WO2020155655A1 - 具有变异衣壳蛋白的腺相关病毒及其用途 - Google Patents

具有变异衣壳蛋白的腺相关病毒及其用途 Download PDF

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WO2020155655A1
WO2020155655A1 PCT/CN2019/107474 CN2019107474W WO2020155655A1 WO 2020155655 A1 WO2020155655 A1 WO 2020155655A1 CN 2019107474 W CN2019107474 W CN 2019107474W WO 2020155655 A1 WO2020155655 A1 WO 2020155655A1
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aav
associated virus
adeno
variant
capsid protein
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谈方志
钟桂生
储岑凤
齐洁玉
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ShanghaiTech University
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Definitions

  • the present invention relates to the field of biotechnology, in particular to an adeno-associated virus with a variant capsid protein and its use.
  • Hearing loss is one of the most common sensory disabilities, affecting more than 6.8% of the world's population (about 500 million people).
  • the most common treatment for hearing loss is to use hearing devices, which can amplify sound, enhance sound transmission, or directly stimulate neurons. This method is currently the best choice for the treatment of hearing loss, but unfortunately, hearing sensitivity and perception of natural sounds still have limitations in a noisy environment. Therefore, better alternative strategies are needed to treat hearing loss.
  • gene therapy has become a promising strategy for the treatment of genetic diseases.
  • Hereditary hearing loss is a typical monogenic disease. A single mutation can cause hearing loss. Therefore, gene therapy is an ideal and promising potential treatment strategy that can maintain or rebuild hearing function.
  • hereditary hearing loss most deafness genes are expressed in hair cells, and mutations in some genes may affect spiral neurons. A large number of gene mutations directly affect various functions of hair cells.
  • GJB2 key deafness genes
  • GJB2 GJB2 mutations that can cause genetic hearing loss. Mutations in GJB2 are the most common cause of human hereditary hearing loss, accounting for about 50% of autosomal recessive hearing loss and 15%-18% of all hereditary hearing loss. Therefore, gene therapy for hereditary hearing loss needs to target both hair cells and supporting cells.
  • AAV is a non-enveloped icosahedral virus with a diameter of 18-26nm.
  • the capsid structure is composed of three capsid proteins (VP1, VP2 and VP3).
  • the capsid contains a 4.7 kb single-stranded DNA (ssDNA), which carries two genes rep and cap.
  • the DNA is flanked by palindromic inverted terminal repeats (ITRs). Both rep and cap have multiple open reading frames (ORFs), which express proteins required for genome replication and packaging.
  • the ITRs sequence can be directly applied to ordinary plasmids, foreign DNA can be directly inserted between ITRs to form a recombinant plasmid, the AAV virus gene is cloned into the second plasmid, and a new recombinant AAV can be packaged under the help of the plasmid Helper. virus.
  • AAVs include AAV-1, AAV-2, AAV-5, AAV-8, AAV-9 and AAV-DJ.
  • AAV-mediated gene therapy has progressed rapidly in the past decade, and more than 170 human trials (7.2%) have used AAV vectors.
  • AAV vectors have excellent safety, low immunogenicity and high transfection efficiency.
  • the US FDA approved the first AAV-mediated gene therapy in 2017 for the treatment of rare hereditary eye diseases.
  • cochlear gene therapy based on AAV vectors. This may be due to the lack of AAV with higher conduction efficiency in different cell types in the cochlea.
  • Several studies have tested AAV-mediated cochlear gene therapy in animal models and have shown promising results.
  • the purpose of the present invention is to provide an adeno-associated virus with a variant capsid protein and its use to solve the problem of low infection rate of AAV on hair cells and supporting cells in the prior art. problem.
  • the constructed variant AAV-ie has stronger hair cells And the infectivity of supporting cells, the applicant proposed the invention.
  • one aspect of the present invention provides a variant adeno-associated virus AAV-ie capsid protein VP1.
  • the variant adeno-associated virus AAV-ie capsid protein VP1 is compared with wild-type AAV -DJ capsid protein VP1, insert an amino acid fragment between N589 and R590, and the amino acid sequence of the amino acid fragment is shown in SEQ ID NO.1.
  • amino acid sequence of the wild-type AAV-DJ capsid protein VP1 is shown in SEQ ID NO.3.
  • amino acid sequence of the capsid protein VP1 of the variant adeno-associated virus AAV-ie is shown in SEQ ID NO.4.
  • Another aspect of the present invention provides an isolated nucleic acid containing a nucleotide sequence encoding the above-mentioned variant adeno-associated virus AAV-ie capsid protein VP1.
  • the construct can usually be constructed by inserting the aforementioned isolated nucleic acid into a suitable expression vector.
  • suitable expression vector may be Rep2-Capsid or the like.
  • the host cell may be a eukaryotic cell and/or a prokaryotic cell, more specifically may be a mouse cell, a human cell, etc., more specifically may be a human embryonic kidney cell, etc., more specifically may be a HEK293FT cell, etc.
  • Another aspect of the present invention provides a variant adeno-associated virus AAV-ie which contains the aforementioned variant adeno-associated virus AAV-ie capsid egg VP1. That is, between N589 and R590 of the wild-type AAV-DJ capsid protein VP1, the amino acid sequence shown in SEQ ID NO.1 is inserted.
  • the variant adeno-associated virus AAV-ie also includes a heterologous nucleotide sequence encoding the target product, and the heterologous nucleotide sequence encoding the target product can be encapsulated and carried by various capsid proteins.
  • the aforementioned heterologous nucleotide sequence encoding the product of interest can usually be a construct, and the construct can usually contain a nucleic acid encoding the product of interest.
  • the construct can usually be constructed by inserting the nucleic acid encoding the product of interest into a suitable expression vector. Those skilled in the art can select a suitable expression vector.
  • the above-mentioned expression vector may include, but is not limited to, pAAV-CAG, pAAV- TRE, pAAV-EF1a, pAAV-GFAP promoter, pAAV-Lgr5 promoter, pAAV-Sox2 promoter expression vector, etc.
  • the target product is a nucleic acid or protein
  • the nucleic acid may be small guide RNA (sgRNA), interfering RNA (RNAi), and the like.
  • the mutant adeno-associated virus AAV-ie can be used as a carrier material to introduce foreign genes into the cells of the subject. Compared with the parental wild-type AAV-DJ, the mutant adeno-associated virus AAV-ie can affect hair cells and The infection rate of supporting cells is significantly increased.
  • Another aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned mutant adeno-associated virus AAV-ie and pharmaceutically acceptable excipients.
  • Another aspect of the present invention provides the use of the above-mentioned mutant adeno-associated virus AAV-ie for delivering the product of interest to hair cells and/or supporting cells of an individual.
  • the target product delivery may be for non-diagnostic and therapeutic purposes, for example, it may be in vitro to deliver the target product to isolated hair cells and/or supporting cells.
  • the hair cells generally include outer hair cells and/or inner hair cells.
