rAAV2/Retro as delivery system for retina photoreceptor cells and application thereof in preparing medicament for treating retina diseases
阅读说明：本技术 rAAV2/Retro作为针对视网膜感光细胞的递送系统及其在制备治疗视网膜疾病药物中的应用 (rAAV2/Retro as delivery system for retina photoreceptor cells and application thereof in preparing medicament for treating retina diseases ) 是由 庄菁 余克明 吴奕辉 蒋自华 于 2021-03-31 设计创作，主要内容包括：本发明属于生物工程和生物医药技术领域,涉及rAAV2/Retro作为针对视网膜感光细胞的递送系统及其在制备治疗视网膜疾病药物中的应用。以rAAV2/Retro为载体,构建携带外源性目的基因或shRNA的表达系统；以rAAV2/Retro作为递送系统,有效感染视网膜感光细胞,并特异性地表达所述目的基因或敲除内源性基因。本发明借助rAAV2/Retro独特的逆向回溯感染方式,特异性地作用于感光细胞,避免了传统腺相关病毒递送系统的非特异性；而且本发明用于治疗视网膜疾病的药物采用安全、全面的玻璃体腔注射,感染范围大于传统的腺相关病毒,具有更好的治疗效果。因此,利用本发明rAAV2/Retro病毒载体制得的用于治疗视网膜疾病的基因药物具有特异性强,安全性高,治疗效果好的优势。(The invention belongs to the technical field of bioengineering and biomedicine, and relates to rAAV2/Retro as a delivery system aiming at retinal photoreceptor cells and application thereof in preparing a medicament for treating retinal diseases. Constructing an expression system carrying exogenous target genes or shRNA by taking the rAAV2/Retro as a vector; rAAV2/Retro is used as a delivery system to effectively infect retinal photoreceptor cells and specifically express the target gene or knock out endogenous genes. According to the invention, the rAAV 2/retrodirective unique reverse retrospective infection mode is used, the specific effect on the photoreceptor cells is achieved, and the non-specificity of the traditional adeno-associated virus delivery system is avoided; moreover, the medicine for treating the retinal diseases adopts safe and comprehensive intravitreal injection, has a larger infection range than the traditional adeno-associated virus, and has better treatment effect. Therefore, the gene medicine for treating the retinal diseases, which is prepared by utilizing the rAAV2/Retro virus vector, has the advantages of strong specificity, high safety and good treatment effect.)
The application of the rAAV2/Retro as a delivery system for the retinal photoreceptor cells is characterized in that the rAAV2/Retro is used as a vector to construct an expression system carrying an exogenous target gene or shRNA, the rAAV2/Retro is used as the delivery system to deliver the target gene or shRNA into the retinal photoreceptor cells, and the exogenous target gene is expressed or the endogenous gene is knocked out in the retinal photoreceptor cells.
2. The use of claim 1, wherein the exogenous gene or shRNA of interest comprises a gene or shRNA associated with a retinal disease.
The application of the rAAV2/Retro in preparing the medicine for treating the retinal disease is characterized in that the rAAV2/Retro is used as a vector to construct a gene or shRNA expression system related to the retinal disease, so as to prepare the medicine for treating the retinal disease.
4. The use of claim 3, wherein the gene related to retinal diseases comprises RS1, CRX, CNGB3, CNGA3, GNAT2, PDE6C, NGF, BDNF, Neurotrophin-3 and Neurotrophin-4 genes, and the shRNA related to retinal diseases comprises shNRL and shPTEN.
5. The use of claim 3, wherein the retinal disease comprises a disease of retinal photoreceptor cells, ganglion cells, bipolar cells, horizontal cells, amacrine cells, or pigment epithelial cytopathy.
6. The use according to claim 5, wherein the retinal diseases include age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa, congenital achromatopsia, congenital retinal detachment, GUCY2D-Leber congenital amaurosis, and pathologies due to glaucoma and ocular trauma.
7. The medicine for treating the retinal disease is characterized by comprising an rAAV2/Retro serving as a vector and constructing an adeno-associated virus carrying genes or shRNA related to the retinal disease.
8. The medicament of claim 7, wherein the genes related to retinal diseases include RS1, CRX, CNGB3, CNGA3, GNAT2, PDE6C, NGF, BDNF, neurotropin-3, neurotropin-4; the shRNA related to the retinal disease comprises shNRL and shPTEN.
9. The medicament of claim 7, wherein the retinal disease comprises a disease of retinal photoreceptor cells, ganglion cells, bipolar cells, horizontal cells, amacrine cells, or pigment epithelial cytopathy.
10. The medicament according to claim 9, wherein the retinal diseases include age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa, congenital achromatopsia, congenital retinal detachment, GUCY2D-Leber congenital amaurosis, and glaucoma and ocular trauma-induced pathologies.
Technical Field
The invention belongs to the technical field of bioengineering and biomedicine, and particularly relates to a novel rAAV2/Retro serving as a delivery system for retinal photoreceptor cells and application of the novel rAAV2/Retro in preparation of a medicament for treating retinal diseases.
Technical Field
The retina comprises a variety of cells, with the outer nuclear layer consisting primarily of photoreceptor cells (see fig. 1). The photoreceptor cell is a special nerve cell with a light signal conversion function in the retina and is the basis for normal vision generation; photoreceptor cytopathy and abnormal gene expression exist commonly in retinal diseases, so that visual function is seriously affected. In addition, retinal diseases caused by gene mutation or gene expression abnormality of photoreceptor cells (such as retinal cleavage, congenital achromatopsia, age-related macular degeneration, diabetic retinopathy, glaucoma and pathological changes caused by ocular trauma) account for a considerable proportion of blinding eye diseases. Therefore, gene therapy targeting photoreceptor cells is an important strategy for retinal diseases. However, the smooth progress of gene therapy faces important problems: there is a lack of expression systems in the eye that deliver high efficiency against photoreceptor cells.
Delivery of expression systems to photoreceptors is often dependent on viral infection, with adeno-associated virus (AAV) being the most widely used, and two broad types of infection strategies are currently implemented for viral infection of photoreceptors: (1) broad-spectrum strong promoter-based infection strategies: the expression purpose is realized by utilizing adeno-associated viruses (such as AAV2, AAV5, AAV7 and AAV8) with better affinity to eye tissues to carry an expression system comprising a CAG or CMV broad-spectrum strong promoter to enter a photoreceptor cell; (2) infection strategy based on photoreceptor-specific promoters: the strategy is based on the adoption of adeno-associated virus (such as AAV2, AAV5, AAV7 and AAV8) with good affinity to eye tissues, and further carries an expression system containing NR2E3, IRBP and other photoreceptor cell specific promoters, wherein the expression system enters photoreceptor cells to realize expression, and the expression system is not expressed in non-target cells.
However, the above infection methods all have the following obvious disadvantages:
(1) risk of retinal damage. The retina mainly comprises 3 layers of architecture: the ganglion cell layer, inner nuclear layer and outer nuclear layer of photoreceptor cells (FIG. 1); a considerable part of these inherited retinal diseases are caused by the damage of the outer nuclear layer caused by the gene defect or gene expression abnormality of photoreceptor cells, thereby causing blindness. In addition, retinal diseases caused by gene mutation or gene expression abnormality of photoreceptor cells (such as retinal cleavage, congenital achromatopsia, age-related macular degeneration, diabetic retinopathy, glaucoma and pathological changes caused by ocular trauma) account for a considerable proportion of blinding eye diseases. At present, the administration mode of the photoreceptor cells aiming at the outer nuclear layer is mainly divided into two modes: firstly, injecting in vitreous cavity (figure 2A), the injection needle head can enter the vitreous cavity only by penetrating the sclera at the outer side and the pars plana of ciliary body, thus not causing retinal detachment, having small retinal cell injury and light inflammatory reaction; but has the disadvantage that the outer nuclear layer is difficult to reach by common viruses. ② injection in the retina inferior cavity: this method requires crossing the vitreous and re-crossing the retina, as shown in fig. 2B, where the subretinal space is a potential compartment for the separation of the neuroepithelial and pigment epithelial layers of the retina, and the spread of the virus is very limited; meanwhile, subretinal injection causes local detachment of the retina, which may cause glial cell proliferation, retinal detachment, photoreceptor degeneration and impaired visual function.
