WO2025065890A1 - Recombinant adeno-associated virus particle, recombinant adeno-associated virus vector system, and use thereof - Google Patents

Abstract

Provided are a recombinant adeno-associated virus particle, a recombinant adeno-associated virus vector system, and a use thereof. A novel recombinant adeno-associated virus efficiently crossing the blood-brain barrier is prepared, named rAAV32.eB, the capsid thereof being a mutant of the capsid protein AAVhu.32, comprising mutations at two sites, A587D and Q588G, and having the 7-amino acid peptide TLAVPFK inserted between amino acids 588 and 589. A corresponding recombinant adeno-associated virus vector system is also constructed, and an AAV32.eB serotype plasmid is constructed. It is also found that rAAV32.eB has a higher efficiency in transducing neural cells across the blood-brain barrier, and using it for more efficient gene delivery can provide better tools for neuroscience research and the treatment of whole-brain systemic genetic diseases, neurodegenerative diseases, etc.

Description

A recombinant adeno-associated virus particle, a recombinant adeno-associated virus vector system and its application Technical Field

The present invention relates to the fields of genetic engineering and biotechnology, and in particular to a recombinant adeno-associated virus particle, a recombinant adeno-associated virus vector system and applications thereof.

Background Art

One of the main challenges in neuroscience research and gene therapy for the nervous system is how to break through the blood-brain barrier to efficiently deliver target genes. Delivery of target genes to the nervous system requires a tool vector. Among the many existing tool vectors, adeno-associated virus (AAV) vectors are widely popular due to their excellent gene delivery efficiency, stable safety, and relatively low cytotoxicity. Adeno-associated viruses are a type of tiny, non-enveloped, icosahedral, single-stranded DNA virus. So far, no AAV-related diseases have been reported, so it is recognized as the safest viral vector and has become a hot topic in gene therapy vector research.

There are many AAV serotypes, and different serotypes have different targeted infection characteristics. Among them, AAV type 9 (AAV9) is one of the few natural serotypes of AAV that can cross the blood-brain barrier to infect neural cells. In addition, studies have reported that other serotypes of AAV can also cross the blood-brain barrier, such as AAVhu.32 (Yoon et al., 2020,143; 2058–2072). However, although AAVhu.32 is more efficient than AAV9 in crossing the blood-brain barrier, its efficiency is still very low and cannot meet the purpose of efficient delivery through the blood-brain barrier. The delivery efficiency needs to be further improved.

Therefore, the prior art still needs to be improved and developed.

Technical issues

In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a recombinant adeno-associated virus particle, a recombinant adeno-associated virus vector system and its application, aiming to solve the problem of low efficiency of current adeno-associated virus vectors crossing the blood-brain barrier.

Technical Solutions

The technical solution of the present invention is as follows:

A recombinant adeno-associated virus particle, comprising an AAVhu.32 capsid protein mutant; relative to the wild-type AAVhu.32 capsid protein, the AAVhu.32 capsid protein mutant includes mutations at two amino acid sites, 587 and 588, and an amino acid fragment as shown in SEQ. ID NO.1 is inserted between amino acids 588 and 589.

The recombinant adeno-associated virus particles, wherein the alanine at the 587th amino acid site of the AAVhu.32 capsid protein mutant is mutated to aspartic acid, and the glutamine at the 588th amino acid site is mutated to glycine.

The recombinant adeno-associated virus particle, wherein the genome of the recombinant adeno-associated virus particle contains an exogenous target gene of a nucleotide sequence encoding a target gene product.

The recombinant adeno-associated virus particles, wherein the target gene product is a protein, a polypeptide or an interfering RNA.

A recombinant adeno-associated virus vector system, comprising one or more vectors, at least one of which is a recombinant adeno-associated virus packaging plasmid, and the recombinant adeno-associated virus packaging plasmid contains a nucleic acid fragment for encoding the AAVhu.32 capsid protein mutant as described above.