  • the target product is a nucleic acid or protein
  • the nucleic acid may be small guide RNA (sgRNA), interfering RNA (RNAi), and the like.
  • Another aspect of the present invention provides the use of the above-mentioned variant adeno-associated virus AAV-ie in the preparation of a medicine for treating hearing disorders caused by cochlear damage in an individual.
  • the hearing impairment disease is a disease related to hair cells and/or supporting cells and/or spiral neuron cells.
  • the hearing impairment disease is a related disease related to gene defects, environmental damage or aging, for example, it may be related diseases caused by gene mutations, or for example, it may be related diseases caused by noise or drugs. For another example, it may be related diseases caused by aging.
  • the hearing impairment disease may be a related disease such as cell damage, specifically it may be cochlear hair cell damage, support cell damage, etc., more specifically it may be cochlear hair cell damage caused by gene mutation, support cell damage caused by gene mutation, etc. Cell damage caused by noise, cell damage caused by drugs, or caused by aging.
  • variant adeno-associated virus AAV-ie serves as a carrier for delivering the target product.
  • the adeno-associated virus with variant capsid protein and its use of the present invention have the following beneficial effects:
  • Figure 1a shows the expression diagram of the green fluorescent protein mNeonGreen after the wild-type AAV-DJ infects HEK 293T cells in Example 1.
  • Figure 1b shows the expression diagram of the green fluorescent protein mNeonGreen after the variant AAV-ie infects HEK 293T cells in Example 1.
  • Figure 1c shows the statistics of the fluorescence ratio of HEK 293T cells infected with wild-type AAV-DJ and variant AAV-ie in Example 1.
  • Figure 2a shows the immunostaining photos of green fluorescent mNeonGreen and Sertoli cell specific marker protein Sox2 after in vitro infection of cochlea tissue cultured in vitro by AAV1, AAV6, AAV9, AAV-DJ, AAV-ie, Anc80L65 in Example 2.
  • Figure 2b shows the statistical results of the data in Figure 2a.
  • Fig. 3a shows the immunostaining photographs of green fluorescent mNeonGreen and the hair cell specific marker protein Myo7a after AAV1, AAV6, AAV9, AAV-DJ, AAV-ie, Anc80L65 infected cochlea tissue cultured in vitro in Example 2.
  • Figure 3b shows the statistical results of the data in Figure 3a.
  • Figure 4a shows the expression of the green fluorescent mNeonGreen and the support cell marker protein Sox2 represented by magenta in three areas of the cochlea under the wide field of view in Example 3: apex, middle and base.
  • the staining result 14 days after the round window injection of AAV-ie virus in neonatal mice, the cochlea was dissected and immunostained with the Sertoli cell-specific marker protein Sox2, and then the green fluorescent mNeonGreen and immunostaining results were photographed.
  • Figure 4b shows the fluorescence photos of the support cells positive for the green fluorescent mNeonGreen and Sox2 after infection of the cochlea tissue by AAV1, AAV6, AAV8, AAV9, DJ8, Anc80L65, AAV-DJ, and AAV-ie in Example 3.
  • Figure 4c shows the statistical results of the data in Figure 4b.
  • Figure 5a shows the expression of AAV-DJ, AAV-ie green fluorescent mNeonGreen and the staining result of the hair cell marker protein Myo7a represented by magenta in Example 3.
  • Figure 5b shows the statistical results of the data in Figure 5a.
  • Figure 6a shows the expression of green fluorescent mNeonGreen in three regions of the cochlea: apex, middle, and base, and the staining result of the spiral neuron marker protein NeurN represented by magenta.
  • Figure 6b is the statistical result of Figure 6a.
  • Figure 7 shows the immunostaining results of the mouse cochlea sample in Example 4.
  • Figure 8a shows the immunostaining result of the mouse utricle sample in Example 5.
  • Figure 8b shows the immunostaining result of the human utricle sample in Example 5.
  • Figure 8c is an enlarged result of Figure 8b.
  • MOLECULAR CLONING A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel, etc., CURRENT PROTOCOLS INOLOGY MOLECULAR BI, John Wiley&Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; ENZYMatinology, 1998; PMWassarman and APWolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (PBBecker, ed.) Humana Press, Totowa, 1999, etc.
  • myosin 7A Myo7a,#25-6790 Proteus Biosciences, 1:1000
  • Sox2 Sox-2,#sc-17320,Santa Cruz Biotechnology, 1:1000
  • Flag Flag,#F3165,Sigma Aldrich, 1:1000
  • NeuN NeuN,#12943S,Cell Signaling Technology,1:500
  • Secondary antibodies secondary antibodies with three different marker colors (TRIC, FITC, Cy5) of the secondary antibody markers anti-rat, mouse, rabbit and goat, from Invitrogen.
  • DMEM Hyclone company
  • fetal bovine serum Lida company
  • N2 ThermoFisher company
  • ampicillin ThermoFisher company
  • double antibody ThermoFisher company
  • Cell and tissue culture consumables common consumables including various types of culture dishes, centrifuge tubes, pipettes, disposable filters, etc. were purchased from Corning.
  • Example 1 Construction of AAV variants and infection of HEK 293T cells
  • AAV variant named AAV-ie
  • Rep-Cap plasmid Construction of AAV variant (named AAV-ie) Rep-Cap plasmid.
  • the packaging of AAV requires three plasmids: the genome plasmid containing the target gene, the Rep-Cap plasmid and the Helper plasmid.
  • the sequence of the Cap protein in the Rep-Cap plasmid determines the different serotypes of AAV, which in turn affects the preference of AAV infected cells. Therefore, modification of the Cap protein can obtain a new type of AAV.
  • the Rep-Cap plasmid of parental AAV-DJ was synthesized by Nanjing Genscript Company (www.genscript.com.cn).
  • the unique NheI endonuclease site was introduced into the wild-type AAV-DJ VP1 capsid protein between amino acids 589 and 590 by polymerase chain reaction (PCR) mutagenesis.
  • the primer was Nanjing Genscript.com (www.genscript.com). .cn) Synthesis. It was then digested with restriction enzyme NheI and recovered (Axygen: AP-GX-250G). The recovered fragments were tested for concentration by Nanodrop 2000.
  • the DNA sequence (SEQ ID NO. 2: gatgggactttggcggtgccttttaag) encoding DGTLAVPFK was synthesized by Nanjing GenScript Company (www.genscript.com.cn). The synthesized fragment was dissolved to 10 ⁇ M in ultrapure water.
  • the recovered backbone vector and the DNA sequence fragment encoding DGTLAVPFK were recombined (Novoprotein: NR001A), and the connection system was as follows: recombination ligation buffer 2 ⁇ L, backbone vector 30ng, DNA fragment encoding DGTLAVPFK 1 ⁇ L, recombinant ligase 0.5 ⁇ L, ddH 2 O supplement Bring to 10 ⁇ L and react at 50°C for 20 minutes.