(2) The infection range is limited. Because the existing adeno-associated virus drugs can not reach the outer nuclear layer through intravitreal injection, only subretinal space injection can be selected for drug administration, the subretinal space is a potential lacuna, and partial subretinal space injection can not enable the virus to fully infect the whole retina (figure 2B), so that the infection range is limited.
(3) The broad-spectrum strong promoter has poor specificity and targeting. The infection of AAV2, AAV5, AAV7 and AAV8 adeno-associated viruses is not specific to the type of neurons, and can infect all types of neurons and glial cells locally injected by the virus, and the infection of non-target cells by the virus can reduce the infection efficiency of target photoreceptor cells and generate potential interference and risk.
(4) The photoreceptor cell specific promoter expression intensity is poor. The expression system carrying NR2E3 and IRBP photoreceptor cell specific promoter expresses the target protein only in photoreceptor cells, but the expression intensity is far less than that of a broad-spectrum strong promoter.
Disclosure of Invention
Based on the problems in the prior art, the invention aims to provide a method for constructing a high-efficiency expression system aiming at retinal photoreceptor cells by using rAAV 2/Retro; by means of the unique reverse retrospective infection mode of rAAV2/Retro (FIG. 3), the gene specifically acts on photoreceptor cells, and avoids the non-specificity of a traditional adeno-associated virus delivery system infected cells. In addition, the invention also provides a method for preparing the medicine for treating the retinal disease by using the high-efficiency expression system, compared with the traditional adeno-associated virus medicine, the medicine can be injected in a safe and comprehensive vitreous cavity, so that the retinal damage caused by the existing injection administration of the retina lower cavity is avoided, and the medicine has the advantages of strong specificity, good treatment effect and high safety.
Based on the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides an application of rAAV2/Retro as a delivery system for retinal photoreceptor cells, and the rAAV2/Retro is used as a vector to construct an expression system carrying exogenous target genes or shRNA; and taking the rAAV2/Retro as a delivery system, delivering the target gene or shRNA into the retinal photoreceptor cell, and expressing an exogenous target gene or knocking out an endogenous gene in the retinal photoreceptor cell.
The rAAV2/Retro is an adeno-associated virus minor capsid protein which is transformed by directed evolution and determines the infection characteristic of the adeno-associated virus, the rAAV2/Retro is used as the minor capsid protein to assemble the adeno-associated virus for ocular infection, and the rAAV2/Retro virus has the following characteristics when infecting eyes: the method has the advantages of fixed point, high efficiency and small wound, namely has reverse backtracking infection capacity, only infects photoreceptor cells, efficiently expresses exogenous genes, and can realize the purpose of delivering an efficient expression system aiming at the retina photoreceptor cells.
Further, the exogenous target gene or shRNA includes a gene or shRNA associated with a retinal disease.
The invention takes rAAV2/Retro as a vector, fully utilizes the reverse retrospective infection capacity of rAAV2/Retro, specifically infects target cells such as retina photoreceptor cells, and then expresses exogenous target genes or shRNA recombined on rAAV2/Retro in the retina photoreceptor cells at high intensity, thereby achieving the purpose of high-efficiency and specific expression.
In a second aspect, the invention provides application of rAAV2/Retro in preparation of a medicament for treating retinal diseases, and the rAAV2/Retro is used as a vector to construct a gene or shRNA expression system related to the retinal diseases so as to prepare the medicament for treating the retinal diseases.
Further, in the above application, the gene related to the retinal disease includes, but is not limited to, RS1, CRX, CNGB3, CNGA3, GNAT2, PDE6C, NGF, BDNF, neurotropin-3, neurotropin-4 gene, and the above shRNA related to the retinal disease includes, but is not limited to, shrnl, shPTEN.
Further, the above-mentioned retinal diseases include, but are not limited to, diseases of retinal photoreceptor cells, ganglion cells, bipolar cells, horizontal cells, amacrine cells, or pigment epithelial cytopathies.
Further, in the above applications, retinal diseases include, but are not limited to, age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa, congenital achromatopsia, congenital retinal cleavage, GUCY2D-Leber congenital amaurosis, and pathologies due to glaucoma and ocular trauma.
In a third aspect, based on the above applications, the present invention provides a medicament for treating a retinal disease, the medicament comprising a rAAV2/Retro vector to construct an adeno-associated virus carrying a gene or shRNA associated with the retinal disease.
Further, in the above drugs, the genes related to retinal diseases include RS1, CRX, CNGB3, CNGA3, GNAT2, PDE6C, NGF, BDNF, Neurotrophin-3, Neurotrophin-4; the shRNA related to the retinal disease comprises shNRL and shPTEN.
Further, among the above drugs, the retinal diseases include diseases of retinal photoreceptor cells, ganglion cells, bipolar cells, horizontal cells, amacrine cells or pigment epithelial cell lesions.
Further, among the above drugs, retinal diseases include age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa, congenital achromatopsia, congenital retinal cleavage, GUCY2D-Leber congenital amaurosis, and pathological changes caused by glaucoma and ocular trauma.
Further, the above-mentioned drug contains at least one of rAAV2/Retro-CNGA3 virus, rAAV2/Retro-RS1 virus, rAAV2/Retro-CRX virus, rAAV2/Retro-CNGB3 virus, rAAV2/Retro-GNAT2 virus, rAAV2/Retro-PDE6C virus, rAAV2/Retro-NGF virus, rAAV2/Retro-BDNF virus, rAAV2/Retro-Neurotrophin-3 virus, rAAV2/Retro-Neurotrophin-4 virus, rAAV2/Retro-shNRL virus, and rAAV2/Retro-shPTEN virus.
Further, the invention provides a medicine for treating retinal diseases by over-expressing a target gene, which is used for treating congenital achromatopsia caused by CNGA3 gene mutation, and the medicine comprises rAAV2/Retro-Cnga3 virus; the rAAV2/Retro-Cnga3 virus contains rAAV2/Retro vector and Cnga3 gene (ORF). The rAAV2/Retro-Cnga3 virus overexpresses Cnga3 protein, and the nucleotide sequence of the Cnga3 gene is shown as SEQ ID NO. 1.
The Cnga3 protein generated by Cnga3 gene expression is mainly expressed in cone cells of the retina outer nuclear layer, when Cnga3 gene mutation causes Cnga3 protein loss, the cone cells are gradually degenerated and die, cone system function is gradually lost, full-color blindness and impaired eyesight are caused, and the generation of congenital full-color blindness is mainly shown as the progressive reduction of b-wave amplitude after the clear adaptation.
Because the Cnga3 gene is used as a key gene of cone cells, when the Cnga3 gene (ORF) is loaded on the rAAV2/Retro adeno-associated virus, the Cnga3 gene is expected to be used for treating the total color blindness and the impaired photopic vision caused by the deletion of the Cnga3 protein.
