Method for screening AAV with high infection ability of eyes

Abstract

According to the invention, capsid screening of the monkey vector is carried out by using a method of inserting AAV polypeptide into a library for screening, and the AAV capsid capable of remarkably improving eye infection is obtained. Through vitreous injection, compared with wild type AAV2, the infection ability is improved by 5-8 times, and potential eye disease treatment value is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene therapy, and specifically relates to a method for screening ocular AAV capsids and an application thereof. Background Art

[0002] The fundus is composed of the retina, fundus blood vessels, optic nerve head, optic nerve fibers, macula on the retina, and choroid behind the retina. Lesions in these parts are collectively referred to as fundus diseases. The "Big Data Report on the Status of Fundus Lesions in Chinese People" shows that the prevalence of fundus diseases in people aged 60 and over 70 is as high as 21.4% and 25.6% respectively. The causes are complex and varied, ranging from decreased vision to blindness, which seriously affects the quality of life of the elderly. At the same time, data show that fundus diseases are the primary cause of blindness, and their patients account for 54.7% of all patients with blinding eye diseases.

[0003] Common fundus lesions in clinical practice include diabetic retinopathy, fundus retinal vascular occlusion, age-related macular degeneration, retinal tears and detachment. Currently, the common treatment methods are mainly laser photocoagulation and the use of anti-VEGF drugs. Retinal laser photocoagulation can make the retinal tissue irradiated by laser lose its function, reduce oxygen demand, and preserve the normal operation of "normal" retinal function at the expense of local non-key areas. Therefore, when doctors perform laser treatment, the laser area is selective, usually in the peripheral area, areas with severe capillary occlusion, and severe retinal ischemia. Such laser has little effect on your vision, and the benefit is that it guarantees the metabolic needs of key areas of the retina, especially the macula. However, the essence of this method is damage to the retina, so it is difficult to maintain long-term improvement in vision. The emergence of anti-VEGF drugs has brought more effective treatments for indications such as wet macular degeneration and diabetic retinopathy, but they need to be injected into the eye, frequently administered, and patients have poor compliance. Multiple administrations are prone to cause intraocular fibrosis and other problems.

[0004] Reducing the frequency of anti-VEGF treatment and being able to significantly improve the symptoms of fundus diseases through a single dose is a pain point in this field. This gave birth to AAV gene therapy for fundus diseases, and many companies at home and abroad are actively developing in this direction, relying on the good safety and long-term expression of AAV. It can achieve long-term treatment effects after a single dose. However, this method has very high requirements for the specificity and infection efficiency of AAV infection, and natural serotypes of AAV are difficult to meet the requirements of efficient delivery in the eye. Therefore, the development of serotypes that can efficiently infect the eye is extremely important. This patent constructs a method for screening AAV polypeptide insertion libraries, and screens AAVs with significantly enhanced infection capabilities in non-human primates. Summary of the invention

[0005] Herein, a system for high-throughput engineering of functional AAV capsids with altered tropism for various tissues is described, and using this system, capsid variants with increased tropism for target tissues such as ocular tissue have been identified.

[0006] In various aspects, the present disclosure provides a viral protein (VP) capsid polypeptide comprising a region 581 to 589 corresponding to residues 581 to 589 of an AAV2 VP1 polypeptide, wherein the region 581 to 589 comprises a sequence of NTMKDWD, EDTQHQR, PKTDQSD, or GPHTLTP. In various aspects, the present disclosure provides a viral protein (VP) capsid polypeptide comprising a region 581 to 589 corresponding to residues 581 to 589 of an AAV2 VP1 polypeptide, wherein the region 581 to 589 comprises a sequence having at least 85% sequence identity to any one of NTMKDWD, EDTQHQR, PKTDQSD, or GPHTLTP.

[0007] In some aspects, the 581 to 589 region comprises a sequence of any one of NSGHYDG, HSSALPN, APEYITW, AKHEELV, EDTQHQR, or HGQSANQ. In some aspects, the 581 to 589 region modification can increase ocular tissue tropism compared to a wild-type AAV2 VP capsid polypeptide. In some aspects, the ocular tropism is at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 75 times, at least 100 times, at least 200 times, or at least 500 times greater than a wild-type AAV2 VP capsid polypeptide.