  • the transformation steps are as follows: take 100 ⁇ L of competent cells (TransGen: CD201) and thaw on ice; mix 10 ⁇ L of ligation product with competent cells and place on ice for 20 minutes; heat shock at 42°C for 60 seconds; place on ice for 2 minutes and add 400 ⁇ L Resuscitate the LB medium (MDBio: L001-1kg), shake for 30 minutes; take 70 ⁇ L of ampicillin-coated plates (50 ⁇ g/ml, 37°C incubator, incubate for 14 hours.
  • Monoclonal bacteria were selected and expanded in 4ml liquid LB medium. After 14 hours, the plasmid was extracted (Axygene: AP-MN-P-250G).
  • the steps are as follows: Centrifuge the bacterial solution at 4000 rpm for 10 minutes, discard the supernatant medium; add 350 ⁇ L of buffer S1, blow off the bacteria, and transfer to a 2 ml centrifuge tube; add 250 ⁇ L of buffer S2, upside down 8 times ;Add 250 ⁇ L of buffer S3, mix upside down 6 times to produce precipitation; centrifuge at 12,000 rpm for 10 minutes, take the supernatant through the column; centrifuge for 1 minute, discard the waste solution, add 500 ⁇ L of W1, centrifuge for one minute, and discard Waste solution; add 750 ⁇ L of W2, centrifuge, and discard the supernatant; add 500 ⁇ L of W2, centrifuge, and discard the supernatant; idle for 1 minute; add 50 ⁇ L of eluent, let stand for 2 minutes, and centrifuge. After the concentration of the obtained plasmid was tested, 10 ⁇ L was sent for sequencing, and the positive plasmid was stored at -20°C. Sequencing
  • the prepared plasmid can express the variant capsid protein VP1, in which the polynucleotide coding sequence of AAV-ie capsid VP1 is shown in SEQ ID NO.5, and the complete Rep-Cap plasmid is constructed. The sequence is shown in SEQ ID NO.6.
  • AAV variant named AAV-ie
  • Rep-Cap plasmid to express a green fluorescent protein mNeonGreen (GenBank: LC279210.1) genome plasmid pAAV-CAG-mNeonGreen (the full sequence of the plasmid is shown in SEQ ID NO.11), and the pHelper plasmid (the full sequence of the plasmid is shown in SEQ ID NO.12) is co-transfected into HEK-293T cells in an appropriate amount, and AAV virus is purified by iododialkanol gradient ultra-high-speed centrifugation. It is appropriate to measure the virus titer at 1E+12-1E+13GC/mL Concentration, put it at -80°C for later use.
  • mNeonGreen GenBank: LC279210.1
  • pHelper plasmid the full sequence of the plasmid is shown in SEQ ID NO.12
  • the AAV virus (AAV-ie and AAV-DJ, respectively) produced by the capsid protein variant and its parent capsid protein was added to HEK 293T cells cultured with DMEM+10% fetal bovine serum.
  • the MOI value of the virus was 1000.
  • the expression of the green fluorescent protein mNeonGreen was observed with a fluorescence microscope, as shown in Figure 1a-1c.
  • Figures 1a and 1b show the signal of green fluorescent protein, the upper right corner is the cell under bright field natural light.
  • Example 2 AAV variants infect mouse cochlea tissue in vitro
  • the cochlea of P3 wild-type C57 mice (Shanghai Lingchang Biotechnology Co., Ltd.) was quickly taken out on ice, attached to a glass slide coated with cell-tak, and placed in 98% DMEM+1% N 2 +1% Amp (5ug/ mL) After the medium is stable for 12 hours, add 1% FBS and 2 ⁇ 10 10 GC AAV to culture for 48-60 hours. Then the cultured samples were identified by immunostaining. The sample is immersed in 4% medium PFA for fixation, and then the sample is immersed in 10% donkey serum and 0.3% Triton X-100 PBS.
  • Myo7a Myosin 7a
  • Sox2 protein antibodies After incubating for 1 hour at room temperature, add Myo7a (Myosin 7a), Sox2 protein antibodies and The corresponding secondary antibody.
  • the samples were mounted with anti-fluorescence quenching agent to mount the media, and confocal was used for observation.
  • the laser power setting for shooting variant infected samples is selected as the standard. All visible green fluorescent protein mNeonGreen signals are captured by the same laser setting.
  • magenta represents supporting cells
  • green represents the green fluorescent protein mNeonGreen expressed by AAV introduced into cells
  • Figure 2b shows the statistical data of Figure 2a.
  • the results show that the AAV variant AAV-ie can efficiently infect the supporting cells of the mouse cochlea tissue cultured in vitro. Compared with other AAVs and parental AAVs, it has a higher percentage of Sox2-positive cells.
  • magenta represents hair cells
  • green represents the green fluorescent protein mNeonGreen expressed by AAV introduced into cells
  • Figure 3b shows the statistical data of Figure 3a, where OHC represents outer hair cells
  • AAV variants can efficiently infect hair cells in mouse cochlea tissue cultured in vitro.
  • AAV1, AAV6, AAV9, Anc80L65 the construction method is the same as in Example 1, the difference lies in the Rep-Cap plasmid used, and the plasmid sequence is shown in SEQ ID NO. 7 to 10).
  • AAV-DJ the variant AAV-ie has a high infection rate for Myo7a-positive hair cells.
  • Example 3 AAV variants can efficiently infect a variety of tissue cells in the cochlea of mice after in vivo injection
  • AAV variant virus (the concentrations of various viruses are marked in Figure 4) were injected into the perilymph of the cochlea.
  • the specific steps are as follows:
  • the anesthesia method of newborn mice adopts the method of low temperature induction anesthesia.
  • P2-3 mice were placed in an ice bath for 2-3 minutes, and then removed on an ice pad for subsequent surgical procedures.
  • the operation was performed only in the left ear of each mouse, and the right ear was a negative control.
  • an incision is made behind the left ear to expose the round window according to the relative position of the temporal bone and facial nerve. Take care to avoid damaging the facial nerve during surgery.
  • a micro-injection system (Nanoliter2000, WPI) was used to inject AAV into the cochlea through a capillary glass electrode (10mm in diameter) from the round window.
  • the cochlea of young mice can hold 2uL of AAV virus solution, and the injected virus volume is 1-2uL.
  • the wound was sutured and analgesics and anti-inflammatory drugs were applied.
  • the mouse strain used was C57/B6. Ten days after the injection, the cochlea was peeled off. The samples were identified by immunostaining. Immerse the sample in 4% medium PFA for fixation, then immerse the sample in 10% donkey serum and 0.3% Triton X-100PBS. After incubating for 1 hour at room temperature, add the antibodies of Myo7a, Sox2, NeurN and the corresponding secondary antibody. The sample was mounted with anti-fluorescence quenching agent to mount the media, and confocal was used for observation.
  • magenta color represents the Sox2-positive Sertoli cells
  • the green color represents the green fluorescent protein mNeonGreen expressed by the AAV introduced into the cells.
