).

[0030] In this application, the term "front" when used to describe the plasmid backbone refers to the region or segment between two ITRs of the target gene region contained in the plasmid backbone; while the term "back" when used to describe the plasmid backbone refers to the region or segment between two ITRs of the target gene region not contained in the plasmid backbone.

[0031] In this application, the term "housekeeping gene" refers to a class of genes that are stably expressed in all cells, and whose products are essential for maintaining basic cellular life activities. Such as tubulin genes, glycolytic enzyme system genes, and ribosomal protein genes.Housekeeping genes are a class of genes that maintain a low level of methylation and remain in an active transcriptional state. In a preferred embodiment of the present invention, the housekeeping gene is selected from HPRT (hypoxanthine phosphoribosyltransferase), which has a GC content of 41% and a relatively small number of CpG motifs, thus not causing a decrease in the expression of the target gene or activation of immune cells, exhibiting good biocompatibility. In a preferred embodiment of the present invention, different fragments of HPRT can be selected as filling sequences, such as HPRT introns or fragments thereof with a length of 0.5 to 20 kb, for example, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 kb. In a preferred embodiment of the present invention, the filling sequence is selected from HPRT introns (SEQ ID NO: 1 or 2) with lengths of 4.1 kb or 5.8 kb, or HPRT intron 1 (SEQ ID NO: 21) or HPRT intron 3 (SEQ ID NO: 22).

[0032] In this application, the term "intron" refers to a non-coding segment in a gene or mRNA molecule. Because it does not have a coding function, introns can be used as "inert" filling sequences in the present invention to expand the backbone (size) of the recombinant plasmid vector. In a preferred embodiment of the present invention, the intron is selected from introns related to or unrelated to the target gene, for example, when the target gene is a CYP4V2 coding gene, the EF1a intron (SEQ ID NO: 3) or the CYP4V2 intron (SEQ ID NO: 4) can be selected.

[0033] In this application, the term "CYP4V2" generally refers to a protein, which is a member 2 of the cytochrome P450 family 4 subfamily V. The term "cytochrome P450," also known as cytochrome P450 or CYP450, generally refers to a family of heme proteins, belonging to the class of monooxygenases, involved in the metabolism of endogenous substances or exogenous substances, including drugs and environmental compounds. Based on the degree of homology in their amino acid sequences, members are further divided into three levels: family, subfamily, and enzyme individual. The cytochrome P450 enzyme system can be abbreviated as CYP, where families are represented by Arabic numerals, subfamilies by uppercase letters, and enzyme individuals by Arabic numerals, such as CYP4V2 in this application. The human CYP4V2 gene (HGNC: 23198) is 19.28 kb in length, located at 4q35, and has 11 exons, playing an important role in fatty acid metabolism (Kumar S.,Bioinformation, 2011, 7:360-365). CYP4V2 is expressed in almost all tissues, but at high levels in the retina and retinal pigment epithelium, and at slightly lower levels in the cornea. Mutations in the CYP4V2 gene may be associated with crystalline retinal degeneration and / or retinitis pigmentosa.

[0034] In this application, the term "promoter" generally refers to a deoxyribonucleic acid (DNA) sequence that enables the transcription of a specific gene. A promoter can be recognized by RNA polymerase and initiate transcription to synthesize RNA. In RNA synthesis, the promoter can interact with transcription factors that regulate gene transcription, controlling the initiation time and extent of gene expression (transcription). The promoter contains a core promoter region and a regulatory region, located in the regulatory sequence that controls gene expression, upstream of the gene transcription start site (5' direction of the DNA antisense strand), and has no translation function itself.

[0035] In this application, the term "polyadenylation signal site" generally refers to a base sequence located at the 3' end of messenger RNA (mRNA) that can be recognized by polyadenylation-associated cleavage factors. It is usually also a cis-regulatory signal on mRNA. Generally, the tailing process (i.e., polyadenylation) begins after transcription termination. Under the regulation of the polyadenylation signal site, polyadenylation-associated cleavage factors add tens to hundreds of single adenosine nucleotides to the 3'UTR of the mRNA. Common tailing signals include SV40, BGH, HSV, TK signals, etc.

