CN115323002A - Application of gene delivery system in retrograde delivery of gene from brain to spinal cord neuron - Google Patents

Abstract

The invention belongs to the field of biotechnology, and provides application of a gene delivery system in retrograde gene delivery from a brain to spinal cord neurons. Use of a gene delivery system for delivering a gene from the brain to the spinal cord or for preparing an agent for delivering a gene from the brain to the spinal cord, wherein the gene delivery system comprises an adeno-associated viral vector rAAV11, and the adeno-associated viral vector rAAV11 comprises a gene encoding a marker protein and a promoter. rAAV11 vectors can efficiently deliver genes retrograde from the brain to spinal cord neurons compared to other vectors. Provides better tools and technical support for neural loop research, disease model establishment, gene therapy and the like related to the spinal cord, and has wide application value and market prospect.

Description

A gene delivery system in the retrograde delivery of genes from the brain to spinal cord neurons application

technical field

The invention belongs to the field of biotechnology, and in particular relates to the application of a gene delivery system in the retrograde delivery of genes from the brain to neurons of the spinal cord.

Background technique

The central nervous system (Central Nervous System) is composed of the brain and spinal cord, which are the central part of various reflex arcs and the main part of the nervous system. The central nervous system receives incoming information from all parts of the body, and after it is integrated and processed, it becomes a coordinated motor efferent, or is stored in the central nervous system to become the neural basis of learning and memory. Brain organs include the cerebrum, cerebellum, and brainstem. The brainstem is located below the cerebrum, between the spinal cord and the diencephalon. It is a smaller part of the central nervous system and has an irregular columnar shape. From bottom to top, the brainstem consists of the medulla, pons, and midbrain, and the medulla is connected to the spinal cord. The parabrachial nucleus is a nerve nucleus in the pons, located in the upper part of the pons, surrounded by the medial and lateral upper cerebellar peduncles, including the medial parabrachial nucleus (MPbN) and the lateral parabrachial nucleus (lateral parabrachial nucleus) , LPbN).

Spinal projection neurons transmit somatosensory information to multiple brain regions, including the thalamus, periaqueductal gray matter, nucleus of the solitary tract, and parabrachial nucleus (Al-Khater and Todd, 2009; Gauriau and Bernard, 2004; Todd, 2010) It is of great significance to understand the mechanisms of pain, itch, and spinal cord injury repair by using the method of virus tracing to analyze the relevant neural circuits. However, tool viral vectors capable of retrogradely transducing spinal cord neurons from the brain are currently lacking and inefficient.

Based on the above problems, the present invention provides the application of a gene delivery system in the retrograde delivery of genes from the brain to the spinal cord neurons to solve the problem of low efficiency of the tool virus vector for retrograde transduction of spinal cord neurons from the brain.

Contents of the invention

In order to solve the problem of low efficiency of viral vectors used for retrograde transduction of spinal cord neurons from the brain, the present invention provides an application of a gene delivery system in the retrograde delivery of genes from the brain to spinal cord neurons.

The application of the gene delivery system for delivering genes from the brain to the spinal cord, or the application in the preparation of reagents for delivering genes from the brain to the spinal cord, wherein the gene delivery system comprises adeno-associated virus vector rAAV11, and the adeno-associated The viral vector rAAV11 contains the gene and promoter encoding the marker protein.

Further, the marker protein in the gene encoding the marker protein is selected from fluorescent protein and/or enzyme reaction chromogenic protein, and the fluorescent protein is selected from BFP, CFP, GFP, YFP, RFP, iRFP, Cerulean, Venus, eGFP , eCFP, eYFP, eBFP, DsRed, dTomato, tdTomato, mCherry, mKate, mApple, mBanana, mCitrine, mOrange, mPlum, tagRFP and tagBFP, the enzyme reaction chromogenic protein is selected from HRP, Fireflyluciferase and one or more of Renilla luciferase;

The promoter is selected from CAG, CMV, hUbC, Ef1α, nEF, hSyn, CaMKIIα, Vgat, Thy1, TRE, UAS, GFAP, gfaABC1D, TH, RPE65, TRE, CBA, PGK, E-SARE, C-fos, RAM, SST, PV, mDlX, NPY, CR, TCAP, SFRP2, ChAT, TPH2, mTH, GAD67, GFAP104, CD68, Nestin, MBP, TRPV1, L7/Pcp2, mOXT, RK, hGRK1, CAR, Grm6, ROH, One or more of Nrl, MCK, dMCK and tMCK.

