An aav capsid protein mutant and uses thereof

Abstract

The application belongs to the field of virus bodies. The application provides an AAV capsid protein mutant and application thereof, wherein at least one of QNDIKNG, GNDLRPT, NGNAIVG and DNNLAKL is inserted between any two amino acids of a surface variable region of a VP1 protein of a wild type or modified AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.74 and AAVrh.10 or a protein having at least 80% sequence identity with the amino acid sequence of the above proteins. Compared with the existing AAV capsid protein, the insertion of the amino acid sequence of the AAV capsid protein mutant of the application causes a significant increase in the transduction efficiency of the adeno-associated virus on peripheral blood-derived macrophages, bone marrow-derived macrophages and microglial cells.

Description

TECHNICAL FIELD

[0001] The present invention is in the field of virosomes and relates in particular to adeno-associated virus capsid protein mutants and uses thereof. BACKGROUND

[0002] Gene delivery to human macrophages remains a major challenge in basic and translational medical research and engineered AAV capsids with high-efficiency transduction capacity represent an attractive solution.

[0003] Adeno-associated virus (AAV)-derived vectors are promising tools for clinical gene transfer due to their non-pathogenic, low immunogenicity profile, low rate of integration into the host genome and ability to achieve long-term transgene expression in non-dividing cells. However, the transduction efficiency of AAV natural variants in certain immune cells is too low for clinical applications, for these reasons, various approaches are currently used to obtain novel capsid variants with enhanced properties. So far, the most significant progress in these testing approaches comes from the directed evolution of AAV capsids, a process that is carried out by using error-prone PCR, various parental serotype shuffling or insertion of fully randomized short peptide capsid sequences at specific positions, followed by selection of capsid variants either in vitro or in vivo.

[0004] Macrophages are innate immune cells that play a role in regulating a variety of homeostatic and host immune responses. In addition, macrophages are involved in many other biological events, including the regulation of endogenous strength of reactive oxygen species (ROS), iron homeostasis, tissue damage repair, and numerous metabolic functions. In addition to this, macrophages have three major important functions, namely immune regulation, phagocytosis and antigen presentation, which play a key role in performing normal immune responses under different pathophysiological conditions. Defects in macrophage function in mice and humans can lead to severe diseases, including neurodevelopmental delay and dementia, skeletal deformities, defects in tissue repair and remodeling, liver, spleen, reproductive system, lung and heart dysfunction, and chronic inflammation and autoimmune diseases.

[0005] Macrophages are a highly heterogeneous population of immune cells that can be classified in a variety of ways based on their origin, activation state, function, and location of distribution. They can be classified as monocyte-derived macrophages and tissue-resident macrophages based on their origin. Monocyte-derived macrophages are differentiated from monocytes in the bone marrow and migrate to specific sites upon inflammation or tissue damage. Tissue-resident macrophages are found in most tissues and have tissue-specific functions that are “auxiliary” in nature, such as microglia in the brain, Kupffer cells in the liver, alveolar macrophages in the lung, osteoclasts in the bone, and adipose tissue-associated macrophages. Resident macrophages integrate signals from the external environment to coordinate adaptive cellular responses that are critical for the growth, remodeling, and homeostasis of specific tissues.

[0006] As resident macrophages in the central nervous system (CNS), microglia constitute about 10% of the total number of cells in the CNS. Initially viewed as “garbage collectors,” microglia are now recognized as key regulators of the CNS in both normal and pathological conditions. Microglia are capable of active surveillance and rapidly initiate innate and adaptive immune responses upon encountering immune challenges. In addition to immune functions, microglia have multifaceted roles in regulating neural circuit development and plasticity. Microglial dysfunction is a key factor in the progression of CNS aging and a variety of CNS diseases, including neurodegenerative diseases and brain tumors. Clinical studies have found risk variants associated with genes highly expressed by microglia, suggesting that microglia play an important role in the progression of CNS diseases and highlighting the potential of therapeutic intervention targeting microglia.

[0007] Macrophages are adept at detecting and responding to foreign nucleic acids, which makes them resistant to genetic manipulation. Although viral and non-viral approaches have been developed to transduce macrophages, their infection capacity is generally low and has serious side effects, especially in targeting macrophages in vivo and achieving efficient gene delivery, which faces the dual challenges of physiological barriers and the immune system. The main non-viral transduction methods currently used include electroporation and lipid nanoparticles, and viral transduction generally uses lentivirus, adenovirus, and adeno-associated virus (AAV).

[0008] Due to the low pathogenicity of adeno-associated virus (AAV), they have become the most commonly used viral vectors in basic research and gene therapy. Although rAAVs can transduce a variety of cell types in mammals, rAAVs packaged with existing AAV capsids do not achieve high transduction efficiency and sufficient transgene expression levels in macrophages, especially in vivo. In addition, viral transduction of macrophages (and microglia) can also face the problem of triggering immune activation. SUMMARY

[0009] To solve the above problems, the present application provides an adeno-associated virus (AAV) capsid protein mutant and its use. Compared with the existing AAV capsid protein, the insertion of the amino acid sequence of the AAV capsid protein mutant of the present application causes a significant increase in the transduction efficiency of adeno-associated virus in peripheral blood-derived macrophages, bone marrow-derived macrophages and microglial cells.

