CN110423778B - Application of retrograde recombinant type 2 adeno-associated virus in infection of spiral neurons - Google Patents
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Abstract
The invention provides an application of retrograde recombinant type 2 adeno-associated virus in infecting spiral neurons, which is characterized in that retrograde recombinant type 2 adeno-associated virus is injected into cochlea by cochlea round window injection, and the retrograde recombinant type 2 adeno-associated virus efficiently and specifically infects spiral neurons. The invention provides a new application of retrograde recombinant type 2 adeno-associated virus in infecting cochlear helical neurons, which has not been reported before, and can efficiently and specifically infect cochlear helical neurons, and has good application prospect in preparing medicines for treating genetic hearing loss and deafness-associated diseases, in particular auditory sensory neurons-helical neuron-associated diseases.
Description
Technical Field
The invention belongs to the field of recombinant adeno-associated viruses and application thereof, and in particular relates to application of retrograde recombinant adeno-associated virus type 2 in spiral neurons.
Background
The injection of genes that can be improved and corrected into our peripheral and sensory organs, spinal cord and brain to achieve long-term stable and effective therapeutic effects is a major concern in current genetic therapies. However, since physiological barrier systems exist from organ to organ or within organs, such as the most familiar Blood-Brain Barriers (BBBs); therefore, it is important to find a technique that can efficiently penetrate the barrier system. The current common technical approach is to go through multiple repeated drug injections into the brain tissue, retina and cochlear system. However, this disadvantage is high tissue damage and short efficacy duration (Hudry E, et al therapeutic AAV Gene Transfer to the Nervous System: A Clinical readiness.neuron.2019Mar6; 101 (5): 839-862.). It is therefore necessary and necessary to find a single, long-lasting innovative approach.
Currently, in neuroscience research, we often use Adeno-associated virus (Adeno-associated Viral Vector, AAV) for nerve projection and loop tracking. The first AAV for the treatment of hereditary retinal diseases (Inherited Retinal Disorders, IRDs) was approved by the FDA in 2018 in the United states for its safety, stability and high tissue compatibility (Strong Tropism). However, for genetic hearing loss and deafness-related diseases, in particular, deafness diseases caused by auditory sensory neurons, spiral neurons (Spiral Ganglion Neurons, SGNs), have been reported successively, and gene therapy for such deafness diseases requires efficient and specific expression of genes in spiral neurons. However, how to specifically infect such neurons needs to be addressed.
In order to improve and enhance the infection efficiency and specificity of AAVs, many technical strategies have achieved a super-efficient infection efficiency of AAVs; synthetic AAVs such as Anc80L65, AAV2.7m8 and AAV8BP2 were produced (Isgrig K, et al AAV2.7m8is a powerful viral vector for inner ear gene treatment. Nat Commun.2019Jan 25;10 (1): 427.). In the recently reported literature, anc80L65 injection through the cochlear round window can have very high infection efficiency for a variety of cell types in cochlear tissue (landeger LD, et al a synthetic AAV vector enables safe and efficient genetransfer to the mammalian Inner ear. Nat biotechnol.2017mar,35 (3): 280-284.), e.g., inner Hair Cells (IHCs), outer Hair Cells (Outer Hair Cells, OHCs), supporting Cells (Supporting Cells, SCs). But how to increase the specificity of AAVs has not been addressed. Until now, synthetic AAVs have been reported to stay in the stage of high-efficiency infection and low specificity.
Disclosure of Invention
The invention aims to provide an application of retrograde recombinant type 2 adeno-associated virus (rAAV 2-retro) in infecting spiral neurons.
In order to achieve the aim, the invention provides an application of retrograde recombinant type 2 adeno-associated virus in infecting spiral neurons.
Preferably, the retroactive recombinant type 2 adeno-associated virus is pAAV2-retro Helper, available from Addgene under the accession number 81070.
Preferably, the retrograde recombinant type 2 adeno-associated virus is a virus wherein 10 amino acid-containing short peptides are inserted into amino acids 587 and 588 of VP1 protein sequence of the viral Capsid protein Capsd of AAV2
LADQDYTKTA achieves a highly efficient retrograde nature (Tervo DG, et al A Designer AAVVariant Permits Efficient Retrograde Access to Projection neurons. Neuron.2016Oct19;92 (2): 372-382.).