  • 4c represents the counting statistics of 4b.
  • the results show: 1.
  • Figure 4a is the three regions of the cochlear tissue taken from a wide field of view: the expression of green fluorescent mNeonGreen in the apex, middle and base and the support cell markers represented by magenta Staining result of protein Sox2. It can be found that AAV-ie can efficiently introduce fluorescent protein into each cell of the cochlea.
  • the results of Figures 4b and 4c show that AAV variants can efficiently infect Sertoli cells in mouse cochlear tissues in vivo. Compared with AAV1, AAV6, AAV8, AAV9, DJ8, Anc80L65 and AAV-DJ, the variant AAV-ie has an effect on Sox2. Positive Sertoli cells have a higher infection rate.
  • magenta color represents Myo7a-positive hair cells
  • green color represents the green fluorescent protein mNeonGreen expressed by AAV introduced into the cells.
  • IHC means inner hair cell
  • OHC means outer hair cell.
  • 5b represents the counting statistics of 5a. The results show that the AAV variant AAV-ie can efficiently infect hair cells in the mouse cochlea tissue in vivo. Compared with the parental AAV, the variant AAV-ie has a higher infection rate for Myo7a-positive hair cells.
  • magenta color represents the NeurN positive spiral neuron cells
  • green color represents the green fluorescent protein mNeonGreen expressed by the AAV introduced into the cells.
  • 6b represents the counting statistics of 6a. The results showed that in three areas of the cochlea: apex, middle and base, the AAV variant AAV-ie can efficiently infect spiral neurons in mouse cochlea tissue cultured in vitro.
  • Example 4 Injecting AAV-ie carrying the Atoh1 gene into the cochlea to regenerate a large number of hair cells
  • AAV-ie-Atoh1 virus (virus concentration 5E+12GC/mL) carrying mouse Atoh1 gene (NCBI Reference Sequence: NM_007500.5) (see Example 1 for the construction method)
  • the plasmid used was replaced with a plasmid expressing the Atoh1 gene (NCBI Reference Sequence: NM_007500.5) and injected into the perilymph of the cochlea.
  • the mouse strain used was C57/B6. The mice used were pups 2-3 days after birth. Ten days after the injection, the cochlea was peeled off. The samples were identified by immunostaining.
  • the sample will be immersed in 4% medium PFA for fixation, and then the sample will be immersed in 10% donkey serum and 0.3% Triton X-100PBS. After incubating for 1 hour at room temperature, the antibodies of Myo7a, Sox2 and the corresponding secondary antibodies are added. The sample was mounted with anti-fluorescence quenching agent to mount the media, and confocal was used for observation. The results are shown in Figure 7. Magenta indicates the immunostaining of the hair cell marker gene Myo7a, green indicates the immunostaining of the Sertoli cell marker gene Sox2, and the white arrow indicates ectopic regenerated hair cells.
  • Example 5 AAV-ie can efficiently infect utricle cells in mice and humans
  • the utricle is an organ in the inner ear that senses gravity and maintains balance.
  • 1.5 uL AAV-ie virus (virus concentration 6E+12GC/mL) was injected into the perilymph of the cochlea.
  • the mouse strain used was C57/B6.
  • the mice used were pups 2-3 days after birth.
  • the samples were identified by immunostaining. The sample will be immersed in 4% medium PFA for fixation, and then the sample will be immersed in 10% donkey serum and 0.3% Triton X-100 PBS.
