WO2023102736A1 - Recombinant adeno-associated virus packaging plasmid, recombinant adeno-associated virus and application thereof - Google Patents

Abstract

Provided are a recombinant adeno-associated virus packaging plasmid comprising a Rep gene of a type 2 adeno-associated virus and a Cap gene of a type 11 adeno-associated virus, a recombinant adeno-associated virus and an application thereof. The recombinant adeno-associated virus is obtained by co-transfection of a packaging cell line by the recombinant adeno-associated virus packaging plasmid, an adeno-associated virus core plasmid, and an adenovirus element helper plasmid, has the characteristics of marking a specific brain region and an upstream connection network thereof, and is higher in retrograde tracing efficiency. The present invention solves the problem of low efficiency of an existing retrograde tracing method, provides better tools and technical supports for neuroscience research, disease model establishment, gene therapy, etc., and has wide application value and market prospect.

Description

A kind of recombinant adeno-associated virus packaging plasmid, recombinant adeno-associated virus and application thereof technical field

The invention belongs to the field of biotechnology, and specifically relates to a recombinant adeno-associated virus packaging plasmid, a recombinant adeno-associated virus and applications thereof.

Background technique

Viral vectors, especially those that allow efficient gene transfer from axon terminals to the central nervous system (CNS), are useful for dissecting the structure and function of neural circuits in specific brain regions, and for delivering piggybacked therapeutic genes to distant sites. It has become one of the most potential and promising therapeutic tools. In contrast to traditional retrograde tracers, viral vectors can express genes for manipulation of neuronal activity or gene therapy. Some neurotropic viruses have retrograde infection ability, such as herpes simplex virus (HSV), pseudorabies virus (PRV), lentivirus (LV), canine adenovirus (CAV), rabies virus (RABV), etc., but the toxicity is high or the preparation process Complicated, not suitable for gene therapy. Recombinant adeno-associated viruses (rAAVs) have important properties such as low toxicity, high-level transgene expression, and minimal host immune response, and have been widely used in neural circuit regulation and gene therapy research. Treatment of many genetically deficient brain diseases requires the delivery of the correct gene to each cell throughout the brain, such as mucopolysaccharidosis, and thus the development of more efficient recombinant adeno-associated viruses capable of labeling multiple brain regions with more efficient retrograde labeling becomes Particularly important. The reported recombinant adeno-associated virus rAAV2/retro has higher retrograde labeling efficiency than other adeno-associated viruses (Neuron,2016,92(2):372-382.), but there is brain region selectivity (Neurosci Bull, 2020,36(3):202-216.), mainly infects cortical neurons, and its retrograde labeling efficiency needs to be further improved.

Contents of the invention

In order to solve the deficiencies in the prior art, the object of the present invention is to provide a recombinant adeno-associated virus packaging plasmid, a recombinant adeno-associated virus and applications thereof.

The first aspect of the present invention provides a recombinant adeno-associated virus packaging plasmid, which includes the Rep gene of type 2 adeno-associated virus and the Cap gene of type 11 adeno-associated virus.

Further, the recombinant adeno-associated virus packaging plasmid uses pAAV-RC2/1 as the backbone.

Further, the nucleotide sequence of the recombinant adeno-associated virus packaging plasmid is shown in SEQ ID NO: 3.

The second aspect of the present invention provides a recombinant adeno-associated virus vector system, which includes the recombinant adeno-associated virus packaging plasmid, adeno-associated virus core plasmid and adeno-associated virus element helper plasmid.

Further, the adeno-associated virus core plasmid comprises two inverted terminal repeat sequences ITR, a promoter, a foreign gene, a transcriptional regulatory element WPRE and a transcriptional termination sequence hGH polyA;

Preferably, the exogenous gene comprises a reporter gene;

Preferably, the reporter gene is EGFP;

Preferably, the adeno-associated virus core plasmid is pAAV-EF1α-EGFP-WPRE-hGH polyA;

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

The third aspect of the present invention provides a recombinant adeno-associated virus, which is 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.

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

Preferably, the number of molecules of the recombinant adeno-associated virus packaging plasmid, adeno-associated virus core plasmid and adenovirus element helper plasmid is 1:1:1.

Further, the adeno-associated virus core plasmid comprises two inverted terminal repeat sequences ITR, a promoter, a foreign gene, a transcriptional regulatory element WPRE and a transcriptional termination sequence hGH polyA;

Preferably, the exogenous gene comprises a reporter gene;

Preferably, the reporter gene is EGFP;

Preferably, the adeno-associated virus core plasmid is pAAV-EF1α-EGFP-WPRE-hGH polyA;

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

The fourth aspect of the present invention provides the recombinant adeno-associated virus packaging plasmid, the recombinant adeno-associated virus vector system or the application of the recombinant adeno-associated virus in neuron retrograde labeling.