[0008] In some aspects, the VP capsid polypeptide comprises an amino acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% identical to a wild-type AAV (AAV1, AAV2, AAV5, AAV8, or AAV9).

[0009] In various aspects, the present disclosure provides a recombinant adeno-associated virus (rAAV) comprising a VP capsid polypeptide assembled into a recombinant viral capsid as described herein and a payload encapsulated by the recombinant viral capsid.

[0010] In some aspects, the rAAV preferentially targets retinal tissue. In some aspects, the rAAV also includes a VP2 polypeptide containing 581 to 589 districts and a VP3 polypeptide containing 581 to 589 districts. In some aspects, the payload encodes therapeutic polynucleotides or therapeutic peptides. In some aspects, the payload encodes guide RNA, tRNA, suppressor tRNA, siRNA, miRNA, mRNA, shRNA, circular RNA or antisense oligonucleotides (ASO), ribozymes, DNA enzymes, aptamers or any combination thereof. In some aspects, the guide RNA is a CRISPR / Cas guide RNA or an ADAR guide RNA. In some aspects, the payload encodes components of the CRISPR / Cas system, adenosine deaminase (ADAR) enzyme acting on RNA, transcriptional activators or transcriptional repressors.

[0011] In various aspects, the present disclosure provides a pharmaceutical composition comprising a VP capsid polypeptide as described herein, a rAAV capsid as described herein, or a rAAV as described herein.

[0012] In various aspects, the present disclosure provides a method of delivering a payload to retinal tissue of a subject, the method comprising administering a recombinant adeno-associated virus (rAAV) to the subject by intravitreal administration, wherein the rAAV encapsulates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising regions 581 to 589.

[0013] In some aspects, the method includes administering the rAAV by intravitreal administration. In some aspects, the method includes administering the rAAV by subretinal injection, topical mucosal administration or systemic administration. In some aspects, the disease is an eye disease. In some aspects, the eye disease is color blindness, macular degeneration, cataract, choroideremia, glaucoma, optic neuropathy, Marfan syndrome, myopia, polypoidal choroidal vasculopathy, retinitis pigmentosa, Stargardt disease, Usher syndrome, Leber's congenital amaurosis, Leber's hereditary optic neuropathy, uveal melanoma or X-linked retinoschisis. In some aspects, the eye disease is Stargardt disease.