[0036] In this application, the term "selectivity marker" refers to a gene that, after gene expression, can confer a selective phenotype on transformed cells, such as antibiotic resistance (e.g., kanamycin).

[0037] In this application, the term "antibiotic resistance gene" refers to a gene encoding a resistance protein that makes the host bacteria or cells resistant to antibiotics (e.g., kanamycin, ampicillin, chloramphenicol, etc.).

[0038] Example 1. Vector Construction

[0039] In order to reduce the residual DNA from mispackaged AAV products, we have two approaches to plasmid modification: one is to increase the capacity of the target genome between ITRs in the main plasmid to make it as close as possible to the packaging limit of 5.2 kb, so that there is no extra space in the AAV capsid to accommodate the impurity residue of genome or plasmid DNA; the other is to increase the skeleton of the main plasmid to exceed the packaging limit of 5.2 kb and thus be unable to be packaged into the capsid as AAV genome.

[0040] The recombinant plasmid vector pAV-CAG-CYP4V2 (Chinese Patent Application No. 202010520246.7, expressionBased on the AAV vector of CYP4V2 and its uses, the above ideas were followed for modification. Referring to Figure 1, the target gene part on the front of the plasmid was expanded using two methods: one was to insert the EF1a intron-A sequence (SEQ ID NO: 3) between the CAG promoter and CYP4V2 CDS, so that the target genome sequence of the two ITRs and the middle part was expanded from 3.3 kb to 4.2 kb (SEQ ID NO: 13); the other was to insert the second intron of CYP4V2 (SEQ ID NO: 4) between the original exon 2 and the original exon 3 inside the CYP4V2 CDS region, so that the sequence of the two ITRs and the middle part was expanded from 3.3 kb to 4.7 kb (SEQ ID NO: 14).

[0041] Referring to Figure 2, the backbone part on the back of the plasmid was expanded. We chose to insert the first intron part of the housekeeping gene HPRT (sequence NG_012329.2), which is about 13 kb in length. The HPRT gene encodes a protein that is a transferase that catalyzes the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate by transferring a 5-phosphate ribosyl-1-pyrophosphate group. Housekeeping genes exhibit relatively stable transcription and translation levels, with expression levels less affected by environmental factors, and show minimal sustained expression variations across almost all tissues at various growth stages. Therefore, we believe that even with a small amount of residue in the final AAV product, the biological risk is low and the safety profile is good. Furthermore, the first intron of HPRT is 13 kb long, with a GC content of 41%, a small number of CpG motifs, and no complex structure, making it suitable for our cloning and amplification. Insertion into the vector will not increase the complexity of the vector sequence, thus avoiding plasmid recombination or reduced plasmid yield. For these reasons, we selected the amplified sequence of the first intron of HPRT as the filler sequence for plasmid backbone expansion. Two HPRT fragments, 4.1 kb and 5.8 kb respectively, were selected as filler sequences (SEQ ID NO: 1 and SEQ ID NO: 2 respectively) and inserted into the backbone portion on the back of the plasmid to generate the constructs ITR-F1-KanR-Ori-HPRT-ITR (SEQ ID NO: 7) and ITR-F1-KanR-Ori-HPRT 5.8K-ITR (SEQ ID NO: 8), thereby obtaining two amplification plasmids pAV-CAG-CYP4V2-HPRT and pAV-CAG-CYP4V2-HPRT 5.8K (see Figure 3). Example 2. Comparison of plasmid expression before and after vector modification

[0042] The modified plasmid in Example 1 and the original plasmid were transfected into 293T cells (ATCC, CRL-3216) and 48 cells respectively.Cells were lysed after hours, and the expression level of the target gene CYP4V2 was detected by Western blotting (detection antibodies: Anti-CYP4V2, Atlas, HPA029122; ACTB rabbit mAb, ABclonal, AC026). As shown in Figure 4, the results indicate that neither expanding the plasmid's dorsal backbone nor inserting introns into the target gene expression frame affected the cellular protein expression of CYP4V2.

