CN-120118956-B - Packaging auxiliary plasmid for high-yield AAV and application

Abstract

The invention belongs to the technical field of genetic engineering, and discloses a packaging auxiliary plasmid for high-yield AAV and application thereof. The packaging auxiliary plasmid for high-yield AAV has a framework plasmid inserted with a recombination sequence, wherein the recombination sequence sequentially comprises a Rep expression cassette, a Cap expression cassette, an E2A sequence of adenovirus, an E4 truncated sequence of adenovirus, a VA RNA sequence of adenovirus and at least one DA ' sequence from 5' to 3 '. Under the condition of adding DA' sequence, the packaging auxiliary plasmid can promote AAV yield of single cell, and provides a new direction for AAV production.

Inventors

• LI HUAPENG
• XIAO DANQING
• BU YE
• CHEN JUNLIN

Assignees

• 广州派真生物技术有限公司

Dates

Publication Date

20260512

Application Date

20250305

Claims (7)

1. 1. The plasmid group for high-yield AAV by two-plasmid transfection is characterized by comprising an AAV plasmid and a packaging auxiliary plasmid, wherein a recombination sequence is inserted into a skeleton plasmid of the packaging auxiliary plasmid, and the recombination sequence sequentially comprises any one of the following from 5 'to 3': i. Rep protein coding sequence, CMV-P40 promoter sequence, intron sequence, cap protein coding sequence, E2A sequence of adenovirus, E4 truncated sequence of adenovirus, VA RNA sequence, DA' sequence and P5 promoter sequence, or Rep truncated sequences, CMV-P40 promoter sequences, intron sequences, cap protein coding sequences, adenovirus E2A sequences, adenovirus E4 truncated sequences, VA RNA sequences, DA' sequences, and P5 promoter sequences; The Rep truncated sequence is shown as SEQ ID No.7, the E4 truncated sequence of the adenovirus is shown as SEQ ID No.10, and the Intron sequence is shown as SEQ ID No. 4; The P5 promoter sequence is shown as SEQ ID No.2, the CMV-P40 promoter sequence is shown as SEQ ID No.3, the DA' sequence is shown as SEQ ID No.5, the Rep protein coding sequence is shown as SEQ ID No.6, the Cap protein coding sequence is shown as SEQ ID No.8, the E2A sequence of adenovirus is shown as SEQ ID No.9, and the VA RNA sequence is shown as SEQ ID No. 11.
2. 2. A reagent for transfecting high-yielding AAV with two plasmids, comprising the plasmid set of high-yielding AAV according to claim 1.
3. 3. A cell transfected with two plasmids for high AAV production, comprising the plasmid set of claim 1.
4. 4. A cell transfected with two plasmids for high AAV production according to claim 3, wherein said cell comprises at least one of HeLa, HEK293, insect Sf9 cells.
5. 5. A kit for the transfection of two plasmids into high-yield AAV comprising the agent of claim 2 or the cell of claim 3 or 4.
6. 6. A method for transfecting a high-yield AAV with two plasmids, wherein the plasmid set according to claim 1 is transformed into a host cell and the host cell is cultured, or the cell according to claim 3 or 4 is cultured.
7. 7. Use of the plasmid set of claim 1, the agent of claim 2, the cell of claim 3 or 4, the kit of claim 5 in the production of recombinant adeno-associated virus.

Description

Packaging auxiliary plasmid for high-yield AAV and application Technical Field The invention relates to the technical field of genetic engineering, in particular to a packaging auxiliary plasmid for high-yield AAV and application thereof. Background Adeno-associated virus (AAV) has many advantages, making it a favored gene therapy delivery vehicle. First, replication of AAV requires helper virus involvement and is therefore considered to be pathogen-free. Secondly, after AAV enters cells, the genome wrapped by the ITR structure can be cyclized to form an episome structure, so that the probability of recombination of AAV and the genome is reduced, and further, the genome toxicity is reduced. Furthermore, AAV has 12 different serotypes and hundreds of variants derived therefrom, greatly expanding its range of applications. RAAV-based vectors can target dividing cells and non-dividing cells, including retina, liver, heart, muscle, and Central Nervous System (CNS), to achieve long-term expression of therapeutic genes. Up to now, more than 200 clinical trials have been conducted for the treatment of various diseases such as hemophilia a and B, parkinson's disease, wet age-related macular degeneration, mucopolysaccharidoses (MPS), barton's disease, etc. Despite the success of rAAV-based gene therapy in clinical trials, high manufacturing costs remain a bottleneck in the field of gene therapy due to low yields and high dose requirements for certain disease indications, thereby limiting patient access to these promising products. This in turn has increased the need to reduce AAV production costs, establish flexible and GMP-level compliant AAV production systems. Thus, the yield bottleneck of AAV is urgently broken. Improvement and innovation in production upstream, and improvement of AAV yield are one of key points for reducing the production cost of gene therapy at present. Plasmid transient transfection of Human Embryonic Kidney (HEK) 293 cells is a common strategy for preparing adeno-associated virus (AAV) vectors, and has the main advantages of short production period, easy operation, strong flexibility and the like. The three-plasmid transfection method is the most commonly used rAAV production method at present, and uses three plasmids to jointly transfect HEK293 cells, namely a helper plasmid PADHELPER for providing adenovirus E2A, E and VA RNA elements, a packaging plasmid pRC for providing the Rep of AAV and Cap protein coding sequences, wherein the Rep protein is responsible for replication of AAV genome and assisting assembly of AAV genome particles, the Cap protein forms an AAV shell, and a plasmid comprising a target sequence, which can be abbreviated as pGOI, is provided with a 5'ITR sequence and a 3' ITR sequence in a natural AAV genome respectively at the upstream and downstream of the target sequence. Grimm D and Tang Q et al combine the packaging plasmid and helper plasmid of the three plasmids on one plasmid, i.e., combine the AAV rep/cap and Ad helper genes in a single helper plasmid pDG, and AAV production can be performed with only two plasmids. The AAV preparation is carried out by using two plasmids to replace three plasmids, so that the transfection step is simplified, the number of components required by AAV production is reduced, and the cost of plasmid production is reduced, thereby saving a great deal of cost in the GMP production of rAAV. Disclosure of Invention The invention aims to overcome the defects of the prior art and provide a packaging auxiliary plasmid for high-yield AAV and application thereof. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: in a first aspect, the present invention provides a packaging helper plasmid for high-yield AAV, wherein a recombinant sequence is inserted into a backbone plasmid, and the recombinant sequence comprises, in order from 5' to 3', a Rep expression cassette, a Cap expression cassette, an E2A sequence of adenovirus, an E4 truncated sequence of adenovirus, a VA RNA sequence of adenovirus, and at least one DA ' sequence. As a preferred embodiment of the packaging helper plasmid for high AAV production according to the present invention, the recombinant sequence comprises any one of the following sequences in order from 5 'to 3': Rep protein coding sequence, cap protein coding sequence, adenovirus E2A sequence, adenovirus E4 truncated sequence, adenovirus VA RNA sequence, DA' sequence and promoter sequence; a Rep protein coding sequence, a first promoter sequence, an Intron sequence, a Cap protein coding sequence, an E2A sequence of adenovirus, an E4 truncated sequence of adenovirus, a VA RNA sequence, a DA' sequence, a second promoter sequence; Rep truncated sequence, first promoter sequence, intron sequence, cap protein coding sequence, E2A sequence of adenovirus, E4 truncated sequence of adenovirus, VA RNA sequence, DA' sequence, and second promoter sequence. More preferably, the promoter c