The rAAV2/Retro-CAG-Cnga3 virus is prepared by recombining a Cnga3 gene on a rAAV2/Retro vector, and is used as a medicament for treating the complete color blindness and the impaired vision caused by the deletion of the Cnga3 protein, and the Cnga3 invades a neuron through the tail end of a long shaft protrusion by virtue of the unique reverse retrospective infection mode of the rAAV2/Retro to specifically act on a photoreceptor cell so as to avoid the potential risk caused by non-specific infection.
Further, the invention provides a medicament for treating retinal diseases by knocking down a target gene, which comprises rAAV2/Retro-shNrl virus; the rAAV2/Retro-shNrl virus contains an rAAV2/Retro vector and an shRNA sequence aiming at Nrl.
Nrl gene expression generates Nrl protein, which is highly expressed in photoreceptor cells of outer nuclear layer of retina in retinitis pigmentosa, and inhibition of Nrl gene expression can significantly reduce photoreceptor cell apoptosis caused by retinitis pigmentosa. When the rAAV2/Retro adeno-associated virus is loaded with Nrl shRNA sequence, the shRNA sequence is expected to be used for treating retinal damage caused by retinitis pigmentosa.
The rAAV2/Retro-U6-shNrl virus is prepared by recombining an shRNA sequence aiming at Nrl gene on an rAAV2/Retro vector, so that photoreceptor cell apoptosis caused by RD1 gene mutation is inhibited, and by means of a unique reverse retrospective infection mode of the rAAV2/Retro, specific shRNA of Nrl invades a neuron through the end of a long axon, so that Nrl in the photoreceptor cell is specifically knocked down, and the potential risk caused by nonspecific infection is avoided.
Further, promoters for recombination on rAAV2/Retro-CNGA3 virus, rAAV2/Retro-shNRL virus, rAAV2/Retro-shPTEN virus, rAAV2/Retro-RS1 virus, rAAV2/Retro-CRX virus, rAAV2/Retro-CNGB3 virus, rAAV2/Retro-GNAT2 virus, rAAV2/Retro-PDE6C virus, rAAV2/Retro-NGF virus, rAAV2/Retro-BDNF virus, rAAV 2/Retro-neurotropin-3 virus, rAAV 2/Retro-neurotropin-4 virus include, but are not limited to, CMV, CAG, U6, SYN, GFAP, EF1 α.
Furthermore, the nucleotide sequence of the CAG promoter is shown as SEQ ID NO. 2.
Compared with the prior art, the invention has the following beneficial effects:
compared with the traditional delivery system taking AAV2, AAV5, AAV7 and AAV8 as vectors, the rAAV2/Retro vector has the following significant advantages:
(1) rAAV2/Retro has the ability to specifically infect photoreceptor cells.
The specific analysis is as follows: the infection form of rAAV2/Retro serotype adeno-associated virus to neurons is a reverse backtracking mode, as shown in figure 3, rAAV2/Retro serotype adeno-associated virus invades neurons through nerve axon terminals, backtracks to neuron cell bodies along axons, enters cell nuclei to play a role in expressing exogenous genes, has no infection capacity to neurons near the cell bodies, and can specifically infect Recoverin positive photoreceptor cells (as shown in figures 4B and 5A) to achieve specific treatment purposes; the traditional AAV2, AAV5, AAV7 and AAV8 serotype adeno-associated viruses do not have the capability of retrospective infection and the capability of specifically infecting photoreceptor cells, need to be injected to the vicinity of a cell body to effectively infect neurons, and can also infect neurons in the vicinity of the cell body (shown in figures 5B and 6), so that the virus has the risk of nonspecific infection (shown by white arrows in figure 5B); therefore, the rAAV2/Retro serotype adeno-associated virus is taken as a vector, so that potential risks caused by non-specific infection of traditional AAV2, AAV5, AAV7 and AAV8 serotype adeno-associated viruses based on a broad-spectrum promoter can be effectively avoided. Meanwhile, as the rAAV2/Retro has good specificity, a broad-spectrum promoter with high expression efficiency can be used, and the defect of insufficient expression efficiency of an adeno-associated virus delivery system based on a cell-specific promoter is overcome.
(2) The rAAV2/Retro can efficiently express functional foreign protein.
Previous studies by applicants showed that rAAV2/Retro-CAG-mGata3-GFP viruses expressing exogenous Gata3 protein and control rAAV2/Retro-CAG-GFP viruses efficiently and specifically infected the outer nuclear layer of the retina, Gata3 protein (red) was specifically localized in the nucleus as a transcription factor (shown in figure 7B), while the control viruses expressed only GFP green fluorescence (shown in figure 7A). Meanwhile, research shows that the expression efficiency of the rAAV2/Retro serotype adeno-associated virus to the exogenous gene is higher than that of the traditional AAV2, AAV5, AAV7 and AAV8, the medicine taking the rAAV2/Retro serotype adeno-associated virus as a vector can achieve the purpose of high-efficiency treatment through low dosage, and can effectively avoid possible immune reaction caused by high-titer virus.
(3) Establishing a simple and effective use way: administration is carried out in the vitreous cavity.
The delivery vector prepared by taking the rAAV2/Retro as the vector can be injected through a vitreous cavity, and achieves the aim of infecting only photoreceptor cells without infecting other cells or tissues by virtue of the reverse retrospective infection capacity (figure 3) of the rAAV 2/Retro; however, the existing AAV2 and AAV8 serotype adeno-associated viruses have no specificity, and must be injected into the subretinal space to achieve the purpose of infection and expression, and the specific analysis is as follows:
the retinal tissue is a multi-layered neuronal structure, as shown in fig. 1, and consists of innermost ganglion cells, intermediate neurons of the inner nuclear layer, and outermost photoreceptor cells; when the photoreceptor cells of the outer nuclear layer need to be infected and treated, the rAAV 2/retroro adeno-associated virus is injected into the vitreous cavity, the rAAV 2/retroro adeno-associated virus can be spread on the whole retinal surface, as shown in FIG. 4A, the rAAV 2/retroro adeno-associated virus with the reverse backtracking function can effectively infect neurons of the outer nuclear layer through axons, and the rAAV 2/retroro adeno-associated virus directionally infects cells of the outer nuclear layer and has no influence on the inner nuclear layer and nodal cells, so when the rAAV 2/retroro adeno-associated virus carrying the foreign gene is injected into the vitreous cavity, the foreign gene can be efficiently expressed in the outer nuclear layer cells, and cannot be expressed in the inner nuclear layer cells and nodal cells, and the purpose of specifically infecting target cells is achieved (as shown in FIGS. 4 and 5A). The intravitreal administration has the advantages of simple operation, small damage and wide virus action range. During injection in the vitreous cavity, the injection needle can enter the vitreous cavity only by penetrating through the sclera, the pigment epithelium layer and the pars plana of the ciliary body on the outer side as shown in figure 2A; and the virus in the vitreous chamber can sufficiently contact and infect the entire retina.
Whereas traditional serotype adeno-associated viruses (AAV2, AAV4, AAV7 and AAV8) must be injected into the sub-retinal space. Subretinal injection also requires crossing the vitreous and re-crossing the retina, as shown in fig. 2B and 6, which causes local detachment of the retina, possibly causing gliosis, retinal detachment, photoreceptor degeneration and impaired visual function; and the subretinal space is a potential cavity space existing when the retinal neuroepithelial layer and the pigment epithelial layer are separated, the spread of the virus is very limited, the virus can only contact local tissues and then infect the outer nuclear layer, as shown in figures 5B and 6, and the inner nuclear layer and the nodal cell layer are infected by cells (as white arrows in figure 5B and green arrows in figure 6B).
As described above, the present invention has advantages that the rAAV 2/retroadenovirus-associated virus can be used as a vector, and the delivery system can be injected through the vitreous cavity, which is easy to handle and has an excellent delivery effect.