[0014] In some aspects, the payload encodes a therapeutic protein, a therapeutic polynucleotide, a guide RNA, a tRNA, a suppressor tRNA, a siRNA, a miRNA, a mRNA, a shRNA, a circular RNA, an antisense oligonucleotide (ASO), a ribozyme, a DNA enzyme, an aptamer, or any combination thereof. In some aspects, the guide RNA is a CRISPR / Cas guide RNA or an ADAR guide RNA. In some aspects, the therapeutic protein is selected from the group consisting of: CNGB3, NOS2A, CFH, CF, C2, C3, CFB, HTRA1 / LOC, MMP-9, TIMP-3, SLC16A8, GEMIN4, CYP51A1, RIC1, TAPT1, TAF1A, WDR87, APE1, MIP, Cx50, GJA3, GJA8, CRYAA, CRYBB2, PRX, POLR3B, XRCC1, ZNF350, EPHA2, REP1, CALM2, MPP-7, Optineurin, LOX1, CYP1B1, CAV1 / 2, MYOC, PITX2, FOXC1, PAX6, LTBP2, Complex I, ND, OPA1, RPE65, FBN1, TGFBR2, MTHFR, MTR, MTR R, HGF, C-MET, UMODL1, MMP-1 / 2, CBS, IGF-1, UHRF1BP1L, PTPRR, PPFIA2, P4HA2, SERPING1, PEDF, ARMS2-HTRA1, FGD6, ABCG1, LOC387715, CETP, NRL, RDH12, PRPH2(RDS), RHO, RPGR, SNRNP2 00, NR2E3, IMPDH1, CRX, HK1, IMPDH2, PRPF3, AGBL5, ABCA1, ABCA4, CRB1, USH2A, NRP1, ND4, RL BP1, PTEN, BAP1, GNAQ, GNA11, DDEF1, SF3B1, EIF1AX, CDKN2A, p14ARF, HERC2 / OCA2, VEGF and RS1.In some aspects, the therapeutic polynucleotide targets an mRNA encoding a protein selected from the group consisting of CNGB3, NOS2A, CFH, CF, C2, C3, CFB, HTRA1 / LOC, MMP-9, TIMP-3, SLC16A8, GEMIN4, CYP51A1, RIC1, TAPT1, TAF1A, WDR87, APE1, MIP, Cx50, GJA3, GJA8, CRYAA, CRYBB2, PRX, POLR3B, XRCC1, ZNF350, EPHA2, REP1, CALM2, MPP-7, Optineurin, LOX1, CYP1B1, CAV1 / 2, MYOC, PITX2, FOXC1, PAX6, LTBP2, Complex I, ND, OPA1, RPE65, FBN1, TGFBR2, MTHFR, MTR, MTRR, HGF, C-MET, UMODL1, MMP-1 / 2, CBS, IGF-1, UHRF1BP1L, PTPRR, PPFIA2, P4HA2, SERPING1, PEDF, ARMS2-HTRA1, FGD6, ABCG1, LOC387715, CETP, NRL, RDH12, PRPH2(RDS), RHO, RPGR, SNR NP200, NR2E3, IMPDH1, CRX, HK1, IMPDH2, PRPF3, AGBL5, ABCA1, ABCA4, CRB1, USH2A, NRP1, ND4, R LBP1, PTEN, BAP1, GNAQ, GNA11, DDEF1, SF3B1, EIF1AX, CDKN2A, p14ARF, HERC2 / OCA2, VEGF or RS1. In some aspects, the payload encodes a therapeutic polynucleotide targeting ABCA1, ABCA4 or CRB1 or a transgene encoding ABCA1, ABCA4 or CRB1. In some aspects, the payload encodes a component of a CRISPR / Cas system, an adenosine deaminase (ADAR) enzyme acting on RNA, a transcriptional activator or a transcriptional repressor. In some aspects, the components of the CRISPR / Cas system include Cas3, Cas8, Cas10, Cas9, Cas4, Cas12, Cas13, a guide RNA or a combination thereof. In some aspects, the ADAR enzyme is ADAR1 or ADAR2. In some aspects, the transcriptional activator is VP64. In some aspects, the transcriptional repressor is KRAB.

[0015] In some aspects, the method further comprises expressing a therapeutic polypeptide or therapeutic polynucleotide encoded by the payload. In some aspects, the method comprises expressing a therapeutic polypeptide or therapeutic polynucleotide at a higher level than a wild-type AAV2 capsid in an ocular tissue. In some aspects, the method comprises expressing a therapeutic polypeptide or therapeutic polynucleotide at a level of at least 3 times, at least 3.5 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 75 times, at least 100 times, at least 200 times, at least 500 times, or at least 1000 times in retinal tissue compared to a wild-type AAV2 capsid. In some aspects, the method comprises producing a toxicity lower than the toxicity produced when a considerable amount of wild-type AAV2 capsid is applied in the subject. In some aspects, the method comprises producing a toxicity lower than the toxicity produced when a considerable amount of payload is applied to a quantity of a wild-type AAV2 capsid that is sufficient to deliver a considerable amount of payload to retinal tissue. In some aspects, the method includes generating a lower concentration of neutralizing antibodies in the subject than the concentration of neutralizing antibodies generated when a considerable amount of wild-type AAV2 capsid is administered. In some aspects, the method includes generating a lower concentration of neutralizing antibodies in the subject than the concentration of neutralizing antibodies generated when a considerable amount of wild-type AAV2 capsid is administered. In some aspects, the method includes generating a lower concentration of neutralizing antibodies in the subject than the concentration of neutralizing antibodies generated when a considerable amount of wild-type AAV8 capsid is administered. In some aspects, the method includes generating a lower concentration of neutralizing antibodies in the subject than the concentration of neutralizing antibodies generated when a considerable amount of wild-type AAV9 capsid is administered. In some aspects, the method includes generating a lower concentration of neutralizing antibodies in the subject than the concentration of neutralizing antibodies generated when a considerable amount of wild-type AAV2 capsid is administered to deliver a considerable amount of payload to ocular tissue. Beneficial Effects