The invention also provides a method for tracing and marking the retrograde recombinant type 2 adeno-associated virus in the cochlear helical neurons of mice.
The invention also provides application of the retrograde recombinant type 2 adeno-associated virus in preparing medicines for treating cochlear helical neuron-associated diseases.
The invention has the advantages that:
the invention provides a new application of retrograde recombinant type 2 adeno-associated virus (rAAV 2-retro) in infecting cochlear helical neurons, which has not been reported before, can efficiently and specifically infect cochlear helical neurons, and has good application prospect in preparing gene therapy for treating genetic hearing loss and deafness-related diseases, in particular to auditory sensory neurons-helical neurons (Spiral Ganglion Neurons, SGNs) -related diseases.
Drawings
FIG. 1 is a schematic diagram of retrograde recombinant AAVs (rAAV 2-retro) packaging, purification, and cochlear injection;
FIG. 2A is a cochlear anatomic view of a mouse;
FIG. 2B is a fluorescent staining pattern of rAAV2-retro versus conventional AAV2-GFP on the cochlea of mice.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it is understood that various changes and modifications may be made by those skilled in the art after reading the teachings of the present invention, and such equivalents are intended to fall within the scope of the claims appended hereto.
1. Experimental materials and methods
A. Obtaining plasmids:
AAV2 destination plasmids and packaging plasmids are all purchased directly from Addgene: pAAV-CAG-EGFP (adedge, 28014); pAAV 2-retrol Helper (Addgene, 81070); pHelper (Addgene, 112867);
the difference between the retrograde recombinant AAV2 and the antegrade AAV2 is that the Cap cDNA sequence of one of the packaging plasmids (pAAV 2-retro Helper) is subjected to Error-prone PCR (Error-clone PCR) genetic screening and modification; specifically, a short peptide comprising 10 amino acids was introduced into the Cap sequence of VP 1: LADQDYTKTA +v708i+n382D; a specific CAP cDNA sequence is shown in SEQ ID No.1, (Tervo DG, et al A Designer AAV Variant Permits Efficient Retrograde Access to Projection, neurons.2016 Oct19;92 (2): 372-382.).
B. Virus packaging and purification concentration:
eztrans cell transfection:
transfection was performed using 293T cells with cell densities of 80% -90%, and fresh DMEM cell culture medium containing 10% fbs was replaced half an hour prior to transfection;
taking a 10cm dish as an example, the DNA concentration ratio (dose ratio) of the three plasmid system was pAAV-CAG-EGFP: pHelper: pAAV2-retro helper=6. Mu.g: 18. Mu.g: 6. Mu.g. EZtranz (Life iLAB bio, C4058L 1082) at 400 μl/10cm dish (see description for specific transfection methods); mixing DNA with EZtranz, standing at normal temperature for 15 min, adding dropwise into a cell culture dish, replacing fresh DMEM cell culture medium containing 10% FBS after transfection for 8 hr, and culturing for 48 hr;
B.2. cell collection and lysis:
after 48 hours of transfection, the supernatant was discarded, 2.5ml of DMEM medium without FBS was added per 10cm dish and resuspended, 293T cells were collected by pipetting or scraping and placed into 50ml centrifuge tubes, a rapid freeze-thawing step was performed, specifically dry ice and absolute ethanol were mixed in a volume ratio of 1:1, dry ice particles were ground to powder if necessary to increase mixing with absolute ethanol, centrifuge tubes with cell suspensions were rapidly inserted into the mixture and kept for 3 minutes, then water bath was performed in 37℃water, the cell suspension to be coagulated was thoroughly liquefied, and the freeze-thawing step was repeated a further time. Then, DNase I enzyme (Sigma, D4263-1 VL) was added at 0.04mg/ml and RNas A enzyme (Roche, 10109169001) at 0.04mg/ml, followed by centrifugation at 3000rpm for 15 minutes at 37℃for subsequent purification and concentration of the supernatant (cell lysate).