Compared with the traditional AAV2 and AAV8 vectors, the rAAV2/Retro vector can express the foreign gene carried by the vector in a target cell more efficiently, so that the virus taking the rAAV2/Retro vector can express the foreign gene efficiently at a low dose when being used as a therapeutic drug, thereby reducing the drug dose and avoiding the high-titer virus from triggering the immune response of an organism.
The rAAV 2/retroro vector has a unique reverse retrospective infection mode, so that a virus medicament constructed based on the rAAV 2/retroro vector only invades neurons through the long axis protruding ends, has no infection capacity on neurons nearby cell bodies, and specifically acts on photoreceptor cells, thereby avoiding potential risks caused by non-specific infection of the traditional AAV2 and AAV8 vectors.
The medicament for treating the retinal diseases has the advantages of simple operation and small damage, and can be injected into the vitreous cavity by only passing through the sclera, the pigment epithelium layer and the pars plana of the ciliary body on the outer side, so that the retinal detachment is avoided, the retinal cells are small in damage, and the inflammatory reaction is light. The side effects that the existing medicine needs to be injected through the subretinal space, and the injection needs to cross the vitreous body and pass through the retina again, so that the partial separation of the retina can be caused, and the glia cell proliferation, the retinal detachment, the degeneration of the photoreceptors and the visual function damage can be caused are avoided.
The medicament for treating the retinal diseases constructed by the invention also has the advantage of wider action range, and the medicament constructed by the invention can be used for retroactively infecting reversely, so that the outer nuclear layer can be infected by injecting the medicament into the vitreous cavity. The vitreous cavity is an anterior retinal cavity, and virus medicaments in the vitreous cavity can fully contact and infect the whole retina, so that the problems that the existing medicaments can only be injected through a lower retinal cavity, the lower retinal cavity is a potential cavity existing when a neural epithelial layer and a pigment epithelial layer of the retina are separated, and the diffusion of the virus medicaments is very limited are solved.
Drawings
FIG. 1 is a view of a retinal tissue map; FIG. 1A is a HE staining pattern, and FIG. 1B is a schematic diagram of retinal tissue structure.
FIG. 2 is a schematic diagram of the main mode of administration of the virus; fig. 2A is a schematic diagram of a vitreous cavity injection and fig. 2B is a schematic diagram of a sub-retinal cavity injection.
FIG. 3 is a diagram of reverse retrospective infection pathways of rAAV2/Retro serotype adeno-associated virus to neurons.
FIG. 4A is a schematic diagram of intravitreal injection and reverse retrospective infection efficacy of rAAV 2/retrotro; FIG. 4B shows that after 6 months after intravitreal injection of rAAV2/Retro-CAG-GFP, the retinal tissue sections were stained with photoreceptor cell marker Recoverin antibody (red), and the GFP (green) gene of interest was highly expressed in the outer nuclear layer.
FIG. 5 is a graph comparing the effects of intravitreal rAAV2/Retro-CAG-GFP and subretinal AAV2/5-CAG-GFP viruses; FIG. 5A shows that the rAAV 2/retrovirus carries the target gene expressed in the whole retina; FIG. 5B shows that the target gene carried by the conventional AAV2/5 virus is expressed only locally.
FIG. 6 is a graph showing the effects of conventional virus (AAV2, AAV5, AAV7, AAV8A) injection and infection; FIG. 6A is a schematic view of a subretinal injection; FIG. 6B is a schematic representation of forward infection, with green arrows representing cells non-specifically infected with the inner nuclear layer and the ganglion cell layer.
FIG. 7 is a graph showing the effect of viral infection by injected empty vector virus rAAV2/Retro-CAG-GFP and rAAV2/Retro-CAG-mGata3-GFP carrying the gene of interest Gata 3; FIG. 7A is an empty vector virus with negative for Gata3 throughout the retina (red triangle); FIG. 7B shows a virus carrying the gene of interest, with Gata3 strongly positive in the outer nuclear layer (white triangles) and negative in both the inner nuclear and nodal cell layers.
FIG. 8 is a schematic diagram of the rAAV2/retro helper plasmid.
FIG. 9 is a schematic diagram of pAAV plasmid.
FIG. 10A is a graph of the apparent adaptation of the retinal Electrophysiology (ERG) peak after gene drug treatment in cpfl5 mice for 6 months; FIG. 10B is a statistical B-wave amplitude plot of the photopic retinal Electrophysiology (ERG) assay of cpfl5 mice before and after gene drug treatment.
FIG. 11 is a graph showing the expression of GFP in the retina of normal C57bl/6j mice at 6 months after intravitreal injection of rAAV 2/Retro.
FIG. 12 is a graph showing the therapeutic effect of the gene drug (AAV 2/Retro-U6-shNrl); FIG. 12A is a graph of mean adaptive retinal Electrophysiology (ERG) peaks following 6 months of gene drug therapy in rd1 mouse; FIG. 12B is a statistical graph of B-wave amplitude of retinal Electrophysiological (ERG) assays performed on rd1 mice before and after gene drug therapy.
FIG. 13 is a graph showing the expression of retinal Nrl after rd1 mice were injected with control drug (AAV2/Retro-U6-shControl) and gene drug (AAV2/Retro-U6-shNrl), respectively; FIG. 13A is a graph of Nrl expression in the outer nuclear layer of retina after control drug treatment; very strong (red triangle); FIG. 13B is a graph showing Nrl expression in the outer nuclear layer of retina after gene drug treatment. Significantly weaker than the control (pink triangle) and yellow arrows mark the recovery of outer nuclear layer nerve fibers.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. The raw materials used in the following examples are all commercially available general-purpose products unless otherwise specified.
Example 1 use of rAAV2/Retro for Assembly of adeno-associated viruses
The HEK293 cell line was cotransformed with a three plasmid system to produce infectious virus particles. HEK293 cells are cell lines obtained by transforming human embryonic kidney cells with modified adenovirus type 5 DNA and express adenovirus E1 protein. The three plasmids included the rAAV2/Retro helper plasmid (purchased from Addgene under code number 81070), the pAdDeltaF6 helper virus plasmid (purchased from Addgene under code number 112867), and the pAAV expression plasmid (purchased from Addgene under code number 37825).
rAAV2/Retro helper plasmid the minor capsid proteins rAAV2/Retro required for viral assembly were generated in the HEK293 cell line as shown in FIG. 8. Wherein 43 to 145 are ITRs; 413 to 2152 is the virus major capsid protein 1(VP 1); 2169 to 4406 are virus minor capsid proteins retro; 5185 to 5698 are M13 ori; 6449 to 6549 is the ampicillin promoter; 6550 to 7410 is ampicillin; 7581 to 8169 are pUC ori; the viral minor capsid protein retro is the key to the specificity of adeno-associated virus infection.
pAAV is shown in FIG. 9 and has been engineered as an expression plasmid. Wherein 182 to 770 are pUC ori; 941 to 1801 are ampicillin; 1932 to 2387 are f1 ori; 2459 to 2588 are AAV2 ITRs; 3095 to 3336 are Multiple Cloning Sites (MCS); 3343 to 3931 is WPRE; 3974 to 4095 is SV40 polyA; 4257 to 4386 are AAV2 ITRs. Various promoters may be inserted within the multiple cloning site, including but not limited to CMV, CAG, U6, SYN, GFAP, EF1 α. Various expression fragments of interest can be inserted thereafter, including but not limited to various common gene fragments, retinal genetic disease-related gene fragments, shRNA.