[0010] The present application aims to provide an AAV capsid protein mutant, in which at least one of the short peptide amino acid sequences QNDIKNG, GNDLRPT, NGNAIVG and DNNLAKL is inserted between any two amino acids in the surface variable region of the capsid protein VP1 protein of at least one of wild-type or modified AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh.74 and AAV rh.10 or a protein having at least 80% sequence identity with the amino acid sequence of the above protein.

[0011] The nucleotide sequences of the above four short peptides QNDIKNG, GNDLRPT, NGNAIVG and DNNLAKL are shown in SEQ ID NO. 10-SEQ ID NO. 13, respectively.

[0012] Further, the surface variable region of the capsid protein VP1 protein is at least one of VR-IV, VR-V and VR-VIII.

[0013] Further, the surface variable region of the capsid protein VP1 protein includes at least one of 262-269, 327-332, 382-386, 452-460, 488-505, 527-539, 545-558, 581-593 and 704-714.

[0014] Preferably, the at least one of the short peptide amino acid sequences is inserted between the 588th and 589th amino acids of the surface variable region of the capsid protein VP1 protein.

[0015] Further, the AAV capsid protein VP1 protein is a wild-type AAV6 capsid protein VP1 protein. The amino acid sequence of the wild-type AAV6 capsid protein VP1 protein is shown in SEQ ID NO. 1.

[0016] Preferably, four kinds of AAV6 capsid protein mutants are obtained by inserting short peptide amino acids QNDIKNG, GNDLRPT, NGNAIVG, and DNNLAKL between amino acids 588 and 589 of the AAV6 capsid protein VP1 protein, respectively, and the amino acid sequences of the four kinds of AAV6 capsid protein mutants are shown in SEQ ID NO. 2-5, respectively. The gene sequences of the four kinds of AAV6 capsid protein mutants are shown in SEQ ID NO. 6-9, respectively.

[0017] A gene encoding the AAV capsid protein mutant described above.

[0018] An expression vector comprising the gene encoding the AAV capsid protein mutant described above.

[0019] A host cell comprising the gene encoding the AAV capsid protein mutant described above or the expression vector described above.

[0020] An adeno-associated virus comprising the AAV capsid protein mutant described above.

[0021] A method for preparing a recombinant adeno-associated virus (rAAV), comprising introducing at least the following components into a host cell:

[0022] (1) the gene encoding the AAV capsid protein mutant described above or the expression vector described above;

[0023] (2) a GOI plasmid comprising a gene of interest.

[0024] The expression product of the gene of interest described above is a protein or RNA.

[0025] The rAAV prepared by the method described above.

[0026] A pharmaceutical composition comprising the rAAV described above and a pharmaceutically acceptable carrier.

[0027] Use of the gene encoding the AAV capsid protein mutant described above, the expression vector, or the rAAV in the preparation of a medicament for delivering a gene product to cells or tissues of a subject.

[0028] A viral particle comprising the AAV capsid protein mutant described above.

[0029] The viral particle further comprises a recombinant polynucleotide encoding a gene of interest, particularly a gene related to the regulation of macrophage function, such as TREM2, CCL4 / CCL3, CD22, or CSF1R, etc.

[0030] The recombinant polynucleotide encoding the gene of interest further comprises a microglia-specific promoter or enhancer, or a macrophage-specific promoter or enhancer.

[0031] wherein the microglia-specific promoter or enhancer is derived from any one of:

[0032] (a) TMEM119;

[0033] (b) CX3CR1; or

[0034] (c) P2Y12 (P2RY12).

[0035] The macrophage-specific promoter or enhancer is derived from any one of:

[0036] (a) CD11b;

[0037] (b) CD68;

[0038] (c) CSF1R; or

[0039] (d) F4 / 80.

[0040] An engineered particle prepared from the viral particle described above, wherein the cargo carrier is capable of modulating macrophage function, treating or preventing a macrophage-related disease or disorder.

[0041] In some embodiments, the capsid protein can be a native serotype capsid protein, a mutant, a modified capsid protein, or a combination of full-length or fragments of two or more capsid protein amino acid sequences.

[0042] In some embodiments, the native serotype capsid protein, a mutant, a modified capsid protein, or a combination of full-length or fragments of two or more capsid protein amino acid sequences includes AAV type 1 (AAV1), type 2 (AAV2), type 3 (AAV3), type 4 (AAV4), type 5 (AAV5), type 6 (AAV6), type 7 (AAV7), type 8 (AAV8), rh8 type (AAVrh8), type 9 (AAV9), PHP.B type (AAVPHP.B) AAV9, AAV9.47, AAV9 (hu14), AAV10, type 11 (AAV11), type 12 (AAV12), rh8 type (AAVrh8), AAVrh10, hu37 type (AAV.hu37), hu31 type (AAV.hu31), hu32 type (AAV.hu32), rh20 type (AAVrh20), rh39 type (AAVrh39), and rh74 type (AAVrh74), myoAAV, AAVmyo, AAVDJ, and AAVretro.