One object of the present invention is to provide a method for marking spinal cord neural networks. The marking method of the spinal cord neural network comprises the following steps:

S1: Injection of adeno-associated virus vector into the lateral parabrachial nucleus of mouse pons;

S2: Observing the distribution of marker proteins in the spinal cord,

Wherein, the adeno-associated virus vector is rAAV11, and the adeno-associated virus vector also includes a gene encoding a marker protein and a promoter.

Further, the marker protein in the gene encoding the marker protein is selected from fluorescent protein and/or enzyme reaction chromogenic protein, and the fluorescent protein is selected from BFP, CFP, GFP, YFP, RFP, iRFP, Cerulean, Venus, eGFP , eCFP, eYFP, eBFP, DsRed, dTomato, tdTomato, mCherry, mKate, mApple, mBanana, mCitrine, mOrange, mPlum, tagRFP and tagBFP, the enzyme reaction chromogenic protein is selected from HRP, Fireflyluciferase and one or more of Renilla luciferase;

The promoter is selected from CAG, CMV, hUbC, Ef1α, nEF, hSyn, CaMKIIα, Vgat, Thy1, TRE, UAS, GFAP, gfaABC1D, TH, RPE65, TRE, CBA, PGK, E-SARE, C-fos, RAM, SST, PV, mDlX, NPY, CR, TCAP, SFRP2, ChAT, TPH2, mTH, GAD67, GFAP104, CD68, Nestin, MBP, TRPV1, L7/Pcp2, mOXT, RK, hGRK1, CAR, Grm6, ROH, One or more of Nrl, MCK, dMCK and tMCK.

An object of the present invention is to provide an application of an adeno-associated virus vector to express a protein of interest or functional RNA in spinal cord neurons, or to prepare a reagent for expressing a protein of interest or functional RNA in spinal cord neurons, Wherein, the adeno-associated virus vector is rAAV11, and the adeno-associated virus vector further includes a gene and a promoter encoding a target protein or functional RNA.

Further, the target protein is selected from marker protein and/or active protein, and the active protein is selected from activating neuron protein, inhibiting neuron protein, calcium ion signal probe protein, small molecule signal probe protein, mediating apoptosis One or more of proteins, disease-associated mutant proteins, normal proteins under physiological conditions, cytokines, antiviral factors, viral infection co-receptors, recombinases and tool proteins, the functional RNA is selected from small RNA, One or more of small interfering RNA, small hairpin RNA, small guide RNA, organelle-localized RNA, and Barcode RNA for RNA sequencing or in situ hybridization analysis.

One object of the present invention is to provide a method for expressing target protein or functional RNA in spinal cord neurons. The method includes the following steps:

The adeno-associated virus vector was injected into the lateral parabrachial nucleus of the mouse pons,

Wherein, the adeno-associated virus vector is rAAV11, and the adeno-associated virus vector further includes a gene and a promoter encoding a target protein or functional RNA.

Further, the target protein is selected from marker protein and/or active protein, and the active protein is selected from activating neuron protein, inhibiting neuron protein, calcium ion signal probe protein, small molecule signal probe protein, mediating apoptosis one or more of apoptosis proteins, disease-associated mutant proteins, normal proteins under physiological conditions, cytokines, antiviral factors, viral infection co-receptors, recombinases and gene editing tool proteins, the functional RNA is selected from One or more of small RNA, small interfering RNA, small hairpin RNA, small guide RNA, organelle-localized RNA, and Barcode RNA for RNA sequencing or in situ hybridization analysis.

An object of the present invention is to provide an application of an adeno-associated virus vector in the preparation of a drug for treating diseases caused by abnormal neurons of the spinal cord.

The adeno-associated virus vector in this application is rAAV11, and the adeno-associated virus vector also contains genes and promoters encoding therapeutic proteins or functional RNAs.

Further, the target protein is selected from marker proteins and/or active proteins, and the active proteins are selected from the group consisting of activating neuronal proteins, inhibiting neuronal proteins, normal proteins under physiological conditions, cytokines, antiviral factors and gene editing tools One or more of proteins, the functional RNA is selected from one or more of small RNA, small interfering RNA, small hairpin RNA and small guide RNA.