Example 2 preparation of rAAV 2/retroviruses for delivery of pAAV-CAG-Cnga3-GFP expression System in retinal photoreceptor cells
When the Cnga3 gene mutation causes the loss of Cnga3 protein, cone cells gradually degenerate and die, cone system function gradually loses, full color blindness and impairment of eyesight are caused, and the gradual reduction of b wave amplitude after the eyesight adaptation is mainly shown. Overexpression of exogenous Cnga3 protein reversed this process.
The rAAV2/Retro-CAG-Cnga3-GFP virus medicine is obtained by pAAV-CAG-Cnga3-GFP, pAdDeltaF6 and rAAV2/Retro helper plasmids through a virus packaging process, and comprises an rAAV2/Retro virus vector, a Cnga3 gene (ORF) and a reporter gene GFP, wherein the GFP can be removed in formal disease treatment gene medicines.
The rAAV2/Retro-CAG-Cnga3-GFP virus medicine is prepared by the following specific preparation process:
1) construction of pAAV-CAG-Cnga3-GFP plasmid
The CAG promoter, full length mouse Cnga3 ORF, GFP were cloned in tandem into pAAV expression plasmid. The BamHI and XbaI sites of pAAV multiple cloning site were used as cloning targets, as shown in FIG. 9, the main fragment to be cloned was full-length mouse Cnga3 ORF, obtained from mouse 661W cell line by PCR amplification reaction, CAG promoter, full-length mouse Cnga3 ORF, GFP were amplified in tandem by overlap PCR technique and BamHI and XbaI sites were introduced. The pAAV expression plasmid and CAG-Cnga3-GFP tandem product obtained by overlap PCR are subjected to plasmid recombination after being cut by BamHI and XbaI, and the recombinant plasmid is propagated in E.coli Stbl3 and subjected to sequence verification in a Seimerland sequencing facility. Wherein, the nucleotide sequence of Cnga3 is shown in SEQ ID NO. 1; the nucleotide sequence of the CAG promoter is shown as SEQ ID NO. 2.
Before generating the rAAV2/Retro-CAG-Cnga3-GFP virus drug, the accuracy of the pAAV-CAG-Cnga3-GFP plasmid DNA sequence was confirmed. Wherein 182 to 770 are pUC ori; 941 to 1801 are ampicillin; 1932 to 2387 are f1 ori; 2459 to 2588 are AAV2 ITRs; 2899 to 3176 are CAG promoter; 3490 to 5499 is m-Cnga 3; 5512 to 6231 is GFP; 6356 to 6844 are WPRE; 6887 to 7008 is SV40 polyA; 7170 to 7299 is AAV2 ITR.
2) rAAV2/Retro-CAG-Cnga3-GFP virus preparation
(ii) viral packaging of rAAV2/Retro-CAG-Cnga3-GFP virus
a. Preparing HEK-293 cells: add 3X 10 DMEM growth medium to each 100mm cell culture dish 10mL6HEK-293 cells were used for transfection after 48 hours. The health of the HEK-293 cells and the density of passages are very important factors in order to obtain higher titers. When the cell confluence rate reaches 50%, the cells need to be passaged. And cryopreserved in large quantities when cells are in low passage and healthy growth. When passagedAnd plating to avoid cell clumping when used for transfection. Cells were grown to higher confluency before transfection with pAAV-CAG-Cnga3-GFP, pAdDeltaF6, rAAV2/Retro helper.
Transfection of HEK-293 cells: although a number of transfection protocols were successfully used for transfection of these vectors, the PEI MAX 40K (available from Polysciences Inc., cat. 49553-93-7) transfection method described below was recommended, and was stably available when used to transfect HEK-293 cells>107Product titer of virus particles/ml. The method comprises the following specific steps:
examination of HEK293 cells passaged two days before transfection, they should reach 70% to 80% confluence;
plasmid to be co-transfected, pAAV-CAG-Cnga3-GFP, pAdDeltaF6, rAAV2/Retro helper, was removed from-20 ℃ freezer and the concentration of plasmid was adjusted to 1mg/mL using pH7.5 TE buffer;
calculating the required transfection system and plasmid dosage according to the number of packaging discs, if one disc is packaged, sucking 5 mu L (or 5 mu g) of each of pAAV-CAG-Cnga3-GFP, pAdDeltaF6 and rAAV2/Retro helper plasmids into a 1.5mL EP centrifuge tube containing 150ul of serum-free DMEM, uniformly mixing, standing at room temperature for 2 minutes, adding 60ul of PEI MAX with the concentration of 1mg/mL, standing at room temperature for 10 minutes after vortex mixing, and adding 7mL of DMEM growth medium to prepare plasmid DNA/PEI MAX/DMEM mixed solution.
Taking out the cell culture disc from the 37 ℃ incubator, removing the original culture medium, and adding the whole plasmid DNA/PEI MAX/DMEM mixed solution;
returning the culture dish to the incubator, and culturing for another 66-72 hours.
② toxic materials are observed
The extent of packaging of rAAV2/Retro-CAG-Cnga3-GFP viral particles was known by observing morphological changes in HEK-293 cells. For convenience, a negative control was made for packaging a virus, and the transfected group without the aforementioned plasmid was used as a negative control. The most obvious sign of the success of virus packaging is that the color of the medium changes from red to orange or yellow compared to the negative control, and as the virus progresses, some cells become rounded and fall off the disc, and can be seen floating in the medium. Three days after transfection is the best time to collect rAAV2/Retro-CAG-Cnga3-GFP virus particles.
Collecting rAAV2/Retro-CAG-Cnga3-GFP virus particles
rAAV2/Retro-CAG-Cnga3-GFP viral particles are present in both the packaging cells and the culture supernatant, so both cells and culture supernatant are collected for best recovery. The method comprises the following specific steps:
preparing a dry ice ethanol bath and a 37 ℃ water bath;
collecting the toxigenic cells and the culture medium into a 15mL centrifuge tube, and when the cells are collected, inclining a culture disc by a certain angle to scrape the cells into the culture medium;
centrifuging the centrifuge tube with the toxigenic cells and the culture medium under the centrifugation condition of 200g and 3 minutes to separate the toxigenic cells and the culture supernatant, storing the culture supernatant additionally, and resuspending the cells with 1mL of PBS to obtain a cell resuspension solution;
repeatedly transferring the cell resuspension in a dry ice ethanol bath and a water bath at 37 ℃, carrying out freeze-thaw lysis for four times, and slightly shaking each time after each time of thawing, wherein each time of coagulation and thawing is approximately ten minutes;
and centrifuging the cell resuspension after freeze-thaw lysis under the centrifugation condition of 10000g for 5 minutes to remove cell debris, and transferring the centrifuged cell freeze-thaw lysis supernatant to a new centrifuge tube.
Concentration of rAAV2/Retro-CAG-Cnga3-GFP virus
Adding 40% PEG8000 to the supernatant in the new centrifugal tube until the final concentration is 8%, standing on ice for 2 hours, wherein, mixing uniformly every 15 minutes, then centrifuging for 30 minutes under the condition of 2500Xg, removing the supernatant, and combining the collected precipitate with the cell freeze-thaw lysis supernatant after being resuspended by PBS;
mix of PBS resuspended pellet and cell lysis supernatant was centrifuged at 3,000xg for 30 minutes and the supernatant was transferred to another clean tube. If the supernatant still has partial debris, centrifuging again and collecting the supernatant;
adding Benzonase nuclease (final concentration of 50U/mL) to the collected supernatant to digest and remove residual plasmid DNA, closing the tube cap, inverting several times to mix well, and incubating at 37 ℃ for 30 minutes;
filtering with 0.45um filter head, and collecting filtrate to obtain concentrated solution of rAAV2/Retro-CAG-Cnga3-GFP virus.