[0043] Embodiments of the present disclosure also provide kits comprising one or more of the polypeptides, polynucleotides, vectors, engineered AAV capsids, engineered AAV particles, cells, and combinations thereof, and pharmaceutical formulations as described herein.

[0044] In certain embodiments, one or more of the polypeptides, polynucleotides, vectors, engineered AAV capsids, engineered AAV particles, cells, and combinations thereof as described herein can be provided in a kit of parts.

[0045] The present disclosure describes various embodiments of engineered viral capsids, such as adeno-associated virus (AAV) capsids, that can be engineered to confer a specific cell tropism, such as macrophage-specific tropism, to an engineered viral particle. The engineered viral capsids can be capsids from a lentivirus, a retrovirus, an adenovirus, or an AAV. These engineered capsids can be included in an engineered viral particle, such as an engineered lentiviral, retroviral, adenoviral, or AAV viral particle, and can confer a cell-specific tropism, reduce immunogenicity, or both to the engineered viral particle.

[0046] The engineered viral capsids described in the present disclosure can comprise one or more artificial viral capsid proteins. The engineered viral capsid proteins can comprise or consist of a macrophage-specific targeting moiety comprising or consisting of an insertion amino acid as described elsewhere in the present disclosure.

[0047] The engineered viral capsids and / or capsid proteins can be encoded by one or more artificial viral capsid polynucleotides. In certain embodiments, the engineered viral capsid polynucleotide is an engineered AAV capsid polynucleotide, lentiviral capsid polynucleotide, retroviral capsid polynucleotide, or adenoviral capsid polynucleotide.

[0048] In certain embodiments, the engineered viral capsid polynucleotide (e.g., engineered AAV capsid polynucleotide, engineered lentiviral capsid polynucleotide, engineered retroviral capsid polynucleotide, or engineered adenoviral capsid polynucleotide) can comprise a 3' polyadenylation signal. The polyadenylation signal can be an SV40 polyadenylation signal.

[0049] In certain embodiments, the engineered viral capsid protein can comprise an insertion n amino acid sequence. In certain embodiments, n can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 15 amino acids.

[0050] In certain embodiments, the engineered AAV capsid can comprise a 7 amino acid motif.

[0051] In certain embodiments, the n-amino acid sequence can be inserted between two amino acids in a wild-type viral capsid protein (or capsid protein). In certain embodiments, the n-amino acid sequence can be inserted between two amino acids in a variable amino acid region of the viral capsid protein.

[0052] In certain embodiments, the one or more n-amino acid sequences can be inserted between two amino acids in one or more of the 12 variable regions of a wild-type AAV capsid protein.

[0053] In certain embodiments, the one or more 7-amino acid sequences can be inserted between two amino acids in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-III, VR-IX, VR-X, VR-XI, VR-XII, or a combination thereof, respectively.

[0054] In certain embodiments, the 7-amino acid sequence can be inserted between two amino acids in VR-III of the capsid protein.

[0055] In certain embodiments, the engineered capsid can insert a 7-mer motif between any two consecutive amino acids between amino acids 262 to 269, between amino acids 327 to 332, or between amino acids 382 to 386, 452 to 460, 488 to 505, 545 to 558, 581 to 593, 704 to 714 of the AAV6 viral protein.

[0056] In certain embodiments, the engineered capsid can insert an n-amino acid sequence between amino acids 588 and 589 of the AAV6 viral protein.

[0057] In certain embodiments, the engineered capsid can insert a 7-mer motif between amino acids 588 and 589 of the AAV6 viral protein.

[0058] SEQ ID NO. 1 is the reference sequence of the AAV6 capsid protein, at least for reference to the insertion sites described above. It can be appreciated that n-amino acid sequences can also be inserted into corresponding positions in viral proteins of other serotypes of AAV. In some embodiments, as previously described, n-mer can be inserted between any two consecutive amino acids in the AAV viral protein, preferably in a variable region (VR).

[0059] Methods of screening engineered AAV capsids

[0060] The present disclosure also provides a method of screening engineered AAV capsids, utilizing a library of AAV capsids comprising one or more desired cell-specific engineered AAV capsid variants. As shown in Figure 1 and Figure 2 The library of AAV capsids can be administered to a variety of non-human animals for a first round of screening based on mRNA. As shown in Figure 1 The process of transduction by AAV and related vectors can yield mRNA molecules that are reflective of the viral genome. As shown by the various examples herein, the mRNA-based screening method is more specific and effective in determining viral particles that are capable of functionally transducing cells, as selection is based on functional product, rather than merely detecting the presence of viral DNA in the cell.

[0061] Following the first round of administration, one or more engineered AAV viral particles having the desired capsid variant can be obtained, forming a screened library of AAV capsids. The desired AAV viral particles can be identified by measuring the mRNA expression level of the capsid variant and determining which variant is highly expressed in the desired cell type. The capsid variant particle that is highly expressed in the target cell, tissue, and / or organ is the desired AAV capsid variant.

[0062] In certain embodiments, the polynucleotide encoding the AAV capsid variant is under the regulation of a tissue-specific promoter that has selective activity in the desired cell, tissue, or organ.