The recombinant adeno-associated virus vector system, wherein at least one vector is an adeno-associated virus core plasmid, and the adeno-associated virus core plasmid comprises inverted terminal repeat sequences ITR at both ends, a promoter, an exogenous target gene, a transcription regulatory element WPRE and a transcription termination sequence hGH polyA.

In the recombinant adeno-associated virus vector system, at least one vector is an adenovirus element helper plasmid.

A pharmaceutical composition, characterized in that it comprises the recombinant adeno-associated virus particles as described in any of the above items, and/or the recombinant adeno-associated virus vector system as described in any of the above items.

Use of a recombinant adeno-associated virus particle as described in any one of the above items and/or a recombinant adeno-associated virus vector system as described in any one of the above items in central nervous system gene delivery.

Use of a recombinant adeno-associated virus particle as described in any of the above items and/or a recombinant adeno-associated virus vector system as described in any of the above items in the preparation of a drug for treating whole-brain systemic genetic diseases or neurodegenerative diseases.

Beneficial Effects

The present invention provides a recombinant adeno-associated virus particle, a recombinant adeno-associated virus vector system and its application. The present invention prepares a new recombinant adeno-associated virus (rAAV) that efficiently crosses the blood-brain barrier, named rAAV32.eB, whose capsid is a mutant of the AAVhu.32 capsid protein. Compared with the wild-type AAVhu32 capsid protein VP1, there are three major position changes, including mutations at two sites, A587D and Q588G, and a 7-amino acid peptide TLAVPFK is inserted between the 588th and 589th amino acids. At the same time, the present invention also constructs a corresponding recombinant adeno-associated virus vector system and an AAV32.eB serotype plasmid. Not only that, the recombinant adeno-associated virus rAAV32.eB prepared by the present invention is infected by intravenous injection. Compared with rAAVhu.32, rAAV32.eB has a higher efficiency in transducing neural cells across the blood-brain barrier. Finally, the present invention obtained a new recombinant adeno-associated virus rAAV32.eB through engineering modification, and used it for more efficient gene delivery, which can provide better tools for neuroscience research and the treatment of whole-brain systemic genetic diseases, neurodegenerative diseases, etc., and has broad application value and market prospects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the pAAV2/32.eB serotype plasmid map constructed in an embodiment of the present invention.

FIG. 2 is a schematic diagram of brain tissue slice imaging results provided by an embodiment of the present invention.

Embodiments of the present invention

The present invention provides a recombinant adeno-associated virus particle, a recombinant adeno-associated virus vector system and its application. In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

An embodiment of the present invention provides a recombinant adeno-associated virus particle, which contains an AAVhu.32 capsid protein mutant; relative to the wild-type AAVhu.32 capsid protein, the AAVhu.32 capsid protein mutant includes mutations at two amino acid sites, 587 and 588, and an amino acid fragment as shown in SEQ. ID NO.1 is inserted between amino acids 588 and 589.

SEQ. ID NO.1: TLAVPFK

In some embodiments, the alanine at the 587th amino acid position of the AAVhu.32 capsid protein mutant is mutated to aspartic acid (A587D), and the glutamine at the 588th amino acid position is mutated to glycine (Q588G).

In the present invention, "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. Unless otherwise required, the term includes subtypes and naturally occurring and recombinant forms. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also known as a recombinant AAV vector. The term "AAV" can be used to refer to adeno-associated viruses of different serotypes, and the genome sequences of different subtypes of AAV, as well as the sequences of natural terminal repeats (ITRs), Rep proteins and capsid subunits are known in the art. Such sequences can be found in literature or public databases such as gene banks. Recombinant adeno-associated virus particles (abbreviated as "AAV virus particles" or "AAV viruses") refer to virus particles composed of at least one AAV capsid protein and a capsid polynucleotide rAAV vector. The recombinant adeno-associated virus particles (named rAAV32.eB) involved in the present invention contain the above-mentioned AAVhu.32 capsid protein mutant.