[0016] The present invention can significantly enhance the infection of AAV to the eye, thereby reducing the amount of AAV used in ocular gene delivery, which can be increased by 5-8 times compared with wild-type AAV. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Plasmid map of TE54 Figure 2 Improvement in the infectivity of the screened peptides inserted into AAV compared to wild-type AAV (expression level verified separately after peptide insertion) DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of this application more clear, the following is a further detailed description of this application in conjunction with specific embodiments. It should be understood that the specific embodiments described here are only used to explain this patent and are not used to limit the present invention.

[0020] Example 1: Construction of AAV library plasmid Add 3 μg TE54 vector (AAV2 serotype, Figure 1 ), 1.3 μL SfiI enzyme, 5.2 μL NEB CutSmart digestion buffer, and the total volume was supplemented with water to 50 μL. After incubation at 50 degrees Celsius for 1 hour, 1% agarose gel electrophoresis was performed at 175V for 40 minutes, and the main bands obtained were recovered.

[0021] The primers and corresponding material volumes used for amplification of the library synthesized from Twist were as follows: Table 1 Primers required for library amplification

[0022] Carry out PCR amplification reaction according to the conditions in Table 2: Table 2 PCR amplification of ligation products

[0023] The products of the PCR amplification reaction were subjected to gel electrophoresis, and the main bands obtained were recovered. The above-mentioned digestion products and PCR products were connected: 50 ng of PCR product, 70 ng of digestion product, 10 μL of 2x HiFi Assembly Mix, and the reaction system was supplemented to 20 μL with ultrapure water. Incubate at 50 degrees Celsius for 30 minutes.

[0024] Digest the ligation product with Plasmid-Safe DNase. Dilute the ligation product to 200 μL with ultrapure water, add 10 μL 25 mM ATP, 25 μL 10X reaction buffer, 10 μL Plasmid-Safe DNase, and 5 μL ultrapure water. Incubate at 37°C for 12 hours and at 70°C for half an hour.

[0025] Purify the above product: add 25ul of NaAC (3M, pH5.2). Add 550ul of anhydrous ethanol precooled at -20℃ for 2h and then precooled at -20℃ for 1h. Centrifuge at 12000r for 30min, carefully remove the supernatant, and aspirate all the droplets on the tube wall. Add 500ul of 70% ethanol precooled at -20℃, centrifuge at 12000r for 10min, carefully remove the supernatant, and aspirate all the droplets on the tube wall. Place the opened EP tube on the laboratory table at room temperature to allow the residual liquid to evaporate completely. Add 20ul of ddH2O to dissolve the DNA precipitate.

[0026] The plasmid was electroporated into competent cells, 200 μL LB medium was added, incubated at 37°C for half an hour, smeared on an ampicillin-resistant solid culture dish, and after culturing for 16 hours, clones were scraped and plasmids were extracted.

[0027] Example 2: Packaging of library AAV AAV virus was packaged by co-transfecting VPC2.0 cells with two plasmids. VPC2.0 cells were cultured in serum-free suspension until the cell density was 1E+6 cells / ml. The constructed library plasmid and pHelper plasmid were mixed at a molar ratio of 1:1, and then the plasmid DNA was mixed with PEIpro transfection reagent at a mass ratio of 1:2 according to the total mass. After incubation at room temperature for 20 minutes, it was slowly added to the cell suspension (the ratio of cells to three plasmids was 1mL:1μg) and mixed. The cells were cultured in a shaker at 37°C and 8% CO2 for 3 days, and the cell suspension was collected.

