AAV capsid protein mutant and application thereof

Abstract

The present invention is in the field of virions. The invention provides an AAV capsid protein mutant and application thereof. At least one of oligopeptide amino acid sequences QNDIKNG, GNDLRPT, NGNAIVG and DNNLAKL is inserted between any two amino acids in a surface variable region of at least one of wild type or modified capsid protein VP1 of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.74 and AAVrh.10 or proteins with at least 80% sequence consistency with the amino acid sequences of the above proteins. Compared with the existing AAV capsid protein, the AAV capsid protein mutant has the advantage that the transduction efficiency of the adeno-associated virus on peripheral blood-derived macrophages, bone marrow-derived macrophages and microglial cells is remarkably improved due to the insertion of the amino acid sequence.

Description

Technical Field

[0001] This invention belongs to the field of virology, specifically relating to adeno-associated virus capsid protein mutants and their applications. Background Technology

[0002] Delivering genes into human macrophages remains a major challenge in basic and translational medicine research, and engineered AAV capsids with efficient transduction capabilities represent an attractive solution.

[0003] Adeno-associated virus (AAV)-derived vectors have become promising tools for clinical gene transfer due to their non-pathogenicity, low immunogenicity, low rate of integration into the host genome, and long-term transgenic expression in non-dividing cells. However, the transduction efficiency of natural AAV variants in certain immune cells is too low for clinical applications. For these reasons, various methods are currently available to obtain novel capsid variants with enhanced properties. To date, the most significant advance in these assays has come from the directed evolution of the AAV capsid, a process that involves using error-prone PCR, various parental serotype shuffling, or insertion of a completely randomized short peptide capsid sequence at a specific location, followed by selection of capsid variants in vitro or in vivo.

[0004] Macrophages are innate immune cells that function by regulating various homeostatic processes and host immune responses. In addition, macrophages participate in many other biological events, including regulating the endogenous strength of reactive oxygen species (ROS), iron homeostasis, tissue damage repair, and numerous metabolic functions. Furthermore, macrophages have three crucial functions: immune regulation, phagocytosis, and antigen presentation, playing a key role in executing normal immune responses under various pathophysiological conditions. In mice and humans, macrophage dysfunction can lead to serious diseases, including neurodevelopmental delay and dementia, skeletal deformities, impaired tissue repair and remodeling, liver, spleen, reproductive system, lung, and heart dysfunction, as well as chronic inflammation and autoimmune diseases.

[0005] Macrophages are a highly heterogeneous group of immune cells, and can be classified in various ways based on their origin, activation state, function, and distribution. According to origin, they can be divided into monocyte-derived macrophages and tissue-resident macrophages. Monocyte-derived macrophages differentiate from monocytes in the bone marrow and migrate to specific sites during inflammation or tissue damage. Tissue-resident macrophages are present in most tissues and have "helper" tissue-specific functions; for example, they are found in microglia in the brain, Kupffer cells in the liver, alveolar macrophages in the lungs, osteoclasts in bone, and fat-associated macrophages in adipose tissue. Resident macrophages integrate signals from the external environment and coordinate adaptive cellular responses, which are crucial for the growth, remodeling, and homeostasis of specific tissue cells.

[0006] As resident macrophages in the central nervous system (CNS), microglia account for approximately 10% of the total number of cells in the CNS. Initially considered "garbage collectors," microglia are now recognized as key regulators of the CNS in both normal and pathological states. Microglia are capable of active surveillance and rapidly initiating innate and adaptive immune responses in response to immune attacks. In addition to their immune functions, microglia also play multifaceted roles in regulating neural circuit development and plasticity. Microglia dysfunction is a key factor in CNS aging and the progression of various CNS diseases, including neurodegenerative diseases and brain tumors. Clinical studies have identified risk variants associated with genes highly expressed in microglia, suggesting a significant role for microglia in the progression of CNS diseases and highlighting the potential for therapeutic interventions targeting microglia.

[0007] Macrophages excel at detecting and responding to exogenous nucleic acids, making them resistant to gene manipulation. Although viral and non-viral methods have been developed to transduce macrophages, their infectivity is generally low and they have serious side effects. In particular, achieving efficient gene delivery to macrophages in vivo faces the dual challenges of physiological barriers and the immune system. Currently, the main non-viral transduction methods include electroporation and lipid nanoparticles, while viral transduction typically uses lentiviruses, adenoviruses, and adeno-associated viruses (AAVs).