Fifthly, the rAAV2/Retro-CAG-Cnga3-GFP virus is primarily purified
Adding solid cesium chloride (CsCl) to the rAAV2/Retro-CAG-Cnga3-GFP virus concentrate until the density is 1.41g/mL, adding 6.5g CsCl to about 10mL rAAV2/Retro-CAG-Cnga3-GFP virus concentrate, and shaking to dissolve;
adding the rAAV2/Retro-CAG-Cnga3-GFP virus concentrated solution dissolved in CsCl into an ultracentrifuge tube, and filling the residual space of the centrifuge tube with a pre-prepared 1.41g/mL CsCl solution;
centrifuge at 175,000g for 24 hours to form density gradients. Collecting samples with different densities step by step in sequence, sampling for titer determination, and collecting components enriched with rAAV2/Retro-CAG-Cnga3-GFP virus particles;
the above procedure was repeated once and the collected rAAV2/Retro-CAG-Cnga3-GFP viral particles were purified again.
Sixth, desalting and purifying by ultrafiltration
Leaching: 4mL of deionized water is added into an Amicon-15 ultrafiltration device; once wetted in the ultrafiltration device, the membrane cannot be left to air dry again. If not used immediately after rinsing, water was left on the membrane until the experiment was started;
adding rAAV2/Retro-CAG-Cnga3-GFP virus solution obtained by density gradient centrifugation into an ultrafiltration device, adding PBS to make the total volume be 4mL, and covering a cover; subsequently, centrifugation was carried out at 1,500Xg for 10 minutes, and the number of remaining volumes was checked every 5 minutes until the final volume was 200 to 250. mu.L, without reducing the volume to below 200. mu.l, because this would lead to aggregation of virus clones. If the titer is higher, the minimum volume is increased accordingly;
collecting the filtered solutions together for sterilization, and returning the filter membrane to the device; the concentrated virus was diluted with 1 × PBS to a volume of 4 mL;
repeat the above process 3 times; centrifuging the ultrafiltration tube to a final volume of about 0.5 mL; glycerol was added to the virus concentrate to give a final concentration of 5%. Subpackaging and storing at-80 ℃.
Measurement of rAAV2/Retro-CAG-Cnga3-GFP virus titer
We used quantitative PCR method to determine the number of viral particles of rAAV2/Retro-CAG-Cnga3-GFP by detecting the genomic copy number of the rAAV2/Retro-CAG-Cnga3-GFP viral vector. The accuracy and reliability of the standard curve absolute quantitative PCR detection titer are the most critical elements of the quality control of the rAAV2/Retro-CAG-Cnga3-GFP, and the result influences the accuracy of downstream experiments. Therefore, in our quality control procedure, the rAAV2/Retro-CAG-Cnga3-GFP virus titer was well designed to ensure its accuracy and stability, such as designing a series of standards, validation samples (viruses and plasmids) and controls (to remove background and DNA contamination) in the assay. The titration primer used in the quality control process is a stock reagent, so that the virus production cycle can be shortened to adapt to the demand of vector construction at a fast pace.
rAAV2/Retro-CAG-Cnga3-GFP virus was generated using the method described above, and viral particles were titrated and resuspended in balanced salt solution containing 0.014% Tween-20 at a concentration of 1.5X 1012Viral vector genome/mL (vg/mL), sterility and endotoxin deficiency were confirmed in the final product.
For comparison, using the above procedure, only the prAAV 2/retror in the three-plasmid transfection system described above was replaced with the conventional pAAV2/5 (addendum cat No. 104964) to generate rAAV2/5-CAG-Cnga3-GFP virus, and the viral particles were titrated and resuspended in balanced salt solution containing 0.014% Tween-20 at a concentration of 1.5X 1012Viral vector genome/mL (vg/mL), sterility and endotoxin deficiency were confirmed in the final product.
EXAMPLE 3 preparation of rAAV2/Retro Virus for delivery of pAAV-U6-shNrl expression System on retinal photoreceptor cells
Nrl gene expression generates Nrl protein, which is highly expressed in photoreceptor cells of outer nuclear layer of retina in retinitis pigmentosa, and inhibition of Nrl gene expression can significantly reduce photoreceptor cell apoptosis caused by retinitis pigmentosa.
The rAAV2/Retro-U6-shNrl virus medicament is obtained by pAAV-U6-shNrl, pAdDeltaF6 and rAAV2/Retro helper plasmids through a virus packaging process, and comprises an rAAV2/Retro virus vector and shRNA aiming at Nrl genes.
The rAAV2/Retro-U6-shNrl virus medicament is prepared by the following specific steps:
1) construction of pAAV-U6-shNrl plasmid
The U6 promoter, shrna against mouse Nrl gene (shnrl) were cloned in tandem into pAAV expression plasmid. The U6 promoter, shNrl, were amplified in tandem and BamHI and XbaI sites were introduced by overlap PCR technique using BamHI and XbaI sites of pAAV multiple cloning site as cloning targets as shown in FIG. 9. The pAAV expression plasmid and the U6-shNrl tandem product obtained by the overlap PCR are digested by BamHI and XbaI, then plasmid recombination is carried out, and the recombinant plasmid is propagated in E.coli Stbl3 and subjected to sequence verification in a Seimer Fei sequencing facility. Wherein, shControl obtained by scrambling the sequence of shNrl is used as a reference to construct pAAV-U6-shControl. The nucleotide sequence of shNrl is shown as SEQ ID NO. 3; the nucleotide sequence of the U6 promoter is shown as SEQ ID NO. 4; the nucleotide sequence of shControl is shown in SEQ ID NO. 5.
The accuracy of the pAAV-U6-shNrl plasmid DNA sequence was confirmed prior to generation of rAAV2/Retro-U6-shNrl viral drug. Wherein 182 to 770 are pUC ori; 941 to 1801 are ampicillin; 1932 to 2387 are f1 ori; 2459 to 2588 are AAV2 ITRs; 3206 to 3341 are U6 promoter; 3452 to 3500 are shNrl; 3529 to 4117 are WPRE; 4160 to 4281 is SV40 polyA; 4443 to 4542 are AAV2 ITRs.
2) rAAV2/Retro-U6-shNrl and rAAV2/Retro-U6-shControl virus preparation
Referring to the packaging procedure for virus export of rAAV2/Retro-CAG-Cnga3-GFP virus in example 2, the pAAV-CAG-Cnga3-GFP plasmid used in the procedure was replaced with pAAV-U6-shNrl plasmid in full equal amount, i.e., rAAV2/Retro-U6-shNrl virus was obtained in the final product.
Referring to the packaging procedure of rAAV2/Retro-CAG-Cnga3-GFP virus in example 2, the pAAV-CAG-Cnga3-GFP plasmid used in the procedure was replaced with pAAV-U6-shControl plasmid in equal amount, i.e., rAAV2/Retro-U6-shControl virus was obtained in the final product.
Example 4 therapeutic Effect of rAAV2/Retro-CAG-Cnga3-GFP Virus
In this example, a congenital achromatopsia mouse was used as an experimental subject to investigate the therapeutic effect of the rAAV2/Retro-CAG-Cnga3-GFP virus prepared in example 2, and the specific experimental protocol was as follows:
experimental animals: normal 10-week-old C57bl/6j mice were purchased from Experimental animals center, Guangdong province; congenital achromatopsia transgenic Mice cpfl5 rice were purchased from viton. The illumination period is 12h, the illumination is dark-12 h, the food intake and the water drinking are freely carried out, and all animal researches are strictly carried out according to the experimental animal management regulations issued by the national science and technology committee.