B.3. Heparin affinity purification and protein concentration:
before purification, the purification column is filled, the bottom opening of the purification column (Sigma, C4669) with the diameter of 2.5cm is connected with a Luer Lock valve (Sigma, S7369), then 8ml of heparin agarose (Shanghai Kogyo/BBI, C600941-0025) is injected into the column, a filter membrane device (Sigma, S7271-1 EA) for purification is added on the agarose surface layer, a water outlet valve is opened, the agarose buffer solution naturally flows out, and then 25ml of PBS buffer solution is added for cleaning;
loading: adding the cell lysate obtained in the step B.2 into the filter membrane port (without overflowing) by using a 1ml pipetting gun, opening a valve to keep the flow rate at 1 second and 1 drop, passing 10 to 15ml of PBS containing 0.1M NaCl through the column after the lysate completely passes through the column, washing the column, eluting with 15 to 20ml of PBS containing 0.4M NaCl, and collecting the eluent; concentrating: 10ml of the eluate was added to a protein concentration tube (Millipore, UFC 910008) and centrifuged at 5,000g for 15 minutes; finally, the virus concentrate is resuspended by PBS, centrifuged for 15 minutes at 5,000g, and finally 200 mu l to 500 mu l of virus concentrate is obtained, and the virus concentrate is packaged and stored in a refrigerator at-80 ℃;
C. cochlear injection:
injecting the virus concentrate into cochlea of young mouse (first day P0, second day P1, third day P2) by using glass tube for microinjection in round window membrane mode;
the glass tube for injection (DRUMMOND, 5-000-1001-X10) was manufactured to have a tip diameter of about 25 μm by using a needle drawing machine (Sutter Instrument, P-2000); before operation, placing young mice on ice for 2-3 min for anesthesia, during operation for about 5-10 min, wiping the injection site with 70% ethanol for 3 times, shearing subcutaneous tissue at the injection site with Venus, tearing subcutaneous adipose tissue with surgical forceps to expose transparent cochlea bone, finding round window, penetrating the small opening with the tip of glass tube, slowly injecting about 0.5 μl virus, and slowly removing the injection needle after standing for 2 min. The wound was then immediately sutured, placed in a heated pad at 37 ℃ until it was revived, finally carefully placed in a female cage, sacrificed at 4-5 weeks of age, and cochlea was taken for histochemical staining.
FIG. 1 is a schematic diagram of retrograde recombinant AAVs (rAAV 2-retro) packaging, purification, and cochlear injection;
fig. 2A is a cochlear anatomic diagram of a mouse, comprising Inner Hair Cells (IHCs), outer Hair Cells (Outer Hair Cells, OHCs) and spiral neurons (Spiral Ganglion Neurons, SGNs);
FIG. 2B is a fluorescent staining pattern of rAAV2-retro versus conventional AAV2-GFP on the cochlea of mice;
rAAV2GFP retro carries a green fluorescent protein that can widely infect different segments through the cochlea round window membrane (upper panel): top-turn (Apex), middle, and bottom-turn (Base); whereas traditional AAV2-GFP only infects hair cells (bottom panel).
According to the invention, retrograde recombinant AAVs (rAAV 2-retros) are utilized, and the rAAV 2-retros carrying green fluorescent proteins can efficiently and specifically infect cochlear helical neurons SGNs through cochlear round window injection, and meanwhile, other cell types of cochlea are not fluorescent.
SEQUENCE LISTING
<110> Shanghai university of science and technology
<120> use of a retrograde recombinant type 2 adeno-associated virus for infecting helical neurons
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 2238
<212> DNA
<213> adeno-associated virus 2
<400> 1
atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga 60
cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120
gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180
aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctacgac 240
cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt 300
caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcggacgagc agtcttccag 360