[0008] Due to their low pathogenicity, adeno-associated viruses (AAVs) have become the most commonly used viral vectors in basic research and gene therapy. Although rAAVs can transduce multiple cell types in mammals, rAAVs packaged in existing AAV capsids have not achieved high transduction rates and sufficient transgene expression levels in macrophages, especially in vivo. Furthermore, viral transduction into macrophages (and microglia) may also face the challenge of triggering immune activation. Summary of the Invention

[0009] To address the aforementioned problems, this invention provides an adeno-associated virus (AAV) capsid protein mutant and its applications. Compared to existing AAV capsid proteins, the AAV capsid protein mutant of this invention exhibits a significant increase in the transduction efficiency of AAV in peripheral blood macrophages, bone marrow macrophages, and microglia due to the insertion of amino acid sequences.

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

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

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

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

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

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

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

[0017] The gene encoding the above-mentioned AAV capsid protein mutant.

[0018] An expression vector containing the gene encoding the above-mentioned AAV capsid protein mutant.

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

[0020] Adeno-associated viruses, including the aforementioned AAV capsid protein mutants.

[0021] A method for preparing recombinant adeno-associated virus (rAAV) includes introducing at least the following components into a host cell: (1) The gene or expression vector encoding the AAV capsid protein mutant mentioned above; (2) GOI plasmids containing the target gene.

[0022] The expression products of the above-mentioned target genes are proteins or RNA.

[0023] rAAV prepared by the above preparation method.

[0024] A pharmaceutical composition comprising the above-described rAAV and a pharmaceutically acceptable carrier.

[0025] The above-mentioned gene encoding the AAV capsid protein mutant, expression vector, or rAAV is used in the preparation of a drug for delivering the gene product to the cells or tissues of a subject.

[0026] A viral particle comprising the aforementioned AAV capsid protein mutant.

[0027] Viral particles also include recombinant polynucleotides that encode genes of interest, especially those related to regulating macrophage function, such as TREM2, CCL4 / CCL3, CD22, or CSF1R.

[0028] Recombinant polynucleotides encoding genes of interest also include microglia-specific promoters or enhancers, or macrophage-specific promoters or enhancers.

[0029] Among them, microglia-specific promoters or enhancers originate from any of the following: (a) TMEM119; (b) CX3CR1; or (c)P2Y12(P2RY12).

[0030] Macrophage-specific promoters or enhancers originate from any of the following: (a) CD11b; (b) CD68; (c) CSF1R; or (d)F4 / 80.

[0031] An engineered particle prepared from the above-mentioned viral particles, the cargo carrier of which can be used to modulate macrophage function and treat or prevent macrophage-related diseases or disorders.

[0032] In some embodiments, the capsid protein may be a natural serum capsid protein, a mutant of the capsid protein, a modified capsid protein, or a combination of full-length or fragmented amino acid sequences of two or more capsid proteins.

[0033] In some embodiments, the natural serum capsid protein, its mutants, modified capsid proteins, or combinations of full-length or fragmented amino acid sequences of two or more capsid proteins include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, rh8, AAVrh8, AAV9, PHP.B, AAV9, AAV9.47, AAV9 (hu14), AAV10, AAV11, AAV12, rh8, AAVrh8, AAVrh10, hu37, hu31, hu32, and rh20. Type (AAVrh20), type rh39 (AAVrh39) and type rh74 (AAVrh74), myoAAV, AAVmyo, AAVDJ and AAVretro.

[0034] Embodiments of this disclosure also provide kits comprising one or more peptides, polynucleotides, carriers, engineered AAV capsids, engineered AAV particles, cells, and combinations thereof and pharmaceutical formulations as described herein.

[0035] In some embodiments, one or more peptides, polynucleotides, vectors, engineered AAV capsids, engineered AAV particles, cells, and combinations thereof as described herein may be provided in the form of a combo kit.

[0036] This disclosure describes embodiments of various engineered viral capsids (such as adeno-associated virus (AAV) capsids) that can be engineered to impart specific cell tropisms to engineered viral particles, such as macrophage-specific tropism. The engineered viral capsids can be derived from lentivirals, retroviruses, adenoviruses, or AAVs. These engineered capsids can be contained within engineered viral particles (e.g., engineered lentiviruses, retroviruses, adenoviruses, or AAV viral particles) and can impart cell-specific tropism, reduce immunogenicity, or both to the engineered viral particles.