In order to solve the problem of low efficiency of rAAVhu.32 crossing the blood-brain barrier, the present invention obtains a new recombinant adeno-associated virus (rAAV) that efficiently crosses the blood-brain barrier through genetic engineering modification. The new recombinant adeno-associated virus is named rAAV32.eB, and its capsid is a mutant of the AAVhu.32 capsid protein. Compared with the AAVhu32 capsid protein VP1, there are three major position changes, including mutations at two sites, A587D and Q588G, and a 7-amino acid peptide TLAVPFK is inserted, and the insertion position corresponds to the 588th and 589th sites of the wild-type AAVhu.32 capsid protein. The recombinant adeno-associated virus rAAV32.eB prepared by the present invention is infected by intravenous injection. Compared with rAAVhu.32, rAAV32.eB has a higher efficiency in transducing neural cells across the blood-brain barrier. Finally, the present invention obtains a new recombinant adeno-associated virus rAAV32.eB (also known as recombinant adeno-associated virus particles) through engineering modification, and uses it for more efficient gene delivery, which can provide better tools for neuroscience research and the treatment of whole-brain systemic genetic diseases, neurodegenerative diseases, etc., and has broad application value and market prospects.

In some embodiments, the genome of the recombinant adeno-associated virus particle contains an exogenous target gene having a nucleotide sequence encoding a target gene product.

"Exogenous" refers to an entity that is derived from a different genotype than the rest of the entity being compared. For example, nucleotides introduced into a plasmid or vector derived from a different species by genetic engineering techniques are exogenous nucleotides. An rAAV comprising an exogenous nucleic acid encoding an exogenous target gene product is an rAAV comprising a nucleic acid that is not generally included in a naturally occurring wild-type AAV, and the encoded exogenous target gene product is a gene product that is generally not encoded by a naturally occurring wild-type AAV.

In some embodiments, the target gene product is a protein.

In some embodiments, the target gene product is a polypeptide.

In some embodiments, the target gene product is interfering RNA.

Specifically, the interfering RNA can be selected from siRNA or shRNA.

The present invention constructs a new rAAVhu.32 mutant (rAAV32.eB) and uses it for gene delivery; compared with the original AAVhu.32, rAAV32.eB is more efficient in delivering exogenous genes across the blood-brain barrier.

An embodiment of the present invention also provides a recombinant adeno-associated virus vector system, which comprises one or more vectors, wherein at least one vector is a recombinant adeno-associated virus packaging plasmid, and the recombinant adeno-associated virus packaging plasmid comprises a nucleic acid fragment for encoding the above-mentioned AAVhu.32 capsid protein mutant.

In some embodiments, the AAVhu.32 capsid protein mutant includes mutations at two amino acid positions 587 and 588, and an amino acid fragment TLAVPFK is inserted between amino acids 588 and 589.

The nucleic acid fragment of the capsid protein is generally represented by the Cap gene fragment in the present invention. The rep and Cap genes of AAV refer to polynucleotide sequences encoding the replication and encapsidation proteins of adeno-associated viruses. The rep and Cap genes can be located on the same or different plasmids.

In some embodiments, the recombinant adeno-associated virus packaging plasmid uses pAAV2/hu.32 as a backbone, and is obtained by synthesizing the full-length gene sequence of the capsid of hu.32 (GenBank: AY530597.1), and then replacing the full-length gene sequence corresponding to the capsid (cap2) on pAAV2/2 (Brinkase (Shenzhen) Biotechnology Co., Ltd.).

In some embodiments, the recombinant adeno-associated virus packaging plasmid comprises a rep gene segment of adeno-associated virus type II.

In some embodiments, the AAV32.eB sequence of the recombinant adeno-associated virus packaging plasmid is obtained by mutating AAVhu.32 and inserting a peptide segment, and its Cap gene sequence is shown in SEQ ID NO.2. Compared with AAVhu.32 Cap , it has mutations at two sites, A587D and Q588G, and a peptide segment TLAVPFK of 7 amino acids is inserted after site 588.

In some embodiments, at least one vector in the recombinant adeno-associated virus vector system is an adeno-associated virus core plasmid.