The fifth aspect of the present invention provides the application of the recombinant adeno-associated virus as a nerve circuit tracer virus.

The beneficial effects of the present invention are:

The recombinant adeno-associated virus rAAV2/11 prepared by the present invention has the characteristics of marking specific brain regions and their upstream connection networks, and has higher retrograde labeling efficiency, which solves the problem of low efficiency of existing retrograde labeling methods. Model building and gene therapy provide better tools and technical support, and have a wide range of application value and market prospects.

Description of drawings

Figure 1 is a map of the expression vector of the recombinant adeno-associated virus packaging plasmid pAAV-RC2/11.

Figure 2 is the signal of rAAV2/11 infected injection site (caudoputum) cells.

Figure 3 shows the signal of rAAV2/11 retrogradely infected the cortex of the upstream brain region.

Figure 4 shows the signal of rAAV2/11 retrograde infection in the thalamus, the upstream brain region.

Figure 5 shows the signal of rAAV2/11 retrogradely infected the amygdala in the upstream brain region.

Figure 6 shows the signal in the ventral hippocampal region of the injection site of rAAV2/11 infection.

Figure 7 shows the signal in the ventral hippocampal region of the injection site of rAAV2/retro infection.

Figure 8 shows the signals in the dorsal hippocampal CA3 region of the upstream brain region marked by rAAV2/11 retrogradely.

Figure 9 shows the signals in the dorsal hippocampal CA3 region of the upstream brain region marked by rAAV2/retro retrogradely.

Figure 10 shows the signals of the interseptal nucleus complex in upstream brain regions marked by rAAV2/11 retrogradely.

Figure 11 shows the signals of the interseptal nucleus complex in upstream brain regions marked by rAAV2/retro retrogradely.

Figure 12 shows the signals of the dorsal lateral nucleus of the thalamus in the upstream brain region marked retrogradely by rAAV2/11.

Figure 13 shows the signals of the dorsal lateral nucleus of the thalamus in the upstream brain region marked by rAAV2/retro retrogradely.

Figure 14 shows the signal of the thalamic junctional tuberculosis in the upstream brain region marked by rAAV2/11 retrogradely.

Figure 15 shows the signals of the thalamic junctional tuberculosis in the upstream brain region marked by rAAV2/retro retrogradely.

Detailed ways

In order to understand the present invention more clearly, the present invention will now be further described with reference to the following examples and accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the examples, each original reagent material can be obtained commercially, and the experimental methods without specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions suggested by the instrument manufacturer.

Example 1: Construction of recombinant adeno-associated virus packaging plasmid

According to the AAV11 genome sequence (GenBank: AY631966.1), the Cap11 gene sequence was synthesized, and as a template, the Cap11 fragment was amplified using Takara Primerstar Polymerase (Takara Company), and the sequence of the forward primer Cap11-F was shown in SEQ ID NO.1, The sequence of the reverse primer Cap11-R is shown in SEQ ID NO.2; the PCR reaction system is 50μl: 5×Reaction Buffer 10μl, 10mM dNTPs 1μl, 10μM forward primer 2.5μl, 10μM reverse primer 2.5μl, template DNA 0.5 μl, DNA Polymerase 0.5 μl, ddH ₂ O 33 μl. Amplification conditions were: 98°C for 3 min, 98°C for 20 s, 60°C for 20 s, 72°C for 2 min, 72°C for 10 min, 16°C for 30 min, 30 cycles; the amplified DNA fragment was recovered with a gel recovery kit (Omega).

The pAAV-RC2/1 vector (purchased from Addgene, number: 112862) and the Cap11 fragment were respectively digested with SwaI and AgeI (New England Biolabs) restriction enzymes, and then the Cap11 fragment was inserted into pAAV-RC2/ 1. Transform the ligation product into competent Stbl3, and inoculate the clones identified as positive by colony PCR into 5 ml of LB liquid medium for culture and extract the plasmid for sequencing. The clone with correct sequencing is named pAAV-RC2/11, and the obtained plasmid can be Encodes AAV11 capsid protein VP1. The map of the constructed recombinant adeno-associated virus packaging plasmid pAAV-RC2/11 expression vector is shown in Figure 1, and its gene sequence is shown in SEQ ID NO.3. In Figure 1, Rep2 represents the Rep gene of type 2 adeno-associated virus, and Cap11 represents the Cap gene of type 11 adeno-associated virus. All PCR primers, gene synthesis and sequencing of the present invention were completed by Sangon Bioengineering (Shanghai) Co., Ltd.