  • the samples were mounted with anti-fluorescence quenching agent to mount the media and observed by confocal.
  • the results are shown in Figure 8a.
  • the magenta color represents the marker proteins Myo7a and Sox2 of hair cells and supporting cells
  • the green color represents the AAV-ie The delivered green fluorescent protein mNeonGreen. It can be found that AAV-ie can efficiently infect the hair cells and supporting cells of the utricles of mice.
  • a human utricle was taken and infected with 5 ⁇ 10 10 GCs of AAV-ie virus. After 7 days, 4% human samples were fixed in 4% PFA, then the samples were immersed in PBS containing 10% donkey serum and 0.3% Triton X-100. After incubating for 1 hour at room temperature, the antibodies of Myo7a and Sox2 and corresponding Of secondary antibodies. The samples were mounted with anti-fluorescence quenching agent to mount the media and observed by confocal. The results are shown in Figure 8b and Figure 8c, where the green color represents the green fluorescent protein mNeonGreen delivered by AAV-ie. Red indicates the marker protein Myo7a of hair cells, and magenta indicates the marker protein Sox2 of support cells. The results show that AAV-ie can also efficiently infect the hair cells and supporting cells of human utricles.

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Abstract

本发明提供一种具有变异型衣壳蛋白的腺相关病毒及其用途。所述变异型腺相关病毒AAV-ie，即在野生型AAV-DJ衣壳蛋白VP1在N589与R590之间插入氨基酸序列DGTLAVPFK。变异型腺相关病毒AAV-ie能够高效感染毛细胞和支持细胞，相比于其亲本具有较大的改善，为科学研究提供了更好的技术支持。

Description

具有变异衣壳蛋白的腺相关病毒及其用途 技术领域
本发明涉及生物技术领域，特别涉及一种具有变异衣壳蛋白的腺相关病毒及其用途。
背景技术
听力损失是最常见的感官残疾之一，影响到世界6.8％以上的人口(约5亿人)。听力损失最普遍的治疗方法是使用听力设备，这种装置可以放大声音、增强声音传递或直接刺激神经元。这种方法目前是治疗听力损失的最佳选择，但不幸的是，在嘈杂的环境中，听力敏感性和对自然声音的感知仍有局限性。因此，需要更好的替代策略来治疗听力损失。近年来，基因治疗已成为治疗遗传性疾病的一种有希望的策略。
感音神经性听力损失的病例有一半是由于毛细胞(Hair cell)和支持细胞(Supporting cell)的基因突变造成的，螺旋神经元中的基因突变也能导致一部分的听力障碍。100多个耳聋基因的突变已被发现。遗传性听力损失是一种典型的单源性疾病，单一的突变会导致听力损失。因此，基因治疗是一种理想的、有希望的潜在治疗策略，可以保持或重建听力功能。对于遗传性听力损失，大多数耳聋基因在毛细胞中表达，一部分基因的突变可能影响螺旋神经元。大量的基因突变直接影响毛细胞的各种功能。但一些关键耳聋基因如GJB2等主要表达在支持细胞中，其突变会影响支持细胞的功能，进而伴随毛细胞的损伤，最终导致听力丧失。据报道，有超过300个GJB2的突变能够导致遗传听力损失。GJB2的突变是人类遗传性听力损失最常见的原因，约占常染色体隐性听力损失的50％，占所有遗传性听力损失的15％-18％。因此，遗传性听力损失的基因治疗需要同时针对毛细胞和支持细胞。
AAV是一种直径为18-26nm的无包膜的二十面体病毒，衣壳结构由三种衣壳蛋白(VP1、VP2和VP3)构成。衣壳内含有一条4.7kb的单链DNA(ssDNA)，携带两个基因rep和cap，DNA两侧是回文倒置末端重复序列(ITRs)。rep和cap都有多个open reading frames(ORFs)，它们表达基因组复制和包装所需的蛋白质。由于ITRs序列可以直接应用于普通质粒中，因此可以直接将外源DNA插入ITRs之间构成重组质粒，将AAV病毒基因克隆进入第二个质粒，在帮助质粒Helper的作用下包装形成新的重组AAV病毒。目前常用的AAV有AAV-1，AAV-2，AAV-5，AAV-8，AAV-9以及AAV-DJ等。
一些病毒和非病毒基因转移策略已在基因治疗中得到测试。在这些方法中，AAV介导的基因治疗在过去十年中进展迅速，已经有170多个人体试验(7.2％)使用了AAV载体。AAV载体具有出色的安全性、低免疫原性和较高的转染效率。美国FDA在2017年批准了首个 AAV介导的基因治疗，其用于治疗罕见的遗传性眼病。然而，迄今为止，还没有基于AAV载体的耳蜗基因治疗。这可能是由于耳蜗中不同细胞类型缺乏具有较高传导效率的AAV所导致。几项研究在动物模型中测试了AAV介导的耳蜗基因疗法，并显示出有希望的结果。然而，大多数研究的重点是毛细胞和螺旋神经元，这可能是由于缺乏安全和有效的针对支持细胞的AAV。一些AAV血清型已在耳蜗中进行了评估，所有检测的AAV对支持细胞的感染效率极低，低于13％。因此，设计新型可以有效地转染支持细胞的AAV，成为进一步应用基因治疗遗传性听力损失的里程碑。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于提供一种具有变异衣壳蛋白的腺相关病毒及其用途，用于解决现有技术中AAV对毛细胞和支持细胞感染率不高的问题。
基于在野生型AAV-DJ的衣壳蛋白VP1的N589与R590之间，插入氨基酸序列如SEQ ID NO.1所示的氨基酸片段之后，构建的获得的变体AAV-ie具有更强的毛细胞和支持细胞的感染性，申请人提出本发明创造。
为实现上述目的及其他相关目的，本发明一方面提供一种变异型腺相关病毒AAV-ie衣壳蛋白VP1，所述变异型腺相关病毒AAV-ie衣壳蛋白VP1，相比于野生型AAV-DJ衣壳蛋白VP1，在N589与R590之间插入氨基酸片段，所述氨基酸片段的氨基酸序列如SEQ ID NO.1所示。
DGTLAVPFK(SEQ ID NO.1)