[0063] The engineered AAV capsid variant particle identified in the first round of screening can then be administered to a different non-human cell.

[0064] In certain embodiments, the cells used for the second round of screening and identification can be different from the cells used in the first round. Similar to the first round, following administration to the cells, the variant that is most highly expressed in the desired cell can be identified by measuring the expression level of viral mRNA in the cell. The top variant identified in the second round can be selectively barcoded and selectively pooled.

[0065] In certain embodiments, the top variant identified in the second round can be administered to a non-human primate to identify the top cell-specific variant, particularly when the top variant is for use in humans.

[0066] Viral vectors

[0067] In certain embodiments, the vector is a viral vector. As used in the art, the term "viral vector" in this context refers to a polynucleotide-based vector comprising one or more viral components, capable of expressing and packaging a polynucleotide, such as an engineered AAV capsid polynucleotide of the application, a cargo, or other composition or molecule described herein, generating a viral particle comprising the polynucleotide, and producing the viral particle when used alone or in combination with one or more other viral vectors (e.g., in a viral vector system).

[0068] The viral vectors and systems thereof can be used to generate viral particles to deliver, express, and / or produce one or more compositions described herein (including, but not limited to, any viral particle and its carried genetic material).

[0069] The viral vectors can form part of a viral vector system comprising a plurality of vectors. In certain embodiments, integrating a plurality of viral vectors into a system can enhance the safety of the system.

[0070] Suitable viral vectors include adenoviral-based vectors, adeno-associated vectors, helper-dependent adenoviral vectors (HdAd), hybrid adenoviral vectors, and the like.

[0071] Other forms of viral vectors and viral particles produced therefrom are described elsewhere herein.

[0072] In certain embodiments, the viral vectors are designed to generate replication-incompetent viral particles to enhance the safety of the system.

[0073] Adenoviral vectors, helper-dependent adenoviral vectors, and hybrid adenoviral vectors

[0074] In certain embodiments, the vector can be an adenoviral vector. In certain embodiments, the viral particles produced using the adenoviral vector or system thereof can be of serotype 2, 5, or 9. In certain embodiments, the polynucleotide delivered by the adenoviral particle can be up to about 8 kb in length. Thus, in certain embodiments, the adenoviral vector can include a DNA polynucleotide to be delivered, which can range in size from 0.001 kb to 8 kb.

[0075] Adenoviral vectors have been successfully used for gene delivery in a number of settings (e.g., see Teramoto et al., 2000, Lancet 355:1911-1912; Lai et al., 2002, DNA Cell. Biol. 21 :895-913; Flotte et al., 1996, Hum. Gene Ther. 7:1145-1159; and Kay et al., 2000, Nat. Genet. 24:257-261).

[0076] The engineered AAV capsid of the application can be included in an adenoviral vector to make an adenoviral particle comprising the engineered AAV capsid.

[0077] In certain embodiments, the vector can be a helper-dependent adenoviral vector or system thereof.

[0078] Such vectors are also referred to in the art as "gutless", "gutted" or "attenuated" vectors, and are a modified form of adenoviral vectors (see, e.g., Thrasher et al., 2006, Nature 443:E5-7).

[0079] In a helper-dependent adenoviral vector system, a first vector (helper) can comprise all the genes required for viral replication, but with a conditional genetic defect in the packaging domain. A second vector then comprises only the ends of the viral genome, one or more engineered AAV capsid polynucleotides of the application, and a native packaging recognition signal, allowing for the selective assembly and release of the viral particle from the cell (see, e.g., Cideciyan et al., 2009, N Engl J Med. 361 :725-727).

[0080] Gene delivery relying on helper-dependent adenoviral vector systems has been successful in a variety of applications (e.g., Simonelli et al., 2010, J Am Soc Gene Ther. 18:643-650; Cideciyan et al., 2009, N Engl J Med. 361 :725-727; Crane et al., 2012, Gene Ther. 19(4):443-452; Alba et al., 2005, Gene Ther. 12:18-S27; Croyle et al., 2005, Gene Ther. 12:579-587; Amalfitano et al., 1998, J. Virol. 72:926-933; Morral et al., 1999, PNAS 96:12816-12821). The techniques and vectors described in the above references can be modified to accommodate the delivery and integration of the engineered AAV capsid polynucleotides described herein.

[0081] In certain embodiments, the polynucleotide delivered by the helper-dependent adenoviral vector or system thereof can be up to about 38 kb in length. Thus, in certain embodiments, the adenoviral vector can include a DNA polynucleotide to be delivered that can range in length from about 0.001 kb to about 37 kb (e.g., see Rosewell et al., 2011. J. Genet. Syndr. Gene Ther. Suppl. 5:001).

[0082] In certain embodiments, the vector is a hybrid adenoviral vector or system thereof. Hybrid adenoviral vectors are generated by combining the high transduction efficiency of gene-deleted adenoviral vectors with the potential long-term gene integration capability of adeno-associated virus (AAV), retrovirus, lentivirus, and transposon-based gene transfer systems.