The present invention discovers a new application of the adeno-associated virus rAAV11 vector. The rAAV11 vector can retrogradely deliver the target gene from the brain to express the target gene in the neurons of the spinal cord. Based on this, the rAAV11 vector can carry the target gene for marking and manipulating the spinal cord nervous system. Can be used as a carrier for gene therapy.

Compared with other vectors, the rAAV11 vector can efficiently deliver genes retrogradely from the brain to spinal cord neurons.

Description of drawings

Figure 1 shows the imaging results of the longitudinal section of the spinal cord of C57BL/6J mice injected with rAAV11-CAG-EGFP-WPRE-hGH polyA virus vector in the lateral lateral arm nucleus.

Figure 2 shows the results of cross-sectional imaging of the spinal cord of C57BL/6J mice after injection of rAAV11-CAG-EGFP-WPRE-hGH polyA virus vector into the lateral lateral arm nucleus.

Detailed ways

In order to make the above-mentioned purpose, features and advantages of the present invention more obvious and understandable, the specific implementation of the present invention will be described in detail below in conjunction with the accompanying drawings, but it should not be construed as limiting the scope of the present invention.

1. Preparation of recombinant adeno-associated virus

For the virus packaging method, refer to AAV11 permits efficient retrograde targeting of projection neurons, Han et al.bioRxiv 2022.01.13.476170; doi: https://doi.org/10.1101/2022.01.13.476170, using the three-plasmid packaging system to package the adeno-associated virus method, that is, using Plasmid pAAV-CAG-EGFP-WPRE-hGH polyA loaded with core elements, pAAV-RC2/11 serotype AAV capsid plasmid, and adenovirus element helper plasmid pAd-Helper were co-transfected into HEK- 293T cells, the supernatant and cell pellet were collected 72 hours after transfection, concentrated and purified by iodixanol gradient centrifugation, and finally the recombinant adeno-associated virus titer was detected by SYBR Green qPCR method, and rAAV11-CAG-EGFP- The titer of WPRE-hGH polyA virus was 5.0×10 ¹³ VG/mL.

2. In vivo application of recombinant adeno-associated virus

The prepared rAAV11-CAG-EGFP-WPRE-hGH polyA (200nL/mouse) virus was injected into the 8-10 week-old C57BL/6 mice (purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.) through brain stereotaxic injection. The brain region of the lateral parabrachial nucleus (LPbN) of the pons was perfused 3 weeks later, and the brain tissue of the mouse was fixed in PFA (paraformaldehyde) solution treated with DEPC (diethylpyrocarbonate) for 4 hours. Then dehydrated with DEPC-treated 30% sucrose-PBS solution for 48 hours, fully embedded the dehydrated brain tissue with tissue embedding agent and cut into 40 μm thick brain slices with a cryostat. Mount and image them using a slide scanner microscope. As can be seen from the in vivo detection results, the green fluorescent signal labeled with rAAV11-CAG-EGFP-WPRE-hGH polyA virus can be seen in the spinal cord neuron cell body, indicating that rAAV11-CAG-EGFP-WPRE-hGH polyA recombinant adeno-associated virus can be efficiently removed from the brain Retrograde transduction of spinal cord neurons to deliver the gene of interest to spinal cord neurons.

Claims (10)

1. Use of a gene delivery system for delivering a gene from the brain to the spinal cord or for preparing an agent for delivering a gene from the brain to the spinal cord, wherein the gene delivery system comprises an adeno-associated viral vector rAAV11, and the adeno-associated viral vector rAAV11 comprises a gene encoding a marker protein and a promoter.

2. The use according to claim 1,

the marker protein in the gene encoding the marker protein is selected from fluorescent protein selected from one or more of BFP, CFP, GFP, YFP, RFP, iRFP, cerulean, venus, eGFP, eFP, eYFP, eBFP, dsRed, dTomato, tdTomato, mCherry, mKate, mApple, mBanana, mCitrine, mOrange, mGlum, tagRFP and tagBFP and/or enzyme reaction chromogenic protein selected from one or more of HRP, firefly luciferase and Renilla luciferase;

the promoter is selected from one or more of CAG, CMV, hUbC, ef1 alpha, nEF, hSyn, caMKII alpha, vgat, thy1, TRE, UAS, GFAP, gfaABC1D, TH, RPE65, TRE, CBA, PGK, E-SARE, C-fos, RAM, SST, PV, mDlX, NPY, CR, TCAP, SFRP2, chAT, TPH2, mTH, GAD67, GFAP104, CD68, nestin, MBP, TRPV1, L7/Pcp2, mOXT, RK, hK 1, CAR, grm6, ROH, NGRrl, MCK, dMCK and tMCK.