在本发明一些实施方式中，所述野生型AAV-DJ衣壳蛋白VP1的氨基酸序列如SEQ ID NO.3所示。
Figure PCTCN2019107474-appb-000001
Figure PCTCN2019107474-appb-000002
在本发明一些实施方式中，所述变异型腺相关病毒AAV-ie衣壳蛋白VP1的氨基酸序列如SEQ ID NO.4所示。
Figure PCTCN2019107474-appb-000003
本发明的另外一个方面提供了一种分离的核酸，所述核酸含有编码上述变异型腺相关病毒AAV-ie衣壳蛋白VP1的核苷酸序列。
本发明的另外一个方面提供了一种构建体，所述构建体含有上述的分离的核酸。所述构建体通常可以通过将上述分离的核酸插入合适的表达载体中构建获得，本领域技术人员可选择合适的表达载体，例如，上述表达载体可以是Rep2-Capsid等。
本发明的另外一个方面提供了一种宿主细胞，所述宿主细胞包含上述的构建体或基因组中整合有外源的上述的分离的核酸，或所述宿主细胞包含上述变异型腺相关病毒AAV-ie。所述宿主细胞可以是真核细胞和/或原核细胞，更具体可以是小鼠细胞、人细胞等，更具体可以是人胚胎肾细胞等，更具体可以是HEK293FT细胞等。
本发明的另外一个方面提供了一种变异型腺相关病毒AAV-ie，所述变异型腺相关病毒AAV-ie含有如上述变异型腺相关病毒AAV-ie衣壳蛋VP1。即在野生型AAV-DJ衣壳蛋白VP1在N589与R590之间，插入氨基酸序列如SEQ ID NO.1所示的氨基酸片段。
进一步地，所述变异型腺相关病毒AAV-ie还包括编码目的产物的异源核苷酸序列，所述编码目的产物的异源核苷酸序可以是各种衣壳蛋白能包裹携带的。上述编码目的产物的异源核苷酸序列通常可以是构建体，所述构建体通常可以含有编码目的产物的核酸。所述构建 体通常可以通过将编码目的产物的核酸插入合适的表达载体中构建获得，本领域技术人员可选择合适的表达载体，例如，上述表达载体可以是包括但不限于pAAV-CAG、pAAV-TRE、pAAV-EF1a、pAAV-GFAP promoter、pAAV-Lgr5 promoter、pAAV-Sox2 promoter表达载体等。
进一步地，所述目的产物为核酸或蛋白质，所述核酸可以是小向导RNA(sgRNA)、干扰RNA(RNAi)等。
所述变异型腺相关病毒AAV-ie可以作为载体材料，将外源的基因导入受试个体的细胞中，相比于亲本野生型AAV-DJ，变异型腺相关病毒AAV-ie对毛细胞和支持细胞的感染率明显提高。
本发明的另外一个方面提供了一种药物组合物，所述药物组合物包含如上所述的变异型腺相关病毒AAV-ie以及药学上可接受的辅料。
本发明的另外一个方面提供了上述变异型腺相关病毒AAV-ie在用于将目的产物递送至个体的毛细胞和/或支持细胞中的用途。所述目的产物递送可以是非诊断治疗目的的，例如，可以是体外的，对离体的毛细胞和/或支持细胞进行目的产物递送。所述毛细胞通常包括外毛细胞和/或内毛细胞。
进一步地，所述目的产物为核酸或蛋白质，所述核酸可以是小向导RNA(sgRNA)、干扰RNA(RNAi)等。
本发明的另外一个方面提供了上述变异型腺相关病毒AAV-ie在制备治疗个体中耳蜗损伤导致的听力障碍疾病的药物中的用途。
进一步地，所述听力障碍疾病为毛细胞和/或支持细胞和/或螺旋神经元细胞相关疾病。
进一步地，所述听力障碍疾病为基因缺陷、环境损伤或衰老的相关疾病，例如，可以是基因突变等所导致的相关疾病，再例如，可以是噪声所致的、药物所导致的相关疾病，再例如，可以是衰老所导致的相关疾病。
进一步地，听力障碍疾病可以为细胞损伤等的相关疾病，具体可以是耳蜗毛细胞损伤、支持细胞损伤等，更具体可以是基因突变导致的耳蜗毛细胞损伤、基因突变导致的支持细胞损伤等、噪声所致的细胞损伤、药物所导致的细胞损伤或者衰老所导致。
进一步地，所述变异型腺相关病毒AAV-ie作为递送目的产物的载体。如上所述，本发明的具有变异衣壳蛋白的腺相关病毒及其用途，具有以下有益效果：
具有更强的毛细胞和支持细胞的感染性，能够高效感染毛细胞和支持细胞，也能高效感染螺旋神经元，相比于其亲本具有较大的改善，为科学研究提供了更好的技术支持。
附图说明
图1a显示为实施例一中野生型AAV-DJ感染HEK 293T细胞后绿色荧光蛋白mNeonGreen的表达图。
图1b显示为实施例一中变异型AAV-ie感染HEK 293T细胞后绿色荧光蛋白mNeonGreen的表达图。
图1c显示为实施例一中野生型AAV-DJ和变异型AAV-ie感染HEK 293T细胞的荧光比例的统计。
图2a显示了实施例二中AAV1、AAV6、AAV9、AAV-DJ、AAV-ie、Anc80L65体外感染体外培养的耳蜗组织后绿色荧光mNeonGreen与支持细胞特异性标记蛋白Sox2的免疫染色照片。
图2b显示了对图2a的数据统计结果。
图3a显示了实施例二中AAV1、AAV6、AAV9、AAV-DJ、AAV-ie、Anc80L65感染体外培养的耳蜗组织后绿色荧光mNeonGreen与毛细胞特异性标记蛋白Myo7a的免疫染色照片。
图3b显示了对图3a的数据统计结果。
图4a显示了实施例三中大视野下，耳蜗三个区域：顶转(apex)、中转(middle)和底转(base)的绿色荧光mNeonGreen的表达和品红色所代表的支持细胞标记蛋白Sox2的染色结果。新生小鼠体内圆窗注射AAV-ie病毒后14天，将耳蜗剖出，进行支持细胞特异性标记蛋白Sox2的免疫染色，然后对绿色荧光mNeonGreen和免疫染色的结果拍照。
图4b显示了实施例三中AAV1、AAV6、AAV8、AAV9、DJ8、Anc80L65、AAV-DJ、AAV-ie感染耳蜗组织后对绿色荧光mNeonGreen和Sox2阳性的支持细胞的荧光照片。
图4c显示了对图4b的数据统计结果。
图5a显示了实施例三中AAV-DJ，AAV-ie绿色荧光mNeonGreen的表达和品红色所代表的毛细胞标记蛋白Myo7a的染色结果。
图5b显示了对图5a的数据统计结果。
图6a显示了耳蜗三个区域：顶转(apex)、中转(middle)和底转(base)的绿色荧光mNeonGreen的表达和品红色所代表的螺旋神经元标记蛋白NeurN的染色结果。
图6b是对图6a的统计结果。
图7显示了实施例四中小鼠耳蜗样品免疫染色结果。
图8a显示了实施例五中小鼠椭圆囊样本免疫染色结果。
图8b显示了实施例五中人的椭圆囊样本免疫染色结果。
图8c为图8b的放大结果。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold Spring Harbor Laboratory Press，1989 and Third edition，2001；Ausubel等，CURRENT PROTOCOLS IN MOLECULAR BIOLOGY，John Wiley&Sons，New York，1987 and periodic updates；the series METHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATIN STRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODS IN ENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，Academic Press，San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，Chromatin Protocols(P.B.Becker，ed.)Humana Press，Totowa，1999等。
材料与来源：
幼鼠：上海灵畅生物科技有限公司
各种AAV：南京金斯瑞公司合成构建
驴血清：上海翊圣生物科技有限公司
各个抗体与染色时的稀释比：