Main reagents and consumables: physiological saline (Zhejiang Tianrui pharmaceutical Co., Ltd.), a disposable syringe needle (Becton Dickinson and Company, USA), goat serum (Sigma), polyethylene glycol octylphenyl ether (Triton X-100) (Sigma), OCT tissue embedding agent (cherry blossom), paraformaldehyde (Sigma), DAPI (Biyuntian), Topiramide (Shenyang Qinjiao Ocular medicine Co., Ltd.), tribromoethane (Sigma), PBS solution (Zhejiang Senrui Biotech Co., Ltd.), tobramycin eye ointment (Tobacil).
The main apparatus is as follows: ophthalmic experimental operating microscope (Nikon corporation, japan); ophthalmic microscopy instruments (suzhou mingren medical instruments ltd); microsyrinths (Hamilton, usa); fluorescence microscope (zeiss, germany), digital fundus colorography and fluorography system (Topcon), ERG system (Roland Consult), cryomicrotome (laika).
Intraocular injection: anesthetizing mouse with tribromoethanol at a dose of 0.14mL/10G, dilating pupil with 0.5% compound tropicamide, puncturing vitreous cavity with 30G capsule-breaking needle inclined at 45 ° in avascular region behind sclera corneal limbus, injecting into vitreous cavity with 33G microinjector from 45 ° needle at the opening of the capsule-breaking needle, and injecting 1 μ L of the injection with concentration of 1.5 × 1012vg/mL of rAAV2/Retro-CAG-Cnga3-GFP virus drug and rAAV2/Retro-CAG-GFP control virus. Slowly pulled out microscope after remaining for 10sSyringe, no liquid leakage was confirmed. The ocular surface was coated with tobramycin ointment to prevent infection and the mice were placed on a 37 ℃ resuscitation blanket until they were awakened. And (5) conventionally feeding the mice after the operation.
ERG examination: ERG examination is carried out at 14 days, 28 days, 2 months, 3 months and 6 months after the operation, and the improvement of the visual function of the gene therapy is confirmed. Mice were anesthetized with tribromoethanol (0.14mL/10g body weight), compound topiramide (0.5%) dilated pupils, and sodium cellulose eye drops were applied to the ocular surface of mice. ERG detection methods are described in the literature [ Yang S, Luo X, Xiong G, So KF, Yang H, Xu Y. the electrotreatin of Mongolian gerbil (Meriones unguiculatus): compare to mouse. Neurosci Lett.2015; 589:7.] the results of electrophysiological measurements of mouse adaptation to retina are shown in fig. 10 before and after 6 months of gene drug treatment (rAAV 2/retroro-CAG-Cnga 3-GFP) in mice, and it was found that the b-wave amplitude of cpfl5 mice was significantly improved over that of mice injected with control viruses in the eye after 6 months of gene drug treatment in cpfl5 mice, and that the b-wave amplitude of cpfl5 mice treated with the gene drug of the present invention was not significantly different from that of normal same-week-old mice.
Then, by carrying out immunofluorescence detection on the cpfl5 mice treated by the gene medicament, the method comprises the following specific steps:
tissue treatment: the materials are taken after 6 months of operation, the animals are firstly subjected to whole body perfusion by PBS to remove blood, then are perfused and fixed by 4 percent paraformaldehyde, the perfusion speed is 5mL/min, the eyeball is taken out, is punctured at the corneal limbus of the sclera, is soaked in 4 percent paraformaldehyde and fixed overnight at 4 ℃, is subjected to OCT embedding after dehydration by 10 percent, 20 percent and 30 percent gradient sucrose, and is placed in liquid nitrogen for quick freezing. Retinal sections with a thickness of 10um were made using a frozen microtome.
And (3) performing immunofluorescence detection: taking an eyeball frozen section, washing for 3 times by using PBS (phosphate buffer solution) and washing away the OCT; nuclei were labeled using DAPI incubation for 5 min. Excess DAPI was removed by washing 3 times with PBS and mounted with an anti-fluorescence quencher. The GFP expression and retinal morphology were observed by fluorescence microscopy and photographed, and the results are shown in fig. 11, which shows that GFP fluorescence is still expressed specifically and efficiently in the outer nuclear layer of retina, and also shows that the gene drug can be expressed stably within 6 months.
Therefore, the rAAV2/Retro-CAG-Cnga3-GFP virus medicament prepared in the example 1 is injected into the vitreous cavity of a cpfl5 mouse, and the photoreceptor cells of the outer nuclear layer are specifically infected, so that the functional Cnga3 protein is re-expressed, the degeneration process of the cone cells of the cpfl5 mouse is effectively inhibited, and the b-wave amplitude of the photopic adaptation of the treated cpfl5 mouse reaches the level of a normal mouse, and therefore, the rAAV2/Retro-CAG-Cnga3-GFP virus medicament prepared in the invention has good curative effect on congenital panchromatosis.
Example 5 therapeutic Effect of rAAV2/Retro-U6-shNrl Virus
In this example, a retinitis pigmentosa mouse was used as an experimental subject to investigate the therapeutic effect of the rAAV2/Retro-U6-shNrl virus prepared in example 3, and the specific experimental protocol was as follows:
experimental animals: normal 10-week-old C57bl/6j mice were purchased from Experimental animals center, Guangdong province; retinitis pigmentosa transgenic mouse rd1 Mice was purchased from Witonglihua. The illumination period is 12h, the illumination is dark-12 h, the food intake and the water drinking are freely carried out, and all animal researches are strictly carried out according to the experimental animal management regulations issued by the national science and technology committee.
Main reagents and consumables: physiological saline (Zhejiang Tianrui pharmaceutical Co., Ltd.), a disposable syringe needle (Becton Dickinson and Company, USA), goat serum (Sigma), polyethylene glycol octylphenyl ether (Triton X-100) (Sigma), OCT tissue embedding agent (cherry blossom), paraformaldehyde (Sigma), DAPI (Biyuntian), Topiramide (Shenyang Qinjiao Ocular medicine Co., Ltd.), tribromoethane (Sigma), PBS solution (Zhejiang Senrui Biotech Co., Ltd.), tobramycin eye ointment (Tobacil).
The main apparatus is as follows: ophthalmic experimental operating microscope (Nikon corporation, japan); ophthalmic microscopy instruments (suzhou mingren medical instruments ltd); microsyrinths (Hamilton, usa); fluorescence microscope (zeiss, germany), digital fundus colorography and fluorography system (Topcon), ERG system (Roland Consult), cryomicrotome (laika).
Intraocular injection: with 0.1 parts of tribromoethanolAnesthetizing mouse at a dose of 4mL/10G, expanding pupil with 0.5% compound tobramide, tilting the 30G capsulorhexis needle at 45 ° in the posterior avascular region of the corneal limbus of sclera to avoid the lens from breaking into the vitreous cavity, inserting the needle at 45 ° from the opening of the capsulorhexis needle with a 33G microinjector to avoid the lens from entering the vitreous cavity, and injecting 1 μ L of the compound tobramide at a concentration of 1.5 × 1012vg/mL of rAAV2/Retro-U6-shNrl virus drug and rAAV2/Retro-U6-shControl control virus. After leaving the vessel for 10 seconds, the microinjector was slowly pulled out to confirm that no liquid was leaked. The ocular surface was coated with tobramycin ointment to prevent infection and the mice were placed on a 37 ℃ resuscitation blanket until they were awakened. And (5) conventionally feeding the mice after the operation.