[0028] The cell suspension was centrifuged at 10,000 g for 10 min, and the supernatant was transferred to a new centrifuge tube. The cell pellet was resuspended in a small amount of PBS solution and repeatedly frozen and thawed to lyse the cells; after freezing and thawing, the cells were centrifuged at 10,000 g for 10 min again, and the supernatant was collected. The supernatants collected from the two centrifugations were mixed together and filtered with a 0.45μm filter to remove impurities. 1 / 2 volume of 1M NaCl and 10% PEG8000 solution were added, mixed evenly, and incubated at 4 degrees overnight. Centrifuged at 12,000 rpm for 2h, the supernatant was discarded, and the virus pellet was dissolved with an appropriate amount of PBS solution. After complete dissolution, it was filtered and sterilized with a 0.22μm filter. Benzonase nuclease was added to digest and remove residual plasmid DNA (final concentration was 50 U / ml). Close the tube cap and invert several times to mix thoroughly. Incubate at 37°C for 30 minutes; filter with a 0.45 μm syringe filter and take the filtrate, which is the concentrated AAV virus.

[0029] Add solid CsCl to the virus concentrate until the density is 1.41 g / ml (refractive index 1.372); add the sample to an ultracentrifuge tube and fill the remaining space of the centrifuge tube with the pre-prepared 1.41 g / ml CsCl solution; centrifuge at 175,000 g for 24 hours to form a density gradient. Collect samples of different densities step by step in sequence and take samples for titer determination. Collect the fractions enriched with AAV particles.

[0030] Repeat the above process once. Load the virus into a 100 kDa dialysis bag and dialyze for desalting overnight at 4 degrees. The dialysis buffer consists of PBS containing 0.001% Pluronic F68, pH 7.2. The harvested dialyzed sample is the purified AAV virus, which can be used for in vivo efficacy verification.

[0031] Example 3: Monkey intraocular injection and tissue separation 1E12 vg of the purified AAV was injected into two male cynomolgus monkeys about 2 years old. Six weeks after the administration, the monkeys were euthanized, and the eyeballs were removed to separate the retina-choroid layer. The muscle tissue of the eyeball was trimmed, and the muscle tissue was stripped before pretreatment: the eyeball was cleaned with 75% ethanol and HBSS-H-buffer (without Hyaluronidase solution) to prepare for dissection; the cornea was opened in a cross shape, and the vitreous was delivered with a little pressure and transferred to a 50mL centrifuge tube. The tissue was digested by immersing it in 10-15mL hyaluronic acid at 37 °C for 45 min; the eyeball tissue was transferred to 10-15mL pre-cooled HBSS-H+ buffer, and the retina and RPE-choroid layers were separated after standing for 40 minutes; the anterior segment was separated along the corneal margin with sterilized surgical instruments, and the retinal layer was cut into 5 parts of 2×2cm in size with the macula as the center: macular, superior, inferior, temporal, and nasal, and the single cell dissociation step was performed.

[0032] Single cell dissociation and sorting steps: 1. Adjust the pH value of EBSS to 7.2-7.4 by passing 95% O2:5% CO2 gas, and prepare papain dissociation solution (250 μL of DNAse in 5 mL) according to the configuration ratio and place it in a 15 mL centrifuge tube.

[0033] 2. All retinal tissues obtained by the above dissection and separation were transferred to a 15 mL centrifuge tube and passed through 95% O2:5% CO2 gas for 2 minutes.

[0034] 3. Incubate in a 37°C water bath for 20 min. Mix up and down every 10 min and observe the digestion of tissue fragments.

[0035] 4. After digestion, grind the mixture with a 10 mL pipette to avoid bubbles. Centrifuge at 300 g, 5 min, 4 °C, and remove the supernatant.

[0036] 5. Digestion was terminated by adding pH-adjusted EBSS solution, albumin-ovomucoid inhibitor solution (9:1) and 250 μL of DNase.

[0037] 6. The cell suspension was placed in albumin-ovomucoid inhibitor solution (upper: lower volume according to 3:5) and purified using density gradient. Centrifuged at 100g, 6 min, 4°C, and the supernatant was removed.

[0038] 7. Reselect the cells using PBS solution containing 1% FBS, transfer to flow cytometry tubes, and place on ice.

[0039] 8. Stain according to the instructions of Live-Dead Cell Staining Kit. Before flow cytometry sorting, the cell suspension needs to be filtered through a 70 μm cell filter and diluted to 1E6 cells / mL.