Example 2: Preparation of recombinant adeno-associated virus

The recombinant adeno-associated virus packaging plasmid pAAV-RC2/11 expression vector was used to package the recombinant adeno-associated virus, and the adeno-associated virus core plasmid pAAV-EF1α-EGFP-WPRE-hGH polyA was combined with pAAV-RC2/11 The viral element helper plasmid pAd-Helper (purchased from Addgene, number: 112867) was co-transfected into HEK-293T cells according to the number of plasmid molecules 1:1:1. After 72 hours of transfection, the supernatant and cell pellet were collected and processed separately: containing AAV The HEK-293T cell pellet of virus particles was resuspended in the lysate (15 dishes plus 9 mL), and after repeated freezing and thawing in liquid nitrogen and 37°C water bath, added Benzonase nuclease (Sigma, E1014-25KU) to digest at 37°C for 1 hour. Then add a final concentration of 150mM NaCl and shake it at 37°C for 30min; after the cell supernatant is treated with nuclease, add PEG8000 (Solebo, 214B0310) and 0.5mol/L NaCl solution and mix well, then place it at 4°C for 16h to aggregate the protein . The next day, the treated supernatant was centrifuged at 10,000 g at 4°C to discard the supernatant, and then the cell sample and the supernatant sediment sample were combined, centrifuged at 3,000 g for 10 min, and the cell debris was discarded to obtain a concentrated virus solution. Prepare a density gradient of 15%, 25%, 40%, and 58% iodixanol solution in a Beckman ultracentrifuge tube, then add 10 mL of virus concentrate, and finally fill up the centrifuge tube with PBS and seal it. After balancing, put it into a Ti70 rotor and centrifuge at 64000rpm at 18°C for 2 hours in a Beckman ultracentrifuge. After the centrifugation, draw with a syringe to recover the solution of the 40% and 58% iodixanol separation layer. Put the rAAV-containing iodixanol layer solution into a dialysis bag, and dialyze in PBS buffer at 4°C for 16 hours; the dialyzed virus solution is filtered and sterilized with a 0.22 μm filter membrane, and then added to the ultrafiltration tube (Minipore, UFC910024), desalted and concentrated to a volume of about 200 μL, and then added Pluronic F68 (Thermo Fisher Scientific, 24040032) with a final concentration of 0.001%, aliquoted and stored in a -80°C refrigerator. Finally, the titer of recombinant adeno-associated virus was detected by SYBR Green qPCR method, and the titer of rAAV2/11-EF1α-EGFP-WPRE-hGH polyA virus was finally obtained as 1.0×10 ¹³ VG/mL.

Example 3: In vivo application of recombinant adeno-associated virus

(1) The prepared rAAV2/11-EF1α-EGFP-WPRE-hGH polyA (300nL/mouse) virus (rAAV2/11) was injected into 8-10 week old C57BL/6 mice (purchased from Hunan The caudoputamen (Caudoputamen, CPu) region of Slack Jingda Experimental Animal Co., Ltd., after 3 weeks of sufficient expression, injected 0.5ml 1% pentobarbital sodium-0.9% sodium chloride solution in the mouse intraperitoneal cavity Anesthesia was performed, and brain tissue was stripped by cardiac perfusion with phosphate buffered saline (Phosphate Buffer Saline, PBS) and 4% paraformaldehyde solution (Paraformaldehyde Solution, PFA). Mouse brain tissue was fixed with PFA solution overnight and then dehydrated with 30% sucrose-PBS solution. The dehydrated brain tissue was fully embedded with tissue embedding medium and cut into 40 μm thick brain slices with a cryostat. Olympus It was imaged with a VS120 slide scanning microscope (Olympus, Japan). In vivo assay results showed that rAAV2/11-EF1α-EGFP-WPRE-hGH polyA could infect cells at the injection site (caudoputum) (Figure 2), and efficiently retrogradely infect neurons in upstream brain regions, including the cortex (Figure 3), Thalamus (Figure 4) and amygdala (Figure 5), etc.