gcgaaaaaga gggttcttga acctctgggc ctggttgagg aacctgttaa gacggctccg 420
ggaaaaaaga ggccggtaga gcactctcct gtggagccag actcctcctc gggaaccgga 480
aaggcgggcc agcagcctgc aagaaaaaga ttgaattttg gtcagactgg agacgcagac 540
tcagtacctg acccccagcc tctcggacag ccaccagcag ccccctctgg tctgggaact 600
aatacgatgg ctacaggcag tggcgcacca atggcagaca ataacgaggg cgccgacgga 660
gtgggtaatt cctcgggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720
accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caaacaaatt 780
tccagccaat caggagcctc gaacgacaat cactactttg gctacagcac cccttggggg 840
tattttgact tcaacagatt ccactgccac ttttcaccac gtgactggca aagactcatc 900
aacaacaact ggggattccg acccaagaga ctcaacttca agctctttaa cattcaagtc 960
aaagaggtca cgcagaatga cggtacgacg acgattgcca ataaccttac cagcacggtt 1020
caggtgttta ctgactcgga gtaccagctc ccgtacgtcc tcggctcggc gcatcaagga 1080
tgcctcccgc cgttcccagc agacgtcttc atggtgccac agtatggata cctcaccctg 1140
aacgacggga gtcaggcagt aggacgctct tcattttact gcctggagta ctttccttct 1200
cagatgctgc gtaccggaaa caactttacc ttcagctaca cttttgagga cgttcctttc 1260
cacagcagct acgctcacag ccagagtctg gaccgtctca tgaatcctct catcgaccag 1320
tacctgtatt acttgagcag aacaaacact ccaagtggaa ccaccacgca gtcaaggctt 1380
cagttttctc aggccggagc gagtgacatt cgggaccagt ctaggaactg gcttcctgga 1440
ccctgttacc gccagcagcg agtatcaaag acatctgcgg ataacaacaa cagtgaatac 1500
tcgtggactg gagctaccaa gtaccacctc aatggcagag actctctggt gaatccgggc 1560
ccggccatgg caagccacaa ggacgatgaa gaaaagtttt ttcctcagag cggggttctc 1620
atctttggga agcaaggctc agagaaaaca aatgtggaca ttgaaaaggt catgattaca 1680
gacgaagagg aaatcaggac aaccaatccc gtggctacgg agcagtatgg ttctgtatct 1740
accaacctcc agagaggcaa cctagcagac caagactaca caaaaactgc taggcaagca 1800
gctaccgcag atgtcaacac acaaggcgtt cttccaggca tggtctggca ggacagagat 1860
gtgtaccttc aggggcccat ctgggcaaag attccacaca cggacggaca ttttcacccc 1920
tctcccctca tgggtggatt cggacttaaa caccctcctc cccagattct catcaagaac 1980
accccggtac ctgcgaatcc ttcgaccacc ttcagtgcgg caaagtttgc ttccttcatc 2040
acacagtact ccacgggaca ggtcagcgtg gagatcgagt gggagctgca gaaggaaaac 2100
agcaaacgct ggaatcccga aattcagtac acttccaact acaacaagtc tattaatgtg 2160
gactttactg tggacactaa tggcgtgtat tcagagcctc gccccattgg caccagatac 2220
ctgactcgta atctgtaa 2238
Claims (3)
1. Use of a retrograde recombinant type 2 adeno-associated virus for infecting a spiral neuron, wherein the retrograde recombinant type 2 adeno-associated virus comprises a packaging plasmid pAAV2-retro Helper.
2. Use of a retrograde recombinant type 2 adeno-associated virus comprising the packaging plasmid pAAV2-retro Helper for tracer labelling in a cochlear helical neuron of a mouse.
3. Use of a retrograde recombinant type 2 adeno-associated virus comprising the packaging plasmid pAAV2-retro Helper in the manufacture of a medicament for the treatment of cochlear helical neuron-associated diseases.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103492574A (en) * | 2011-02-22 | 2014-01-01 | 加州理工学院 | Delivery of proteins using adeno-associated virus (aav) vectors |
| CA3126951A1 (en) * | 2015-07-30 | 2017-02-02 | Massachusetts Eye And Ear Infirmary | Ancestral virus sequences and uses thereof |
| CN109310745A (en) * | 2015-12-11 | 2019-02-05 | 马萨诸塞眼科耳科诊所 | For by the material and method of delivery of nucleic acids to cochlea and vestibular cell |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103492574A (en) * | 2011-02-22 | 2014-01-01 | 加州理工学院 | Delivery of proteins using adeno-associated virus (aav) vectors |
| CA3126951A1 (en) * | 2015-07-30 | 2017-02-02 | Massachusetts Eye And Ear Infirmary | Ancestral virus sequences and uses thereof |
| CN109310745A (en) * | 2015-12-11 | 2019-02-05 | 马萨诸塞眼科耳科诊所 | For by the material and method of delivery of nucleic acids to cochlea and vestibular cell |
Non-Patent Citations (2)
| Title |
|---|
| A designer AAV variant permits efficient retrograde access to projection neurons;D. Gowanlock R. Tervo et al.;《Neuron》;20161019;第92卷(第2期);第372-373页第3段 * |
| 经圆窗膜鼓阶显微注射腺相关病毒转染小鼠耳蜗的研究;陈嘉伟等;《听力学及言语疾病杂志》;20190621;第27卷(第3期);摘要 * |
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