[0043] Example 3. Comparison of AAV virus yield and impurity content before and after vector modification. The modified plasmid and the original plasmid in Example 1 were co-transfected into 293T cells (ATCC, CRL-3216) with RC8 plasmid (providing the REP2 gene and CAP8 gene for AAV packaging; the plasmid was fully synthesized by General Biotechnology (Anhui)) and Helper plasmid (providing the Ad5 adenovirus E2A gene, E4 gene and VA RNA gene for AAV packaging; the plasmid was fully synthesized by General Biotechnology (Anhui)). The packaging process was described in Chinese Patent Application No. 202010520246.7. We performed TaqMan qPCR analysis on the viral titer (primers designed in the CYP4V2 CDS region of the target gene) and plasmid impurity content (primers designed in the kana resistance region) in the purified AAV product. The analysis procedure is based on [Cristina Martinez-Fernandez de la Camara et al., Accurate Quantification of AAV Vector Genomes by Quantitative PCR, Genes 2021, 12, 601. https: / / doi.org / 10.3390 / genes12040601].

[0044] CYP4V2 primers (viral titer quantification) (5'→3'): Instructions for use, page 6 / 34, CN 121794376 A

[0045] The results are shown in the table below. Data shows that compared to AAV8-CYP4V2 virus (packaged from the original plasmid pAV-CAG-CYP4V2), AAV8-HPRT virus (with a 4.1 kb HPRT intron sequence inserted into the back backbone of the pAV-CAG-CYP4V2 plasmid) and AAV8-5.8K virus (with a 5.8 kb HPRT intron sequence inserted into the back backbone of the pAV-CAG-CYP4V2 plasmid) both significantly reduced the residual plasmids in AAV products due to mispackaged plasmids. AAV8-inA virus (with an EF1a intron-A sequence inserted between the CAG promoter and the CYP4V2 CDS in the pAV-CAG-CYP4V2 plasmid) showed a slight improvement. AAV8-in2 virus (with an EF1a intron-A sequence inserted between the CAG promoter and the CYP4V2 CDS in the pAV-CAG-CYP4V2 plasmid) showed a slight improvement.The amount of plasmid residue (the second intron of CYP4V2 inserted between the original exon 2 and the original exon 3 inside the CYP4V2 CDS region) is increased.

[0046]

[0047] Example 4. Expression comparison of AAV8-HPRT and AAV8-inA viruses As shown in Example 3, after modification, the content of mispackaged impurities in AAV8-HPRT, AAV8-5.8K and AAV8-inA virus products decreased. We compared the infectivity and expression level of the three viruses. HEK293T cells were treated with 10 μg / ml mitomycin C (CST, 51854S) for 2 h, and then infected with AAV8-HPRT, AAV8-5.8K, and AAV8-inA viruses with an MOI of 3e5, respectively. Cells were lysed after 72 hours, and the expression level of the target gene CYP4V2 was detected by Western blotting (Anti-CYP4V2, Atlas, HPA029122; ACTB rabbit mAb, ABclonal, AC026). As shown in Figure 5, the mRNA expression levels of AAV8-HPRT, AAV8-5.8K, and AAV8-inA were consistent after infection, but the protein expression level of the target gene in AAV8-inA was significantly lower than that in AAV8-HPRT and AAV8-5.8K. Instruction manual, page 7 / 34, 10 CN 121794376 A

[0048] Example 5. AAV master plasmid ITR sequence repair. One side of our original AAV packaging plasmid had a complete ITR sequence, while the other side of the ITR lacked a 11bp C loop sequence, as shown in the dashed box in Figure 6. We repaired the missing 11bp.

[0049] We performed ITR repair on both ends of the original AAV packaging plasmid pAV-CAG-CYP4V2 and pAV-CAG-CYP4V2-HPRT plasmid, obtaining the repaired plasmids pAV-CAG-CYP4V2-ITRres and pAV-CAG-CYP4V2-HPRT-ITRres (SEQ ID NO: 11) (in this document, "itrres" or "ITRres" refers to the repair of the missing ITR sequence). These plasmids were used for virus packaging, obtaining viruses AAV8-CYP4V2, AAV8-HPRT, AAV8-CYP4V2-ITRres, and AAV8-HPRT-ITRres (ZVS101e), respectively. The packaging yield and viral infectivity of each virus were compared.