[0083] In some embodiments, such hybrid vector systems enable stable transduction of genes and at limited integration sites. See, e.g., Balague et al., 2000, Blood 95:820-828; Morral et al., 1998, Hum. Gene Ther. 9:2709-2716; Kubo and Mitani, 2003, J. Virol. 77(5):2964-2971; Zhang et al., 2013, Plos One 8(10) e76771; and Cooney et al., 2015, Mol. Ther. 23(4):667-674. The techniques described in these references can be adapted and modified for use with the engineered AAV capsid systems of the present application.

[0084] In some embodiments, the hybrid adenoviral vector can comprise one or more features from a retrovirus and / or an adeno-associated virus. In certain embodiments, the hybrid adenoviral vector can comprise one or more features from a spumaretroviral or foamy virus (FV). See, e.g., Ehrhardt et al., 2007, Mol. Ther. 15:146-156; Liu et al., 2007, Mol. Ther. 15:1834-1841. The techniques and vectors described in these references can also be used with the engineered AAV capsid systems of the present application. Advantages of incorporating one or more features from a FV into a hybrid adenoviral vector or system thereof include that the resulting viral particles can infect a broad spectrum of cells; they have a larger packaging capacity than other retroviruses; and they can persist in quiescent (non-dividing) cells.

[0085] Adeno-associated viral vectors

[0086] In certain embodiments, the engineered vector or system can be an adeno-associated viral vector (AAV vector). See, e.g., West et al., Virology 160:38-47 (1987); U.S. Patent No. 4,797,368; PCT Patent Publication WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); and Muzyczka, J. Clin. Invest. 94:1351 (1994).

[0087] Although adeno-associated viruses are similar to adenoviral vectors in some respects, they have certain deficiencies in replication and / or pathogenicity, and thus can be safer than adenoviral vectors.

[0088] In certain embodiments, the adeno-associated virus can integrate into a specific site of chromosome 19 of a human cell, and the formulation can be used to treat without significant side effects.

[0089] In certain embodiments, the AAV vector, system thereof, and / or AAV particle has a loading capacity of about 4.7 kb. The AAV vector or system thereof can comprise one or more engineered capsid polynucleotides described herein.

[0090] The AAV vector or system thereof can comprise one or more regulatory molecules. In certain embodiments, the regulatory molecules can be promoters, enhancers, repressors, and the like, which are described in more detail elsewhere herein.

[0091] In certain embodiments, the AAV vector or system thereof can comprise one or more polynucleotides encoding one or more regulatory proteins.

[0092] In certain embodiments, the one or more regulatory proteins can be selected from Rep78, Rep68, Rep52, Rep40, variants thereof, combinations thereof, and the like. In certain embodiments, the promoter can be a tissue-specific promoter discussed supra.

[0093] In certain embodiments, the tissue-specific promoter can drive expression of the engineered AAV capsid polynucleotides described herein.

[0094] The AAV vector or system thereof can comprise one or more polynucleotides encoding one or more capsid proteins, such as the engineered AAV capsid proteins described herein. The engineered capsid proteins are capable of assembling to form a protein shell of an AAV viral particle.

[0095] The engineered capsid can achieve tropism to specific cells, tissues, and / or organs.

[0096] In certain embodiments, the AAV vector or system thereof can further comprise one or more adenovirus helper factors or polynucleotides encoding the same.

[0097] The adenovirus helper factors can include, but are not limited to, E1A, E1B, E2A, E4 ORF6, and VA RNAs.

[0098] In certain embodiments, the host cell line producing the AAV particle expresses one or more adenovirus helper factors.

[0099] In certain embodiments, the AAV vector or system thereof can be designed to generate AAV particles of a particular serotype.

[0100] In certain embodiments, the serotype can be AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, AAV-9, or any combination of these serotypes.

[0101] In certain embodiments, the AAV can be AAV1, AAV-2, AAV-5, AAV-9, or any combination thereof.

[0102] The skilled person can select a particular AAV serotype depending on the cell type that is to be targeted. For example, for targeting brain and / or neuronal cells, AAV serotype 1, 2, 5, 9 or any combination of the hybrid capsids AAV1, AAV2, AAV5, AAV9 can be selected;

[0103] For targeting myocardial tissue, AAV4 can be selected.

[0104] For targeting the liver, AAV8 can be selected.

[0105] Thus, in certain embodiments, an AAV vector and system thereof capable of generating AAV particles targeting brain and / or neuronal cells can be designed to generate AAV having AAV1, AAV2, AAV5 or hybrid capsid particles thereof.

[0106] In certain embodiments, an AAV vector system capable of generating AAV particles targeting myocardial tissue can be designed to generate AAV particles of AAV type 4.

[0107] In certain embodiments, an AAV vector system capable of generating AAV particles targeting the liver can be designed to generate AAV particles of AAV type 8. See also Srivastava (2017, Curr. Opin. Virol. 21:75-80).

[0108] It is to be understood that although different serotypes can provide cell, tissue and / or organ specificity to some extent, each serotype is still multi-tropic, and thus can cause tissue toxicity if used to target a tissue in which it has a low transduction efficiency.