Specifically, the adeno-associated virus core plasmid comprises inverted terminal repeat sequences ITR at both ends, a promoter, an exogenous target gene, a transcriptional regulatory element WPRE and a transcriptional termination sequence hGH polyA.

More specifically, the exogenous target gene includes a reporter gene;

More specifically, the reporter gene is EGFP;

In a specific embodiment, the adeno-associated virus core plasmid is pAAV-CMV-EGFP-WPRE-hGH polyA.

In some embodiments, at least one vector in the recombinant adeno-associated virus vector system is an adenovirus element helper plasmid.

Specifically, the adenovirus element helper plasmid is pAd-Helper.

In some embodiments provided by the present invention, the recombinant adeno-associated virus particles are obtained by co-transfecting a packaging cell line with the recombinant adeno-associated virus packaging plasmid, adeno-associated virus core plasmid and adenovirus element helper plasmid.

In some embodiments, the packaging cell line is selected from HEK-293 cells, HEK-293T cells or HEK-293FT cells;

In some embodiments, the molecular numbers of the recombinant adeno-associated virus packaging plasmid, the adeno-associated virus core plasmid, and the adenovirus element helper plasmid are 1:1:1.

The present invention constructs a recombinant adeno-associated virus packaging plasmid, AAV32.eB serotype plasmid, and uses it to package recombinant adeno-associated virus particles, prepares recombinant adeno-associated virus rAAV32.eB-CMV-EGFP-WPRE-hGH polyA, and verifies that the recombinant adeno-associated virus rAAV32.eB prepared by the present invention is infected by intravenous injection, and compared with rAAVhu.32, rAAV32.eB has a higher efficiency of transducing nerve cells across the blood-brain barrier. It can provide better tools for neuroscience research and the treatment of whole-brain systemic genetic diseases, neurodegenerative diseases, etc., and has broad application value and market prospects.

An embodiment of the present invention further provides a pharmaceutical composition, which comprises the recombinant adeno-associated virus particles and/or the recombinant adeno-associated virus vector system as described above.

In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.

Specifically, the carriers such as additives and flavoring agents are made into various dosage forms, including powders, tablets, pellets, capsules, microcapsules, granules or liquids.

The embodiments of the present invention also provide a use of the recombinant adeno-associated virus particle and/or the recombinant adeno-associated virus vector system as described above in gene delivery to the central nervous system.

The embodiments of the present invention also provide a use of the recombinant adeno-associated virus particles and/or the recombinant adeno-associated virus vector system as described above in the preparation of drugs for treating whole-brain systemic genetic diseases or neurodegenerative diseases.

The present invention provides a recombinant adeno-associated virus vector system for efficiently delivering exogenous genes across the blood-brain barrier to targeted nerve cells and a recombinant adeno-associated virus packaged therefrom, wherein the recombinant adeno-associated virus is a mutant of the hu.32 adeno-associated virus (rAAVhu.32), named rAAV32.eB. The present invention injects rAAV32.eB carrying the target gene into mice through the tail vein, which can efficiently transduce the central nervous system across the blood-brain barrier. Finally, the present invention constructs a new rAAVhu.32 mutant (rAAV32.eB) and uses it for gene delivery, and rAAV32.eB is more efficient in delivering exogenous genes across the blood-brain barrier than the original AAVhu.32, providing a new viral vector for the study of neural structure and function in the whole brain and gene delivery, etc., and has a wide range of application value and broad market prospects in the fields of central nervous system network analysis, disease model establishment and gene therapy.

The following is a further explanation of a recombinant adeno-associated virus particle, a recombinant adeno-associated virus vector system and its application according to the present invention through specific examples:

Generally, the nomenclature used in and techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references.

Example 1 Construction of rAAV32.eB vector

The AAV32.eB sequence was obtained by mutating AAVhu.32 and inserting a peptide segment. Its Cap gene sequence is shown in SEQ. ID NO.2. Compared with AAVhu.32 Cap , it has mutations at two sites, A587D and Q588G, and a peptide segment TLAVPFK of 7 amino acids is inserted after site 588.