ERG examination: ERG examination is carried out at 14 days, 28 days, 2 months, 3 months and 6 months after operation, and the improvement of the visual function of gene drug treatment is confirmed. Mice were anesthetized with tribromoethanol (0.14mL/10g body weight), compound topiramide (0.5%) dilated pupils, and sodium cellulose eye drops were applied to the ocular surface of mice. ERG detection methods are described in the literature [ Yang S, Luo X, Xiong G, So KF, Yang H, Xu Y. the electrotreatin of Mongolian gerbil (Meriones unguiculatus): compare to mouse. Neurosci Lett.2015; 589:7.] the results of electrophysiological measurements of the retina in response to photopic adaptation of mice before and after 6 months of gene drug treatment (rAAV2/Retro-U6-shNrl) in the Rd1 mice are shown in fig. 12, and it was found that the b-wave amplitude of the Rd1 mice was significantly improved over that of the mice in the control virus group injected into the eyes 6 months after gene drug treatment in the Rd1 mice.
Then, the rd1 mouse treated by the gene medicine of the invention is subjected to immunofluorescence detection, and the specific steps are as follows:
tissue treatment: the materials are taken after 6 months of operation, the animals are firstly subjected to whole body perfusion by PBS to remove blood, then are perfused and fixed by 4 percent paraformaldehyde, the perfusion speed is 5mL/min, the eyeball is taken out, is punctured at the corneal limbus of the sclera, is soaked in 4 percent paraformaldehyde and fixed overnight at 4 ℃, is subjected to OCT embedding after dehydration by 10 percent, 20 percent and 30 percent gradient sucrose, and is placed in liquid nitrogen for quick freezing. Retinal sections with a thickness of 10um were made using a frozen microtome.
And (3) performing immunofluorescence detection: taking an eyeball frozen section, washing for 3 times by using PBS (phosphate buffer solution) and washing away the OCT; retinas infected with the gene drug rAAV2/Retro-U6-shNrl and control virus AAV2/Retro-U6-shControl were labeled with an antibody specific for marker Nrl, and the nuclei were labeled with DAPI incubation for 5 min. Excess DAPI was removed by 3 washes with PBS and mounted with an anti-fluorescence quencher. Nrl expression and retinal morphological structure were observed by fluorescence microscope and photographed, and the results are shown in FIG. 13, FIG. 13A is a graph of Nrl expression in the outer nuclear layer of retina after control virus treatment, as indicated by red triangle in the figure, it can be seen that Nrl expression in the outer nuclear layer of retina is very strong; FIG. 13B is a graph showing Nrl expression in the outer retinal nucleus layer after gene drug treatment, in which Nrl expression was significantly reduced in the outer retinal nucleus layer after gene drug treatment as compared with that in the control group, FIG. 13A, as indicated by the yellow arrow mark, and it can be seen that the treatment of gene drug treatment was helpful in promoting the recovery of outer nuclear layer nerve fibers.
Therefore, the rAAV2/Retro-U6-shNrl virus medicament prepared in example 3 is injected into the vitreous chamber of an rd1 mouse, the photoreceptor cell of an outer nuclear layer is specifically infected, and the expression of Nrl gene is knocked down, so that the degeneration process of the photoreceptor cell of an rd1 mouse is effectively inhibited, and the b-wave amplitude of ERG of an rd1 mouse after treatment is obviously improved, therefore, the rAAV2/Retro-U6-shNrl virus medicament prepared in the invention has good curative effect on congenital retinitis pigmentosa.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
<110> Zhongshan ophthalmic center of Zhongshan university
<120> rAAV2/Retro serving as delivery system for retinal photoreceptor cells and preparation of delivery system for treating retinal diseases
Application of medicine
<130> 0220062-
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 2007
<212> DNA
<213> Cnga3
<400> 1
atggcaaagg tgaacaccca gtgttcacag ccctccccga cccaactttc aataaagaat 60
gcggacagag atctcgacca cgtagagaac ggcctgggca ggtctcactc accatgtgag 120
gagacatctt ccacactgca gcaagggatc gccatggaga cccgaggact ggctggatcc 180
gcacggagcg tagtcacaag ccagggacca gccagggtgt cacgcctcat catctcgatt 240
cgtgcgtggg cctccaggca cttacacgat gaagaccaga cacctgactc ctttttggat 300
cgatttcatg gatctgagct taaggaagtc tccacccggg aaagcaatgc ccagcccaac 360
ccaggagaac agaagccacc agacggaggg gaaggcagga aggaggagcc cattgtggtg 420
gacccctcca gcaacatcta ctaccgctgg ctgactgcca tcgcgctccc agtcttctat 480
aactggtgtc tacttgtatg cagggcctgt tttgatgagc tacaatcaga gcacctgacg 540
ctgtggctgg tcttggacta ctctgcagat gtcctttatg ttttagacat gctggttcga 600
gcccggacag gtttccttga gcaaggccta atggtcagag ataccaagag gctgtggaaa 660
cattacacaa agaccttgca cttcaagctg gacatcctgt ctctcatccc cacagacctg 720
gcttatttaa agttgggcgt gaactaccca gaactgaggt tcaaccgcct cctgaagttc 780
tctcggctct ttgaattctt tgaccgcaca gagacaagga ccaactaccc caacgtgttc 840
aggataggga acttggtcct ttacacactc atcatcatcc actggaatgc ctgcatctac 900
tttgccattt ccaagttcat tggttttggg acagactcct gggtctatcc aaacacctcc 960
aagccggagt atgcacgact ctcccggaag tacatttaca gtctctactg gtccaccctg 1020
accctgacca ccattggtga gaccccaccc cccgtgaaag atgaagagta tctctttgtg 1080
gtcatcgact tcctggtggg tatcctgatc tttgccacca tagtgggcaa cgtaggctcc 1140
atgatttcca acatgaacgc tccccgggta gagttccagg ctaagatcga ctccgtcaag 1200
cagtacatgc agttccggaa ggtaaccaag gacttggaga ctcgggttat ccggtggttt 1260
gactatctgt gggccaacag gaagacggtg gatgaaaagg aagtgctcaa aaacctcccg 1320
gacaagctga aggctgagat cgccatcaac gtgcacctgg acacgctgaa gaaggtccga 1380
atcttccagg actgcgaggc ggggctgctg gtggagctgg tgctgaagct ccgccccact 1440
gtgttcagcc ccggggacta catatgcaaa aagggggaca ttggaagaga gatgtacatc 1500
atcaaggagg gcaagctggc tgtggtggct gatgacgggg tcacccagtt tgtggtcctc 1560
agtgacggca gttactttgg ggagattagc atcttaaaca ttaaggggag caagtctggg 1620
aaccgcagga cggccaacat caggagtatc ggctactcag acctgttctg cctctccaag 1680
gatgacctaa tggaagccct caccgagtac ccagacgcta agagggctct ggaggaaaag 1740
ggccgtcaga ttctgatgaa ggacaaccta attgatgagg acctagtcgc ggccagggta 1800
gataccaggg acgttgagga gaaggtggag tacctggagt cgtccctgga catcctgcag 1860
acgaggtttg cccgactcct ggctgagtac agtgcctccc agatgaagct gaaacagcgg 1920
ctcactcggc tggagagcca gatgaacagg aggtgttgtg gcttctcacc tgacagggag 1980
aattccgagg atgcttcaaa gactgac 2007
<210> 2
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<212> DNA
<213> CAG promoter
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tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa 60
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ggcgcgcgcc rggsggggsg gggsggggsg rggggsgggg cggggcgagg cggagaggtg 180
cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc 240
ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcg 278
<210> 3
<211> 49
<212> DNA
<213> shNrl
<400> 3
ggtcctgtct ctatggaagg tcgagccttc catagagaca ggacccttt 49
<210> 4
<211> 241
<212> DNA
<213> U6 promoter
<400> 4
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttgggtt tatatatctt gtggaaagga 240
c 241
<210> 5
<211> 47
<212> DNA
<213> shControl
<400> 5
ggtctgcgtt cgcgtgataa tcaagagtta tcacgcgaac gcagacc 47
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