一抗：myosin 7A(Myo7a,#25-6790 Proteus Biosciences,1:1000),Sox2(Sox-2,#sc-17320,Santa Cruz Biotechnology,1:1000),Flag(Flag,#F3165,Sigma Aldrich,1:1000),NeuN(NeuN,#12943S,Cell Signaling Technology,1:500)。
二抗：二抗标记物anti-rat、mouse、rabbit及goat的各三种不同标记颜色(TRIC,FITC,Cy5)的二抗，来源于invitrogen公司。
细胞和组织培养试剂：DMEM(Hyclone公司)，胎牛血清(Lensa公司)，添加物N2(ThermoFisher公司)，氨苄西林(ThermoFisher公司)，双抗(ThermoFisher公司)。
细胞与组织培养耗材：包括各类培养皿、离心管、移液管，一次性滤器等常用耗材购于Corning公司。
实施例一构建AAV变异体并感染HEK 293T细胞
AAV变异体(命名为AAV-ie)Rep-Cap质粒的构建。
AAV的包装需要三个质粒：含有目的基因的基因组质粒、Rep-Cap质粒和Helper质粒。
其中Rep-Cap质粒中的Cap蛋白的序列决定了AAV的不同血清型，进而影响了AAV感染细胞的偏好性。因此，改造Cap蛋白能够得到新的AAV型。
(1)载体的构建、酶切和纯化
亲本AAV-DJ的Rep-Cap质粒由南京金斯瑞公司(www.genscript.com.cn)合成。
首先，通过聚合酶链式反应(PCR)诱变将独特NheI内切酶位点引入野生型AAV-DJ VP1衣壳蛋白氨基酸589和590之间，引物南京金斯瑞公司(www.genscript.com.cn)合成。然后用限制性内切酶NheI消化，并回收(Axygen:AP-GX-250G)。回收后的片段经过Nanodrop 2000检测浓度。
编码DGTLAVPFK的DNA序列(SEQ ID NO.2：gatgggactttggcggtgccttttaag)由南京金斯瑞公司(www.genscript.com.cn)合成。将合成的片段用超纯水溶解到10μM。
(2)载体的连接、转化与质粒提取。
回收的骨架载体和编码DGTLAVPFK的DNA序列片段经过重组连接(Novoprotein:NR001A)，连接体系如下：重组连接buffer 2μL，骨架载体30ng，编码DGTLAVPFK的DNA片段1μL，重组连接酶0.5μL，ddH 2O补齐到10μL，50℃反应20分钟。
转化步骤如下：取100μL感受态细胞(TransGen:CD201)在冰上解冻；10μL的连接产物与感受态细胞混合，冰上放置20分钟；42℃热激60秒；冰上放置2分钟，加入400μL复 苏LB培养基(MDBio:L001-1kg)，摇床30分钟；取70μL涂氨苄平板(50μg/ml，37℃培养箱，培养14个小时。
挑选单克隆菌，在4ml液体LB培养基中扩大培养，14小时后提取质粒(Axygene:AP-MN-P-250G)。
步骤如下：菌液经过4000转/分钟离心10分钟，倒掉上清培养基；加入350μL的buffer S1，将菌体吹散，转移到2ml离心管中；加入250μL的buffer S2，上下颠倒8次；加入250μL的buffer S3，颠倒混匀6次，产生沉淀；12000转/分钟离心10分钟，取上清过柱；离心1分钟，倒掉废液，加入500μL的W1，离心一分钟，倒掉废液；加入750μL的W2，离心，倒掉上清；加入500μL的W2，离心，倒掉上清；空转1分钟；加入50μL的洗脱液，静置2分钟，离心。获得质粒经过浓度检测，取10μL送测序，阳性质粒保存在-20℃。测序结果表明，获得质粒能够编码变异型衣壳蛋白VP1。
进一步的实验结果表明，制备获得的质粒能够表达变异型衣壳蛋白VP1，其中，AAV-ie capsid VP1的多核苷酸编码序列如SEQ ID NO.5所示，构建获得的Rep-Cap质粒的全序列如SEQ ID NO.6所示。
2.AAV变异体(命名为AAV-ie)病毒的包装与纯化。
将得到的Rep-Cap质粒，表达一个绿色荧光蛋白mNeonGreen(GenBank:LC279210.1)的基因组质粒pAAV-CAG-mNeonGreen(质粒全序列如SEQ ID NO.11所示)，pHelper质粒(质粒全序列如SEQ ID NO.12所示)以合适的量共转于HEK-293T细胞中，采用碘二烷醇梯度超高速离心纯化AAV病毒，测量病毒滴度在1E+12-1E+13GC/mL为合适浓度，放置-80℃备用。
在用DMEM+10％胎牛血清培养的HEK 293T细胞中加入衣壳蛋白变异体和其亲本衣壳蛋白产生的AAV病毒(分别为AAV-ie和AAV-DJ)。病毒加入的MOI值为1000。48小时后，用荧光显微镜观察了绿色荧光蛋白mNeonGreen的表达，如图1a-1c。图1a，1b大图表示的绿色荧光蛋白的信号，右上角的是明场自然光条件下的细胞。
结果显示：AAV变异体与其亲本AAV感染HEK 293T细胞的比例相似。
实施例二AAV变异体体外感染小鼠耳蜗组织
在冰上迅速取出P3野生型C57小鼠(上海灵畅生物科技有限公司)耳蜗，贴于涂有cell-tak的玻片上，置于98％DMEM+1％N 2+1％Amp(5ug/mL)培养基中稳定12h后，加入1％FBS和2×10 10GC AAV培养48-60h。然后对培养的样品进行免疫染色鉴定。样品浸入4％中PFA 中进行固定处理,然后样品浸入含有10％的驴血清和0.3％Triton X-100PBS中，室温下孵育1小时后,加入Myo7a(肌球蛋白7a)、Sox2蛋白的抗体以及相应的二级抗体。样品采用抗荧光淬火剂安装介质封片，并采用共聚焦进行观察。
在利用共聚焦方法拍摄图像时，选择拍摄变异体感染样品的激光功率设置作为标准。所有可见的绿色荧光蛋白mNeonGreen的信号都是通过相同的激光设置捕获的。
在数据处理方面，通过计算mNeonGreen阳性的支持细胞和毛细胞的数量，在耳蜗上手动量化了mNeonGreen的百分比，Myo7a阳性的是毛细胞，Sox2蛋白阳性的是支持细胞。
其中如图2a-2b，品红色表示的是支持细胞，绿色表示的是AAV导入细胞内表达出来的绿色荧光蛋白mNeonGreen,图2b显示的为图2a的统计数据。结果显示：AAV变异体AAV-ie能高效感染体外培养的小鼠耳蜗组织的支持细胞，与其他AAV及亲本AAV相比，其感染Sox2阳性的细胞比例更高。
其中如图3a-3b，品红色表示的是毛细胞，绿色表示的是AAV导入细胞内表达出来的绿色荧光蛋白mNeonGreen，图3b显示的为图3a的统计数据，其中OHC表示外毛细胞，IHC表示内毛细胞。结果显示：AAV变异体能高效感染体外培养的小鼠耳蜗组织中的毛细胞。与AAV1,AAV6,AAV9,Anc80L65(构建方法与实施例一相同，区别仅在于所使用的Rep-Cap质粒不同，质粒序列详见SEQ ID NO.7～10)和AAV-DJ相比，变异体AAV-ie对Myo7a阳性的毛细胞感染率很高。
上述结果表明：AAV变异体AAV-ie能高效感染Myo7a阳性的毛细胞、Sox2蛋白阳性的支持细胞。
实施例三AAV变异体体内注射后能高效感染小鼠耳蜗中的多种组织细胞
利用耳蜗圆窗注射技术，将1.5uL AAV变异体病毒(各种病毒的浓度如图4中标注)注射入耳蜗的外淋巴液中。具体步骤如下：新生小鼠的麻醉方法采用低温诱导麻醉的方法。P2-3小鼠置于冰浴中2-3min，取出在冰垫上进行后续手术过程。手术仅在每只小鼠的左耳中进行，右耳为阴性对照。手术时，在左耳耳后切口，根据颞骨和面神经的相对位置关系，暴露圆窗。手术时注意避免损伤面神经。接下来，使用微量进样系统(Nanoliter2000，WPI)将AAV通过毛细玻璃电极(直径10mm)从圆窗注射进入耳蜗中。幼鼠耳蜗可容纳2uL的AAV病毒溶液，注射病毒体积为1-2uL。手术后缝合伤口并涂抹止痛药和消炎药剂。