In order to construct the AAV32.eB serotype plasmid, the primers Mu-32.eB-F (SEQ. ID NO.3) and Mu-32.eB -R (SEQ. ID NO.4) were synthesized in this example, and the point mutation method was used, with the plasmid pAAV2/hu.32 as the template (the plasmid was obtained by synthesizing the full-length gene sequence of the capsid of hu.32 (GenBank: AY530597.1) and then replacing the full-length gene sequence corresponding to the capsid (cap2) on pAAV2/2 (Brinkas (Shenzhen) Biotechnology Co., Ltd.)), and the Fast Mutagenesis System kit was used to obtain pAAV2/32.eB by PCR reaction. The details are as follows:

SEQ. ID NO.3

GATGGGACTTTGGCGGTGCCTTTTAAGGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATA

SEQ. ID NO.4

CTTAAAAGGCACCGCCAAAGTCCCATCACTCTGGTGGTTTGTGGCCACTTGTCCA

The PCR reaction system is:

2 × TransStart FastPfu Fly PCR SuperMix, 25 μl;

10 µM forward primer, 1 μl;

10 μM reverse primer, 1 μl;

Template pAAV2/hu.32, 2 μl;

_ddH2O , 31 μl.

The reaction program was: 98°C, 5 min; 36 cycles of {98°C, 30 s; 60°C, 30 s; 72°C, 5 min; 72°C, 10 min}; 16°C, 30 min.

The PCR product needs to be added with 1 μl of template digestion enzyme DMT, and the template is digested at 37°C for 1 hour. Take 5 μl of the digested product to transform competent Stbl3. After 16 hours, pick a single colony to activate and inoculate it into 15 ml of LB liquid culture medium. After culture and amplification, the plasmid is extracted and sequenced. The map of the constructed recombinant adeno-associated virus serotype packaging plasmid pAAV2/32.eB expression vector is shown in Figure 1. All PCR primers, gene synthesis and sequencing were completed by commercial companies.

Example 2 Preparation of recombinant adeno-associated virus

The virus packaging method was referred to AAV13 Enables Precise Targeting of Local Neural Populations, Han et al. ; Int. J. Mol. Sci, 2022, 23(21), 12806; doi: 10.3390/ijms232112806. The adeno-associated virus was packaged using a three-plasmid packaging system. The core component plasmid pAAV-CMV-EGFP-WPRE-hGH polyA, the adenovirus component auxiliary plasmid pAd-Helper and the pAAV2/32.eB (or pAAV2/hu.32) serotype plasmid were co-transfected into HEK-293T cells at a ratio of 1:1:1. After 72 hours of transfection, the supernatant and cell pellet were collected, concentrated and purified by iodixanol gradient centrifugation, and finally purified by SYBR Green The titer of recombinant adeno-associated virus was detected by qPCR. The titer of rAAV32.eB-CMV-EGFP-WPRE-hGH polyA virus was 1.0×10 ¹³ VG/mL, and the titer of rAAVhu.32-CMV-EGFP-WPRE-hGH polyA virus was 1.0×10 ¹³ VG/mL.

Example 3 Application of recombinant adeno-associated virus

The prepared rAAV32.eB-CMV-EGFP-WPRE-hGH polyA and rAAVhu.32 -CMV-EGFP-WPRE-hGH polyA (as a control) viruses were injected into 8-10 week old C57BL/6 mice (purchased from a commercial company) by tail vein injection. The virus titer was 1.0×10 ¹³ VG/mL, and the injection dose for each mouse was 100 μL. Brain tissue was obtained by perfusion 3 weeks later. The mouse brain tissue was fixed with DEPC-treated PFA solution for 4 hours and then dehydrated with DEPC-treated 30% sucrose-PBS solution for 48 hours. The dehydrated mouse brain was fully embedded with tissue embedding agent and cut into 40 μm thick slices with a freezing microtome. The slices were mounted and microscopically imaged using a slide scanner. After C57BL/6 mice were injected with rAAV32.eB-CMV-EGFP-WPRE-hGH polyA and rAAhu.32-CMV-EGFP-WPRE-hGH polyA virus vectors through the tail vein, the imaging results of brain tissue sections are shown in Figure 2. It can be seen that in the whole brain area, the rAAV32.eB virus infection showed stronger fluorescence and more positive cells than the rAAVhu.32 virus.