[0050]

[0051] The above four viruses were used to infect 293T cells, and the infection procedure was the same as in Example 4. Referring to Figure 7, the results show that the four viruses...After infection of 293T cells with the virus, there was no significant difference in the mRNA expression level and protein expression level of the target gene.

[0052] Example 6. Application of pAV-HPRT-ITR master plasmid backbone for the virus packaging of other target genes. We used the modified pAV-HPRT-ITR master plasmid backbone (with 4.1 kb HPRT intron inserted; considering that there was no significant difference in the expression level, packaging yield and impurity residue of AAV8-HPRT and AAV8-5.8K, the pAV-HPRT-ITRres sequence was used as the backbone sequence) (SEQ ID NO: 9, which is different from pAV-CAG-CYP4V2-HPRT in that it does not include the CAG promoter and CYP4V2 element and the ITR was repaired as in Example 5) for the construction of master plasmids for ZVS101e and ZVS203e. In the ZVS101e plasmid, the CAG promoter and CYP4V2 coding region were inserted into the target gene region between the ITRs on the front side of the pAV-HPRT-ITR main plasmid backbone, with a genome length of 3.3 kb, thus obtaining pAV-CAG-CYP4V2-HPRT-ITRres (SEQ ID NO: 11). In ZVS203e, the CAG promoter and Cas9 coding region (sequence reference CN113038972B) were inserted into the target gene region between the ITRs on the front side of the pAV-HPRT-ITR main plasmid backbone, with a genome length of 4.6 kb. The analysis methods for viral packaging and impurity residue were the same as in Example 3.

[0053] The results in Table 3 demonstrate that the pAV-HPRT-ITR main plasmid backbone is applicable not only to CYP4V2 but also to Cas9. Therefore, the empty vector of the present invention (i.e., the vector without the target gene) can be extended to the packaging of other target genes and can significantly reduce the residue of plasmid impurities in viral packaging. Instruction manual, page 8 / 34, 11 CN 121794376 A

[0054]

[0055] (Note: ZVS101e is the same as "AAV8-HPRT-ITRres" above, indicating that the virus is packaged using the modified vector (pAV-CAG-CYP4V2-HPRT-ITRres) and produced using the same non-GMP (non-GMP) production process as the unmodified AAV8-CYP4V2. The difference between ZVS101e-LOT1 (GMP), ZVS101e-LOT2 (GMP), and ZVS101e-LOT3 (GMP) and ZVS101e is that they use GMP production processes. The same applies to AAV8-cas9, ZVS203e-LOT1 (GMP), and ZVS203e-LOT2 (GMP).) Those skilled in the art should understand that although the present invention has been specifically described with reference to the above embodiments,This invention is not limited to these specific embodiments. Based on the methods and technical solutions taught in this invention, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of this invention, and the resulting equivalent embodiments are all within the scope of this invention.

[0056] Specification page 9 / 34, 12 CN 121794376 A

[0057]

[0058] Specification page 10 / 34, 13 CN 121794376 A

[0059]

[0060] Specification page 11 / 34, 14 CN 121794376 A

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[0105]

[0106] Specification 34 / 34 pages 37 CN 121794376 A Figure 1 Figure 2 Specification Figure 1 / 4 pages 38 CN 121794376 A Figure 3 Figure 4 Instruction manual, Figures 2 / 4, page 39, CN 121794376 A, Figures 5 and 6; Instruction manual, Figures 3 / 4, page 40, CN 121794376 A, Figure 7; Instruction manual, Figures 4 / 4, page 41, CN 121794376 A

Claims

1. A recombinant plasmid vector comprising a target gene expression cassette and a filler sequence, wherein the target gene expression cassette is located between two inverted terminal repeat (ITR) sequences of adeno-associated virus (AAV), and the filler sequence is located outside the two ITR sequences, wherein the recombinant plasmid vector fragment comprising the target gene expression cassette (including the two ITR sequences) between the two ITR sequences is referred to as a first fragment, and the remaining portion of the recombinant plasmid vector including the filler sequence other than the first fragment is referred to as a second fragment, wherein the length of the second fragment is greater than or equal to 5.2 kb.