[0037] The engineered viral capsids described in this disclosure may comprise one or more artificial viral capsid proteins. These engineered viral capsid proteins may include a macrophage-specific targeting portion, which comprises or is composed of inserted amino acids as described in other parts of this disclosure.

[0038] The engineered viral capsid and / or capsid protein may be encoded by one or more artificial viral capsid polynucleotides. In some embodiments, the engineered viral capsid polynucleotide is an engineered AAV capsid polynucleotide, lentiviral capsid polynucleotide, retroviral capsid polynucleotide, or adenoviral capsid polynucleotide.

[0039] In some embodiments, this engineered viral capsid polynucleotide (e.g., engineered AAV capsid polynucleotide, engineered lentiviral capsid polynucleotide, engineered retroviral capsid polynucleotide, or engineered adenoviral capsid polynucleotide) may include a 3′ polyadenylation signal. This polyadenylation signal may be an SV40 polyadenylation signal.

[0040] In some embodiments, the engineered viral capsid protein may include an inserted n-amino acid sequence. In some embodiments, n may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 15 amino acids.

[0041] In some embodiments, the engineered AAV capsid may contain a 7-amino acid motif.

[0042] In some embodiments, the n-amino acid sequence may be inserted between two amino acids in the wild-type viral capsid protein (or capsid protein). In some embodiments, the n-amino acid sequence may be inserted between two amino acids in a variable amino acid region of the viral capsid protein.

[0043] In some embodiments, one or more n-amino acid sequences may be inserted between two amino acids in one or more of the 12 variable regions of the wild-type AAV capsid protein.

[0044] In some embodiments, these one or more 7-amino acid sequences may 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 combinations thereof.

[0045] In some embodiments, the 7-amino acid sequence can be inserted between two amino acids of VR-III in the capsid protein.

[0046] In some embodiments, the engineered capsid may insert a 7-mer motif between any two consecutive amino acids between amino acids 262 and 269 in the AAV6 viral protein; between any two consecutive amino acids between amino acids 327 and 332; or between amino acids 382 and 386, 452 and 460, 488 and 505, 545 and 558, 581 and 593, and 704 and 714.

[0047] In some embodiments, the engineered capsid may insert an n-amino acid sequence between amino acids 588 and 589 of the AAV6 viral protein.

[0048] In some embodiments, the engineered capsid may insert a 7-mer motif between amino acids 588 and 589 of the AAV6 viral protein.

[0049] SEQ ID NO. 1 is a reference sequence of the AAV6 capsid protein, at least for reference to the insertion site described above. It is understood that the n-amino acid sequence can also be inserted at the corresponding position in other serotypes of AAV viral proteins. In some embodiments, as previously described, the n-mer can be inserted between any two consecutive amino acids in the AAV viral protein, preferably in the variable region (VR).

[0050] Methods for screening engineered AAV caps This disclosure also provides a method for screening engineered AAV capsids using an AAV capsid library containing one or more desired cell-specific engineered AAV capsid variants. Figure 1 and Figure 2 As shown, the AAV capsid library can be administered to various non-human animals for initial mRNA-based screening. Figure 1As shown, the transduction process of AAV and its related vectors can yield mRNA molecules that reflect the viral genome. Based on several examples in this paper, mRNA-based screening methods are more specific and effective in identifying viral particles capable of functionally transducing cells because they select based on functional products, rather than simply detecting the presence of viral DNA in the cell.

[0051] Following the initial dosing, a screened AAV capsid library can be formed using one or more engineered AAV viral particles with the desired capsid variant. 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. Capsid variant particles that are highly expressed in target cells, tissues, and / or organs are the desired AAV capsid variants.

[0052] In some embodiments, the polynucleotide encoding the AAV capsid variant is regulated by a tissue-specific promoter that is selectively active in the desired cell, tissue, or organ.

[0053] The engineered AAV capsid variant particles identified in the first round of screening can then be administered to different non-human cells.