所用的小鼠品系是C57/B6。注射10天后，将耳蜗剥离出来。对样品进行免疫染色鉴定。将样品浸入4％中PFA中进行固定处理，然后样品浸入含有10％的驴血清和0.3％Triton  X-100PBS中，室温下孵育1小时后,加入Myo7a、Sox2、NeurN的抗体以及相应的二级抗体。样品采用抗荧光淬火剂安装介质封片,并采用共聚焦进行观察。
如图4a-4c，品红色表示的是Sox2阳性的支持细胞，绿色表示的是AAV导入细胞内表达出来的绿色荧光蛋白mNeonGreen。4c表示的是对4b的计数统计。结果显示：1.图4a是大视野拍摄的耳蜗组织的三个区域：顶转(apex)、中转(middle)和底转(base)的绿色荧光mNeonGreen的表达和品红色所代表的支持细胞标记蛋白Sox2的染色结果。可以发现AAV-ie能高效的在耳蜗各个细胞中导入荧光蛋白。2.图4b和4c的结果显示AAV变异体能高效感染体内小鼠耳蜗组织中的支持细胞，与AAV1、AAV6、AAV8、AAV9、DJ8、Anc80L65和AAV-DJ相比，变异体AAV-ie对Sox2阳性的支持细胞感染率更高。
如图5a-5b，品红色表示的是Myo7a阳性的毛细胞，绿色表示的是AAV导入细胞内表达出来的绿色荧光蛋白mNeonGreen。IHC表示内毛细胞，OHC表示外毛细胞。5b表示的是对5a的计数统计。结果显示AAV变异体AAV-ie能高效感染体内小鼠耳蜗组织中的毛细胞。与亲本AAV相比，变异体AAV-ie对Myo7a阳性的毛细胞感染率更高。
如图6a-6b,品红色表示的是NeurN阳性螺旋神经元细胞，绿色表示的是AAV导入细胞内表达出来的绿色荧光蛋白mNeonGreen。6b表示的是对6a的计数统计。结果显示在耳蜗三个区域：顶转(apex)、中转(middle)和底转(base)，AAV变异体AAV-ie能高效感染体外培养的小鼠耳蜗组织中的螺旋神经元。
上述结果表明：采用圆窗注射的方法AAV变异体能高效感染Myo7a阳性的毛细胞、Sox2阳性的支持细胞以及NeurN阳性的螺旋神经元。
实施例四体内注射携带Atoh1基因的AAV-ie进入耳蜗使得大量的毛细胞再生
利用耳蜗圆窗注射技术，将携带小鼠Atoh1基因(NCBI Reference Sequence:NM_007500.5)的1.5uL AAV-ie-Atoh1病毒(病毒浓度为5E+12GC/mL)(构建方法参照实施例一，其中所使用的质粒替换为表达Atoh1基因(NCBI Reference Sequence:NM_007500.5)的质粒)注射入耳蜗的外淋巴液中，具体方法参照实施例三。所用的小鼠品系是C57/B6。所用的小鼠为出生2-3天后的幼鼠。注射10天后，将耳蜗剥离出来。对样品进行免疫染色鉴定。样品将浸入4％中PFA中进行固定处理,然后样品浸入含有10％的驴血清和0.3％Triton X-100PBS中，室温下孵育1小时后,加入Myo7a、Sox2的抗体以及相应的二级抗体。样品采用抗荧光淬火剂安装介质封片,并采用共聚焦进行观察。结果如图7所示,品红色表示的是毛细胞标记基因Myo7a的免疫染色，绿色表示的是支持细胞的标记基因Sox2的免疫染色， 白色箭头表示异位再生的毛细胞。
上述结果表明：AAV-ie-Atoh1病毒导入Atoh1基因到支持细胞后，在sensory region和GER区域能够显著再生毛细胞，
实施例五AAV-ie能高效感染小鼠和人的椭圆囊(utricle)细胞
椭圆囊(utricle)是内耳中感知重力和维持平衡的器官。利用耳蜗圆窗注射技术，将1.5uL AAV-ie病毒(病毒浓度为6E+12GC/mL)注射入耳蜗的外淋巴液中，具体方法参照实施例三。所用的小鼠品系是C57/B6。所用的小鼠为出生2-3天后的幼鼠。注射10天后，将椭圆囊剥离出来。对样品进行免疫染色鉴定。样品将浸入4％中PFA中进行固定处理,然后样品浸入含有10％的驴血清和0.3％Triton X-100 PBS中，室温下孵育1小时后,加入Myo7a、Sox2的抗体以及相应的二级抗体。样品采用抗荧光淬火剂安装介质封片,并采用共聚焦进行观察，结果如图8a所示，其中品红色分别表示了毛细胞和支持细胞的标记蛋白Myo7a和Sox2，绿色表征了由AAV-ie传递的绿色荧光蛋白mNeonGreen。可以发现AAV-ie能够高效感染小鼠的椭圆囊的毛细胞和支持细胞。
取人的椭圆囊，用5×10 10GCs的AAV-ie病毒感染。7天后，4％人样本在4％PFA中进行固定处理,然后样品浸入含有10％的驴血清和0.3％Triton X-100 PBS中，室温下孵育1小时后,加入Myo7a、Sox2的抗体以及相应的二级抗体。样品采用抗荧光淬火剂安装介质封片,并采用共聚焦进行观察，结果如图8b和图8c所示，其中绿色表征了由AAV-ie传递的绿色荧光蛋白mNeonGreen。红色表示毛细胞的标记蛋白Myo7a，品红色支持细胞的标记蛋白Sox2。结果显示，AAV-ie也能够高效感染人的椭圆囊的毛细胞和支持细胞。
以上的实施例是为了说明本发明公开的实施方案，并不能理解为对本发明的限制。此外，本文所列出的各种修改以及发明中方法、组合物的变化，在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述，但应当理解，本发明不应仅限于这些具体实施例。事实上，各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。

Claims (14)

  1. 一种变异型腺相关病毒AAV-ie衣壳蛋白VP1，所述变异型腺相关病毒AAV-ie衣壳蛋白VP1，相比于野生型AAV-DJ衣壳蛋白VP1，在N589与R590之间插入氨基酸片段，所述氨基酸片段的氨基酸序列如SEQ ID NO.1所示。
  2. 如权利要求1所述的一种变异型腺相关病毒AAV-ie衣壳蛋白VP1，其特征在于，所述变异型腺相关病毒AAV-ie衣壳蛋白VP1的氨基酸序列如SEQ ID NO.4所示。
  3. 一种分离的核酸，其特征在于，所述核酸含有编码如权利要求1中所述的变异型腺相关病毒AAV-ie衣壳蛋白VP1的核苷酸序列。
  4. 一种构建体，所述构建体含有如权利要求3所述的分离的核酸。
  5. 一种宿主细胞，其特征在于，所述宿主细胞包含如权利要求4所述的构建体或基因组中整合有外源的如权利要求3所述的分离的核酸。
  6. 一种变异型腺相关病毒AAV-ie，其特征在于，所述变异型腺相关病毒AAV-ie含有如权利要求1-2任一权利要求所述的变异型腺相关病毒AAV-ie衣壳蛋白VP1。
  7. 如权利要求6所述的变异型腺相关病毒AAV-ie，其特征在于，所述变异型腺相关病毒AAV-ie还包括编码目的产物的异源核苷酸序列。
  8. 如权利要求6所述的变异型腺相关病毒AAV-ie，其特征在于，所述目的产物为核酸或者蛋白质，所述核酸优选选自小向导RNA、干扰RNA。
  9. 一种药物组合物，其特征在于，所述药物组合物包含如权利要求6～8任意一项所述的变异型腺相关病毒AAV-ie以及药学上可接受的辅料。
  10. 如权利要求6～8任意一项所述的变异型腺相关病毒AAV-ie在用于将目的产物递送至个体的毛细胞和/或支持细胞中的用途。
  11. 根据权利要求10所述的用途，其特征在于，所述目的产物为核酸或蛋白质，所述核酸优选为小向导RNA、干扰RNA。
  12. 如权利要求6～8任意一项所述的变异型腺相关病毒AAV-ie在制备治疗个体中耳蜗损伤导致的听力障碍疾病的药物中的用途。
  13. 如权利要求12所述的用途，其特征在于，所述听力障碍疾病为基因缺陷、环境损伤或衰老的相关疾病。
  14. 根据权利要求12所述的用途，其特征在于，所述听力障碍疾病为细胞损伤的相关疾病。
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