[0040] 9. CytoFLE vertical sorting, 24-well to collect single retinal living cells.

[0041] 10. Recount the collected cell suspension, centrifuge at 1300 rpm, 4°C, 3 min, remove excess liquid and proceed with subsequent testing.

[0042] Example 4: Cell extraction and DNA sequencing RNA was extracted using the Trizol method. The cells obtained above were divided into 8 portions. 1 ml Trizol was added to each sample, and the mixture was repeatedly blown with a 1 ml pipette tip until dissolved, and then allowed to stand for 5 min. 200 μl chloroform was added to each 1 ml Trizol homogenate, vortexed for 30 s, and allowed to stand for 5 min. Centrifuged at 12000 g at 4 °C for 15 min, and it was visible that it was divided into 3 layers. The upper aqueous phase was removed. An equal volume of isopropanol was added, inverted to mix, and allowed to stand for 10 min. Centrifuged at 12000 g at 4 °C for 10 min, the supernatant was discarded, and the RNA precipitate was collected. Washed twice with 75% alcohol, centrifuged at 12000 g at 4 °C for 5 min, the supernatant was discarded, and the RNA precipitate was placed in an ultra-clean workbench to air dry. RNase-free water was added to dissolve the RNA precipitate. RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814), and the reaction system was prepared according to Table 3: Table 3 Preparation of RNA reversal system

[0043] The reaction conditions were: 25°C, 10 min; 37°C, 2 h; 85°C, 5 min; 4°C, ∞.

[0044] The above reverse transcription products were subjected to subsequent NGS PCR for a total of two rounds of PCR. The design rules for the 1st Round NGS PCR primers are shown in Table 4, the reaction system is shown in Table 5, and the reaction steps are shown in Table 6. The design rules for the 2nd Round NGS PCR primers are shown in Table 7, the reaction system is shown in Table 8, and the reaction steps are shown in Table 9. After the 2nd Round NGS PCR, if there are experimental repetitions, according to the 2nd Round NGS PCR primer P5 / P7index, the non-overlapping N experimental repetition PCR products are mixed together. Take 50 μl of the product and mix it with loading buffer, and perform 1% agarose gel electrophoresis (marker DL2000). According to the 2nd Round NGS PCR primer P5 / P7index, non-repeated PCR products can be mixed and recovered and sent to the sequencing company for NGS sequencing. Each sample is sequenced 4 times. The sequencing results of the top 50 peptides ranked by the number of times they appear in the sequencing results are shown in Table 10.

[0045] Table 4 1st Round NGS PCR Primer Design Rules

[0046] Table 5 1st Round NGS PCR reaction system

[0047] Table 6 1st Round PCR reaction steps

[0048] Table 7 2nd Round NGS PCR Primer Design Rules

[0049] Table 8 2nd Round NGS PCR reaction system

[0050] Table 9 2nd Round PCR Steps

[0051] Table 10 Top 50 sequences enriched in monkey

[0052] Example 5: Validation of Targets The top50 polypeptides were inserted into AAV for packaging, and the capsid of wild-type AAV2 was mixed at a dose of 2E10 vg per virus per eye, and the mixed AAV was injected into the eye of a cynomolgus monkey according to the method of Example 3-4, and the monkey eye was taken for cell isolation and sequencing. Finally, the results were compared with wild-type AAV2. Figure 2 AAV inserted polypeptide sequences that are highly enriched in the eye are shown.

Claims

1. A modified adeno-associated virus AAV capsid protein, wherein the length of the modified AAV capsid inserted in the GH-loop is 7 amino acids relative to the parent AAV capsid protein, and the sequence of the inserted amino acids is as shown in SEQ ID NO: 1-6.

2. As described in claim 1, the inserted amino acid in AAV is an amino acid with a similarity of more than 85% with SEQ ID NO: 1-6.

3. As described in claims 1-2, the AAV capsid serotype is AAV1, AAV2, AAV5, AAV8 or AAV9.

4. As described in claims 1-2, the use of modified AAV is for ocular delivery.

5. As described in claims 1-2, the use of modified AAV is for the treatment of ophthalmic diseases.

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