2. The recombinant plasmid vector according to claim 1, wherein the length of the first fragment is less than or equal to 5.2 kb and preferably greater than or equal to 3.0 kb.

3. The recombinant plasmid vector according to claim 1 or 2, wherein the length of the filling sequence is greater than 3.0 kb, more preferably greater than 4.0 kb, and even more preferably greater than 6.0 kb, for example 5.0 kb to 10.0 kb; preferably the filling sequence is selected from introns, non-coding gene sequences or housekeeping gene sequences related to or unrelated to the target gene, more preferably selected from HPRT introns (SEQ ID NO: 1, 2, 21 or 22), EF1a introns (SEQ ID NO: 3) and CYP4V2 introns (SEQ ID NO: 4), and even more preferably HPRT introns (SEQ ID NO: 1 or 2).

4. The recombinant plasmid vector according to any one of claims 1 to 3, wherein the target gene encodes a therapeutic protein, preferably CYP4V2 or Cas9.

5. The recombinant plasmid vector according to any one of claims 1 to 4, wherein the recombinant plasmid vector comprises an adeno-associated virus (AAV) vector.

6. The recombinant plasmid vector according to claim 5, wherein the filling sequence is selected from HPRT introns (SEQ ID NO: 1 or 2), and the target gene encodes a protein CYP4V2 (SEQ ID NO: 5), preferably the nucleotide sequence of the target gene is shown in SEQ ID NO:

6.

7. The recombinant plasmid vector according to any one of claims 1 to 6, wherein the target gene expression cassette includes a promoter, enhancer, Kozak sequence, regulatory element, and / or polyadenylate signaling site for expressing the target gene.

8. The recombinant plasmid vector according to any one of claims 1 to 7, wherein the recombinant plasmid vector further comprises a selective marker and / or a replication site in the second fragment, preferably the selective marker being an antibiotic resistance gene, such as a kanamycin resistance gene.

9. The recombinant plasmid vector according to any one of claims 1 to 8, comprising a sequence selected from the group consisting of (5'→3'): ITR-F1-KanR-Ori-HPRT-ITR (SEQ ID NO:7); ITR-F1-KanR-Ori-HPRT 5.8K-ITR (SEQ ID NO:8); ITR-F1-KanR-Ori-HPRT-ITR (11bp repaired) (SEQ ID NO:9); and ITR-F1-KanR-Ori-HPRT 5.8K-ITR (11bp repaired) (SEQ ID NO:10).

10. The recombinant plasmid vector according to any one of claims 1 to 9, wherein the recombinant plasmid vector is pAV-CAG-CYP4V2-HPRT-ITRres (ITR 11bp repaired) (SEQ ID NO: 11) or pAV-CAG-CYP4V2-HPRT 5.8K-ITRres (ITR 11bp repaired) (SEQ ID NO: 12).

11. A cell comprising a recombinant plasmid vector according to any one of claims 1 to 10.

12. The cell of claim 11, wherein the cell provides AAV Rep and / or Cap proteins.

13. Viral particles produced by culturing cells according to claim 11 or 12, wherein the AAV is selected from AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV2 / 5, AAV2 / 8, AAV2 / 1, AAV2 / 9, AAV2 / 6, AAV2 / 4, AAV2 / 6, AAV5 / 2, AAV8 / 1, AAV8 / 2, AAV2 / 7, AAV2 / 12, and AAV2 / 10, preferably AAV2 and AAV8, more preferably AAV2 / 8.

14. A pharmaceutical composition comprising: a) a recombinant plasmid vector according to any one of claims 1 to 10, a cell according to claim 11 or 12, or a viral particle according to claim 13; and b) a pharmaceutically acceptable adjuvant.

15. The pharmaceutical composition of claim 14, wherein the excipient comprises a stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier, preservative, or any combination thereof.

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