[0109] Thus, in addition to achieving certain tissue targeting capabilities by selecting a particular AAV serotype, the tropism of an AAV serotype can also be modulated by the engineered AAV capsids described herein. As described elsewhere herein, any serotype wild-type AAV variant can be generated by the methods described herein and determined to have a particular cell tropism, which can be the same as or different from the wild-type tropism of that serotype.

[0110] In certain embodiments, the tropism of a wild-type serotype for a particular cell type can be enhanced (e.g., made more selective or specific for a particular cell). For example, wild-type AAV6 is biased towards muscle and liver in humans. By including the engineered AAV capsids described herein and / or capsid protein variants of wild-type AAV6, the tropism for liver can be reduced or eliminated and the tropism for muscle increased, thereby increasing the specificity for muscle compared to wild-type AAV6.

[0111] As described previously, the engineered capsids and / or capsid protein variants of a wild-type AAV serotype can be included that have a tropism different from that of the wild-type reference serotype.

[0112] In certain embodiments, the AAV vector is a hybrid AAV vector or system thereof.

[0113] A hybrid AAV refers to a virus that has a portion of the genome extracted from one AAV serotype and packaged into a capsid derived from another, different serotype. For example, to produce rAAV2 / 5 particles using the helper-free, transient transfection method described above, the first and third plasmids (the adenovirus helper plasmid) would be identical to those discussed in the context of rAAV2 production; however, the second plasmid, pRepCap, would be different and would be referred to as pRep2 / Cap5, where the Rep gene is derived from AAV2 and the Cap gene is derived from AAV5.

[0114] The production protocol would be identical to the AAV2 production method described above. The resulting rAAV would be referred to as rAAV2 / 5, where the genome is based on rAAV2 and the capsid is based on AAV5. It is expected that the cell or tissue tropism exhibited by this AAV2 / 5 hybrid virus would be identical to that of AAV5. It should be understood that such wild-type hybrid AAV viruses would also exhibit the specificity issues discussed previously for non-hybrid wild-type serotypes.

[0115] The advantages of wild-type hybrid AAV systems can be combined with the enhanced and customizable cell specificity provided by the engineered AAV capsids described herein by generating hybrid AAV vectors incorporating the engineered AAV capsids.

[0116] It is to be understood that a hybrid AAV can also comprise an engineered AAV capsid wherein its genome comprises elements of different serotypes (i.e. the engineered AAV capsid is a variant of a reference wild-type serotype).

[0117] For example, a hybrid AAV can be generated wherein the engineered AAV capsid is a variant of AAV serotype 9 and is used to package rep elements of AAV serotype 2. As previously mentioned, the tropism of the hybrid AAV particle generated by the engineered AAV capsid will depend on the properties of the engineered capsid itself.

[0118] The present application has the beneficial effects of:

[0119] The AAV capsid protein mutant of the present application, the insertion of the amino acid sequence causes a significant increase in the transduction efficiency of peripheral blood-derived macrophages, bone marrow-derived macrophages and microglial cells by the adeno-associated virus. The adeno-associated virus AAV capsid protein mutant of the present application, for the first time, targets human macrophages, realizes efficient and specific delivery of nucleic acids into macrophages, and shows excellent characteristics of cross-species transduction, not limited by species, and has broad basic and clinical application prospects. This provides a more flexible and efficient solution for macrophage-related basic and clinical translation research. The present application develops an AAV vector with an engineered capsid through directed evolution, and realizes efficient gene delivery to macrophages and microglial cells. BRIEF DESCRIPTION OF DRAWINGS

[0120] Figure 1 The first screening process for AAV variant library;

[0121] Figure 2 The second screening process for AAV capsid;

[0122] Figure 3 Cell morphology observation after 7 days of CD14 positive cell culture;

[0123] Figure 4 Flow cytometry identification of human PBMC CD14+ cells;

[0124] Figure 5 In the middle, A is the ratio of the top 100 sequences to the total number of Count, and B is the insert feature analysis of the top 200 Count;

[0125] Figure 6 In the middle, A is the ratio of the Barcode sequence in the parent library and macrophages, respectively, and B is the transduction ability of the AAV variant relative to wild-type AAV6;

[0126] Figure 7EGFP fluorescence observation of human macrophage transduction for 4 AAV6 variants and control AAV9, AAV6, AAV9-HGTAASH and AAV9-WPPKTTS;

[0127] Figure 8 Flow cytometry analysis of EGFP for each group;

[0128] Figure 9 EGFP fluorescence observation of mouse bone marrow-derived macrophage transduction for 4 AAV6 variants and control AAV9, AAV6, AAV9-HGTAASH and AAV9-WPPKTTS;

[0129] Figure 10 Flow cytometry analysis of EGFP for each group. DETAILED DESCRIPTION

[0130] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0131] AAV screening process:

[0132] We started with wild-type AAV6 or AAV9 capsid and generated a library of AAV6 or AAV9 capsids, in which each variant has a random heptapeptide sequence inserted between amino acids 588 and 589 of AAV6 or AAV9 VP1 protein. The insertion site is located at the protrusion of the three-fold symmetry axis of the capsid, which helps the inserted peptide segment to interact with molecules on the target cell membrane. We transduced the rAAV of the capsid library into cultured human monocyte-derived macrophages and recovered the capsid variants that successfully mediated transduction. Through next-generation sequencing technology, we identified the highly enriched capsid variants after screening, such as Figure 1 shown.