In summary, the present invention provides a recombinant adeno-associated virus particle, a recombinant adeno-associated virus vector system and its application. The present invention prepares a new recombinant adeno-associated virus (rAAV) that efficiently crosses the blood-brain barrier, named rAAV32.eB, whose capsid is a mutant of the AAVhu.32 capsid protein. Compared with the wild-type AAVhu32 capsid protein VP1, there are three major position changes, including mutations at two sites, A587D and Q588G, and a 7-amino acid peptide TLAVPFK is inserted between the 588th and 589th amino acids. At the same time, the present invention also constructs a corresponding recombinant adeno-associated virus vector system and an AAV32.eB serotype plasmid. Not only that, the recombinant adeno-associated virus rAAV32.eB prepared by the present invention is infected by intravenous injection. Compared with rAAVhu.32, rAAV32.eB has a higher efficiency in transducing neural cells across the blood-brain barrier. Finally, the present invention obtained a new recombinant adeno-associated virus rAAV32.eB through engineering modification, and used it for more efficient gene delivery, which can provide better tools for neuroscience research and the treatment of whole-brain systemic genetic diseases, neurodegenerative diseases, etc., and has broad application value and market prospects.

It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims (10)

A recombinant adeno-associated virus particle, characterized in that it contains an AAVhu.32 capsid protein mutant; relative to the wild-type AAVhu.32 capsid protein, the AAVhu.32 capsid protein mutant includes mutations at two amino acid sites, 587 and 588, and an amino acid fragment as shown in SEQ. ID NO.1 is inserted between amino acids 588 and 589. The recombinant adeno-associated virus particle according to claim 1 is characterized in that the alanine at the 587th amino acid site of the AAVhu.32 capsid protein mutant is mutated to aspartic acid, and the glutamine at the 588th amino acid site is mutated to glycine. The recombinant adeno-associated virus particle according to claim 1 is characterized in that the genome of the recombinant adeno-associated virus particle contains an exogenous target gene with a nucleotide sequence encoding a target gene product. The recombinant adeno-associated virus particle according to claim 3, characterized in that the target gene product is a protein, a polypeptide or an interfering RNA. A recombinant adeno-associated virus vector system, characterized in that it comprises one or more vectors, wherein at least one vector is a recombinant adeno-associated virus packaging plasmid, and the recombinant adeno-associated virus packaging plasmid comprises a nucleic acid fragment for encoding the AAVhu.32 capsid protein mutant as described in claim 1. The recombinant adeno-associated virus vector system according to claim 5, characterized in that at least one vector is an adeno-associated virus core plasmid, and the adeno-associated virus core plasmid comprises inverted terminal repeat sequences ITR at both ends, a promoter, an exogenous target gene, a transcription regulatory element WPRE and a transcription termination sequence hGH polyA. The recombinant adeno-associated virus vector system according to claim 5 is characterized in that at least one vector is an adenovirus element helper plasmid. A pharmaceutical composition, characterized in that it comprises the recombinant adeno-associated virus particles described in any one of claims 1 to 4, and/or the recombinant adeno-associated virus vector system described in any one of claims 5 to 7. A use of the recombinant adeno-associated virus particle according to any one of claims 1 to 4 and/or the recombinant adeno-associated virus vector system according to any one of claims 5 to 7 in central nervous system gene delivery. A use of the recombinant adeno-associated virus particle according to any one of claims 1 to 4 and/or the recombinant adeno-associated virus vector system according to any one of claims 5 to 7 in the preparation of a drug for treating whole-brain systemic genetic diseases or neurodegenerative diseases.