3. A method for marking a spinal nerve network, comprising the steps of:

s1: injecting the adeno-associated virus vector into lateral brachial paranucleus of mouse pons;

s2: observing the distribution of the marker protein in the spinal cord,

wherein the adeno-associated virus vector is rAAV11, and the adeno-associated virus vector also comprises a gene and a promoter for coding a marker protein.

4. The marking method according to claim 3,

the marker protein in the gene encoding the marker protein is selected from fluorescent protein selected from one or more of BFP, CFP, GFP, YFP, RFP, iRFP, cerulean, venus, eGFP, eFP, eYFP, eBFP, dsRed, dTomato, tdTomato, mCherry, mKate, mApple, mBanana, mCitrine, mOrange, mGlum, tagRFP and tagBFP and/or enzyme reaction chromogenic protein selected from one or more of HRP, firefly luciferase and Renilla luciferase;

the promoter is selected from one or more of CAG, CMV, hUbC, ef1 alpha, nEF, hSyn, caMKII alpha, vgat, thy1, TRE, UAS, GFAP, gfaABC1D, TH, RPE65, TRE, CBA, PGK, E-SARE, C-fos, RAM, SST, PV, mDlX, NPY, CR, TCAP, SFRP2, chAT, TPH2, mTH, GAD67, GFAP104, CD68, nestin, MBP, TRPV1, L7/Pcp2, mOXT, RK, hGRK1, CAR, grm6, ROH, nrl, MCK, dMCK and tMCK.

5. Use of an adeno-associated viral vector for expressing a target protein or functional RNA in spinal cord neurons, or for preparing a reagent for expressing a target protein or functional RNA in spinal cord neurons, wherein the adeno-associated viral vector is rAAV11, and further comprises a gene encoding a target protein or functional RNA and a promoter.

6. The use according to claim 5, wherein the protein of interest is selected from the group consisting of a marker protein and/or an active protein selected from one or more of an activating neuronal protein, an inhibiting neuronal protein, a calcium signaling probe protein, a small molecule signaling probe protein, a mediating apoptotic protein, a disease-associated mutein, a normal protein under physiological conditions, a cytokine, an antiviral factor, a viral infection co-receptor, a recombinase and a tool protein, and the functional RNA is selected from one or more of a small RNA, a small interfering RNA, a small hairpin RNA, a small guide RNA, an organelle localization RNA and a Barcode RNA for RNA sequencing or in situ hybridization analysis.

7. A method for expressing a protein of interest or a functional RNA in spinal cord neurons, comprising the steps of:

injecting the adeno-associated virus vector into lateral arm paranucleus of mouse pons,

wherein the adeno-associated viral vector is rAAV11, and the adeno-associated viral vector also comprises a gene and a promoter for coding a target protein or functional RNA.

8. The method of claim 7, wherein the protein of interest is selected from a marker protein and/or an active protein selected from one or more of an activating neuronal protein, an inhibiting neuronal protein, a calcium signaling probe protein, a small molecule signaling probe protein, a mediating apoptotic protein, a disease-associated mutein, a normal protein under physiological conditions, a cytokine, an antiviral factor, a viral infection helper receptor, a recombinase, and a gene editing tool protein, and wherein the functional RNA is selected from one or more of a small RNA, a small interfering RNA, a small hairpin RNA, a small guide RNA, an organelle localization RNA, and a Barcode RNA for RNA sequencing or in situ hybridization analysis.

9. An application of an adeno-associated virus vector in preparing a medicament for treating diseases caused by abnormal spinal cord neurons,

the adeno-associated virus vector is rAAV11, and the adeno-associated virus vector also comprises a gene and a promoter for coding therapeutic protein or functional RNA.

10. The use as claimed in claim 9, wherein the protein of interest is selected from a marker protein and/or an active protein selected from one or more of an activating neuronal protein, a suppressing neuronal protein, a normal protein under physiological conditions, a cytokine, an antiviral factor and a gene editing tool protein, and the functional RNA is selected from one or more of a small RNA, a small interfering RNA, a small hairpin RNA and a small guide RNA.

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