[0133] Next, the first round of highly enriched capsid variants were packaged with labeled EGFP to prepare a barcode library, then transduced into human monocyte-derived macrophages again, and subjected to next-generation DNA sequencing to finally determine the truly highly enriched capsid variants, such as Figure 2 shown.

[0134] Finally, we packaged the EGFP reporter vector into rAAV of the candidate capsid variants, and transduced cultured human monocyte-derived macrophages, mouse bone marrow-derived macrophages and microglia cells with these AAV variants, respectively. Their transduction ability was evaluated and compared with parental AAV9, AAV6, AAV9-HGTAASH and AAV9-WPPKTTS, which were reported to be able to efficiently transduce cultured mouse microglia cells (Lin et al,. Nat Methods. 2022, 19(8): 976-985).

[0135] Screening of AAV6 variants with high efficiency of targeting macrophages

[0136] 1. Induction and identification of human macrophages

[0137] In order to obtain human macrophages that can be used for AAV capsid screening, isolation and induced differentiation of human PBMC CD14+ cells were performed, and the whole experiment process is as follows:

[0138] Isolation of CD14+ cells → M-CSF induced differentiation → cell identification

[0139] Specifically:

[0140] (1) Experimental method: A. Reconstitution of commercialized PBMC cells; B. Isolation of CD14 positive cells by using Easy Human CD14 positive selection kit II; C. Culture in cell culture medium containing M-CSF for 7 days to induce cell differentiation into macrophages; D. Flow cytometry identification by using anti-CD11b and anti-CD45.

[0141] (2) Experimental results: By microscopic observation, the cells after 7 days of culture showed fusiform or pancake shape, as shown in Figure 3 . By flow cytometry analysis, the ratio of CD11b+CD45+ cells was 96.1%, as shown in Figure 4 , indicating that most of the cells were human macrophages.

[0142] 2. Screening of AAV6-7NNK library

[0143] This is the first screening of the macrophage-targeting AAV6 variant library, and the screening process is shown in Figure 1 .

[0144] (1) Experimental method: A new AAV library construction strategy was used to insert random heptapeptide sequences between positions 588 and 589 of the VP1 protein, constructing an AAV variant library. Since the insertion site is located in the common region of VP1, VP2, and VP3 proteins, the inserted peptide segment will also exist in VP2 and VP3 proteins. The AAV6 library was used to transduce CD14+ induced macrophages at 1E+5, and RNA samples were collected after 48 hours. The RNA samples were reverse transcribed into cDNA, and then the variable region of the Capsid was amplified by PCR. Finally, the PCR products were subjected to NGS sequencing.

[0145] Through bioinformatics analysis, the AAV variants were ranked from high to low according to the copy number, and the candidate AAV variants were obtained for the second screening.

[0146] (2) Experimental results: The AAV6 variant library constructed by the new strategy can maximize the avoidance of cross-packaging problems between different AAV capsids. NGS sequencing analysis found that the AAV6 mother library contains about 1E+6 unique nucleotide sequences, which is consistent with the sequence number obtained by most libraries in the first round of screening reported. Through NGS sequencing analysis of human macrophages infected with AAV6-7NNK library, it was found that this sample contains about 1E+5 unique nucleotide sequences.

[0147] By comparing with the AAV6 mother library, more than 27700 DNA sequences were significantly enriched. The top 200 sequences accounted for 16.7% of the total count, and the top 20 sequences accounted for 3.8% of the total count. The highest count reached more than 38000, accounting for more than 0.4% of the total count, as shown in Figure 5 A.

[0148] In the amino acids from position 588 to 595 of VP1, the top 200 sequences of count number showed certain DNxxxxG characteristics, as shown in Figure 5 B. Considering that the copy number of AAV detected in macrophages is closely related to the yield of AAV, the top 20 count numbers will be selected for subsequent screening.

[0149] 3. AAV6-barcode library screening

[0150] In this study, the top 20 count numbers in the first screening were selected for the second screening, and the screening process is shown in Figure 2 .

[0151] (1) Experimental method: In this study, EGFP with barcode was used as GOI, and three-plasmid method was used to package AAV6 variants and control AAV9, AAV6, AAV9-HGTAASH and AAV9-WPPKTTS. CD14+ cells were 4.2E6 cells, and barcode-containing AAV was added at a MOI of 1.9E3. RNA samples were collected after 48 hours, reverse transcribed into cDNA, then PCR amplified the barcode region, and finally NGS sequencing. Through bioinformatics analysis, the proportion of each barcode in the total count was obtained.

[0152] (2) Experimental results: NGS analysis of AAV6 barcode master library, the master library includes 23 barcodes, except that 3 barcodes are distributed high or low in the master library, other barcodes are evenly distributed.

[0153] NGS analysis of the experimental group found that two barcodes accounted for the majority of the count, as shown in Figure 6 A.