(2) Mix rAAV2/11-EF1α-EGFP-WPRE-hGH polyA virus (rAAV2/11) with the same titer of rAAV2/retro-EF1α-mCherry-WPRE-hGH polyA virus (rAAV2/retro) in equal volumes ( A total of 300 nL) was injected into the ventral hippocampus of 8-10-week-old C57BL/6 mice (purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.), and the brain was perfused and imaged 3 weeks later. In vivo detection results showed that rAAV2/11 retrogradely transduced more neurons in the upstream brain region than rAAV2/retro, indicating that rAAV2/11 retrograde labeling efficiency was significantly higher than rAAV2/retro. Figure 6 shows the signal in the ventral hippocampus at the injection site of rAAV2/11 infection; Figure 7 shows the signal in the ventral hippocampus at the injection site of rAAV2/retro infection; Figure 8 shows the dorsal hippocampus CA3 in the upstream brain region of rAAV2/11 retrogradely labeled Regional signal; Figure 9 is the signal of the dorsal hippocampus CA3 region of the upstream brain region retrogradely labeled by rAAV2/retro; Figure 10 is the signal of the middle compartment nucleus complex in the upstream brain region retrogradely labeled by rAAV2/11; Figure 11 is the signal of the retrogradely labeled rAAV2/retro The signal of the interseptal nucleus complex in the upstream brain region; Figure 12 is the signal of the dorsal lateral nucleus of the upstream brain region marked by rAAV2/11 retrogradely; Figure 13 is the signal of the dorsal lateral nucleus of the upstream brain region marked by rAAV2/retro retrogradely; Figure 14 is the signal of the dorsal lateral nucleus of the upstream brain region marked by rAAV2/retro /11 retrogradely labeled thalamic tuberculosis signal in the upstream brain region; Figure 15 shows rAAV2/retro retrogradely labeled thalamic tuberculosis signal in the upstream brain area.

Apparently, the above-mentioned embodiments are only examples for clear description, rather than limiting the implementation. For those of ordinary skill in the art, other changes or changes in different forms can be made on the basis of the above description. It is not necessary and impossible to exhaustively list all the implementation manners here. And the obvious changes or changes derived therefrom are still within the scope of protection of the present invention.

The nucleotide sequence of the present invention is as follows:

SEQ ID NO: 1

SEQ ID NO: 2

SEQ ID NO: 3

Claims (10)

1. A recombinant adeno-associated virus packaging plasmid, characterized in that the recombinant adeno-associated virus packaging plasmid includes the Rep gene of type 2 adeno-associated virus and the Cap gene of type 11 adeno-associated virus.
2. The recombinant adeno-associated virus packaging plasmid according to claim 1, wherein the recombinant adeno-associated virus packaging plasmid uses pAAV-RC2/1 as the backbone.
3. The recombinant adeno-associated virus packaging plasmid according to claim 1, wherein the nucleotide sequence of the recombinant adeno-associated virus packaging plasmid is as shown in SEQ ID NO:3.
4. A recombinant adeno-associated virus vector system, characterized in that it comprises the recombinant adeno-associated virus packaging plasmid according to claim 1, the adeno-associated virus core plasmid and the adeno-associated virus element auxiliary plasmid.
5. The recombinant adeno-associated virus vector system according to claim 4, wherein the adeno-associated virus core plasmid comprises an inverted terminal repeat sequence ITR at both ends, a promoter, an exogenous gene, a transcriptional regulatory element WPRE and a transcriptional termination sequence hGH polyA;

   Preferably, the exogenous gene comprises a reporter gene;

   Preferably, the reporter gene is EGFP;

   Preferably, the adeno-associated virus core plasmid is pAAV-EF1α-EGFP-WPRE-hGH polyA;

   Preferably, the adenovirus element helper plasmid is pAd-Helper.
6. A recombinant adeno-associated virus, characterized in that it is obtained by co-transfecting a packaging cell line with the recombinant adeno-associated virus packaging plasmid, the adeno-associated virus core plasmid and the adeno-associated virus element helper plasmid according to claim 1.
7. The recombinant adeno-associated virus according to claim 6, wherein the packaging cell line is selected from HEK-293 cells, HEK-293T cells or HEK-293FT cells;

   Preferably, the number of molecules of the recombinant adeno-associated virus packaging plasmid, adeno-associated virus core plasmid and adenovirus element helper plasmid according to claim 1 is 1:1:1.
8. The recombinant adeno-associated virus according to claim 6, wherein the adeno-associated virus core plasmid comprises two inverted terminal repeat sequences ITR, a promoter, a foreign gene, a transcriptional regulatory element WPRE and a transcriptional termination sequence hGH polyA ;

   Preferably, the exogenous gene comprises a reporter gene;

   Preferably, the reporter gene is EGFP;

   Preferably, the adeno-associated virus core plasmid is pAAV-EF1α-EGFP-WPRE-hGH polyA;

   Preferably, the adenovirus element helper plasmid is pAd-Helper.
9. The recombinant adeno-associated virus packaging plasmid described in any one of claims 1-3, the recombinant adeno-associated virus vector system described in claim 4 or 5, or the recombinant adeno-associated virus described in any one of claims 6-8 in the nerve Application of meta retrograde markers.
10. The use of the recombinant adeno-associated virus according to any one of claims 6-8 as a nerve circuit tracer virus.

Images