[0154] By comparing with the control AAV6, it was found that the transduction ability of 20 AAV variants was enhanced, especially DC-11 (corresponding heptapeptide sequence QNDIKNG), DC-16 (corresponding heptapeptide sequence GNDLRPT), DC-25 (corresponding heptapeptide sequence NGNAIVG) and DD-2 (corresponding heptapeptide sequence DNNLAKL) performed best, their transduction ability reached 1215 times, 499 times, 820 times and 524 times of AAV6 parent respectively, as shown in Figure 6 B. Subsequent verification experiments will also focus on these four AAV6 variants, the amino acid sequences of the VP1 of the four AAV6 variants are respectively as shown in SEQ ID NO. 2-5, and the gene sequences of the VP1 of the four AAV6 variants are respectively as shown in SEQ ID NO. 6-9.

[0155] Verification of AAV variant transduction ability on macrophages

[0156] 1. AAV variant transduction on human macrophages

[0157] A series of AAV variants with better transduction ability than the parent were obtained through secondary screening, and the above four better ones were selected for further verification.

[0158] (1) Experimental method: EGFP with barcode as GOI, three-plasmid method was used to package the four AAV6 variants screened and control AAV9, AAV6, AAV9-HGTAASH and AAV9-WPPKTTS. CD14+ cells were plated in 12-well plates at 4E5 / well, and transduction test was performed at MOI 5E4. After 48h, cell fluorescence was observed and EGFP expression was detected by flow cytometry.

[0159] (2) Experimental results: Through microscopic observation, except for wild-type AAV9, other AAV groups had EGFP-positive macrophages, as shown in FIG. 1. Through flow cytometry analysis, the positive rate of AAV6 variants was significantly higher than that of wild-type AAV6 (wild-type AAV6 was 24.6%), among which the positive rate of variant AAV6-QNDIKNG was the highest, reaching 51.8%. As shown in FIG. 2. Figure 7 Figure 8

[0160] 2. Transduction of mouse bone marrow-derived macrophages by AAV variants

[0161] A series of AAV variants with better transduction capacity than the parent were obtained through secondary screening, and the better 4 of them were selected for further verification.

[0162] (1) Experimental method: EGFP with barcode as GOI, three-plasmid method was used to package the four AAV6 variants screened and control AAV9, AAV6, AAV9-HGTAASH and AAV9-WPPKTTS. CD14+ cells were plated in 12-well plates at 4E5 / well, and transduction test was performed at MOI 5E4. After 48h, cell fluorescence was observed and EGFP expression was detected by flow cytometry.

[0163] (2) Experimental results: Through microscopic observation, except for wild-type AAV9, other AAV groups had EGFP-positive macrophages, as shown in FIG. 1. Through flow cytometry analysis, the positive rate of AAV6 variants was significantly higher than that of wild-type AAV6 (wild-type AAV6 was 24.6%), among which the positive rate of variant AAV6-QNDIKNG was the highest, reaching 51.8%. As shown in FIG. 2. Figure 9 Figure 10

[0164] As can be seen from the above, at the same dose, relative to the wild-type AAV parent, the AAV variants screened can more efficiently transduce macrophages, and this transduction capacity is applicable in macrophages of different species. These efficient AAV variants can not only promote basic research on macrophages, but also can be applied to CAR-M cell therapy and other diseases related to macrophages.​​​​

[0165] The above examples are only for illustrating the present application, but not limiting the present application. Those skilled in the art can make various changes or modifications without departing from the spirit and scope of the present application. Therefore, all equivalent technical solutions should belong to the scope of the present application, which should be defined by the claims.

Claims

1. An AAV capsid protein mutant, characterized in that, Short peptide amino acids QNDIKNG, GNDLRPT, NGNAIVG, and DNNLAKL were inserted between amino acids 588 and 589 of the wild-type AAV6 capsid protein VP1, respectively, to obtain four AAV6 capsid protein mutants, the amino acid sequences of which are shown in SEQ ID NO.2-5.

2. The gene encoding the AAV capsid protein mutant of claim 1.

3. An expression carrier, characterized in that, It contains the gene encoding the AAV capsid protein mutant as described in claim 2.

4. A host cell, characterized in that, It comprises the gene encoding the AAV capsid protein mutant as described in claim 2 or the expression vector as described in claim 3.

5. Adeno-associated virus, characterized in that, It includes the AAV capsid protein mutant as described in claim 1.

6. A method for preparing recombinant adeno-associated virus (rAAV), characterized in that, This includes introducing at least the following components into the host cell: (1) The gene encoding the AAV capsid protein mutant as described in claim 2 or the expression vector as described in claim 3; (2) GOI plasmids containing the target gene.

7. A pharmaceutical composition, characterized in that, It includes rAAV prepared by the preparation method of claim 6 and a pharmaceutically acceptable carrier.

8. The gene encoding the AAV capsid protein mutant as described in claim 2, the expression vector as described in claim 3, or the rAAV prepared by the preparation method as described in claim 6, for use in the preparation of a medicament for delivering the gene product to the cells or tissues of a subject.

9. A viral particle, characterized in that, It includes the AAV capsid protein mutant as described in claim 1.

10. The virus particle according to claim 9, characterized in that, It also includes recombinant polynucleotides that encode genes of interest.

11. An engineered particle prepared from the virus particles of claim 10.

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