[0054] In some embodiments, the cells used in the second round of screening and identification may differ from those used in the first round. Similar to the first round, after cell administration, the variants with the highest expression in the desired cells can be identified by measuring the expression level of viral mRNA in the cells. The top variants identified in the second round can be selectively barcoded and selectively mixed.

[0055] In some embodiments, the top variant identified in the second round can be given to non-human primates to identify top cell-specific variants, especially when the top variant is used in humans.

[0056] Viral vector In some embodiments, the vector is a viral vector. As used in the art, "viral vector," in this context, refers to a polynucleotide-based vector containing one or more viral components, capable of expressing and packaging a polynucleotide, such as the engineered AAV capsid polynucleotide of this invention, a cargo, or other components or molecules described in this invention, to generate viral particles containing said polynucleotide, and capable of producing said viral particles when used alone or in conjunction with one or more other viral vectors (e.g., in a viral vector system).

[0057] The viral vector and its system can be used to generate viral particles to deliver, express, and / or produce one or more of the components described herein (including, but not limited to, any viral particles and the genetic material they carry).

[0058] The viral vector can form part of a viral vector system that includes multiple vectors. In some embodiments, integrating multiple viral vectors can enhance system security.

[0059] Suitable viral vectors include adenovirus vectors, adeno-associated virus vectors, helper-dependent adenoviral vectors (HdAd), and hybrid adenoviral vectors.

[0060] Other forms of viral vectors and the viral particles they produce are described in other parts of this article.

[0061] In some embodiments, the viral vector is designed to generate viral particles that do not have the ability to replicate, thereby improving the security of the system.

[0062] Adenovirus vectors, helper-dependent adenovirus vectors, and hybrid adenovirus vectors In some embodiments, the vector may be an adenovirus vector. In some embodiments, the viral particles generated using the adenovirus vector or its system may be serotype 2, 5, or 9. In some embodiments, the polynucleotide delivered by the adenovirus particles may be up to about 8 kb in length. Therefore, in some embodiments, the adenovirus vector may include DNA polynucleotides to be delivered, ranging in size from 0.001 kb to 8 kb.

[0063] Adenoviral vectors have been successfully used for gene delivery in multiple scenarios (see Teramato 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).

[0064] The engineered AAV capsid of the present invention can be incorporated into an adenovirus vector to prepare adenovirus particles containing the engineered AAV capsid.

[0065] In some embodiments, the vector may be a helper-dependent adenovirus vector or a system thereof.

[0066] Such vectors are also known in the art as "sterile", "helper virus component-free" or "attenuated" vectors, and are a modified form of adenovirus vector (see, for example, Thrasher et al., 2006, Nature 443:E5-7).

[0067] In helper-dependent adenovirus vector systems, the first type of vector (helper vector) may contain all the genes required for viral replication, but has conditional gene defects in the packaging domain. The second type of vector contains only the ends of the viral genome, one or more of the engineered AAV capsid polynucleotides of this invention, and native packaging recognition signals, thereby allowing selective assembly and release of the viral particle from the cell (see, for example, Cideciyan et al., 2009, NEngl J Med. 361:725-727).

[0068] Gene delivery using helper-dependent adenovirus 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 literature can be modified to adapt to the delivery and integration of the engineered AAV capsid polynucleotides described in this invention.

[0069] In some embodiments, the length of the polynucleotide delivered by the helper-dependent adenovirus vector or its system may be about 38 kb. Therefore, in some embodiments, the adenovirus vector may include DNA polynucleotides to be delivered, the length of which may range from about 0.001 kb to about 37 kb (see, for example, Rosewell et al., 2011. J. Genet. Syndr. Gene Ther. Suppl. 5:001).

[0070] In some embodiments, the vector is a hybrid adenovirus vector or a system thereof. Hybrid adenovirus vectors are generated by combining the high transduction efficiency of gene-deleted adenovirus vectors with the potential long-term gene integration capabilities of adeno-associated virus (AAV), retrovirus, lentivirus, and transposon-like gene transfer systems.

[0071] In some embodiments, this hybridization vector system enables stable transduction of genes at limited integration sites. For example, see Balague et al., 2000, Blood 95:820-828; Morral et al., 1998, Hum. GeneTher. 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 and vectors described in these documents can be adapted and modified for use in the engineered AAV capsid system of this invention.

[0072] In some embodiments, the hybrid adenovirus vector may contain one or more features from retroviruses and / or adeno-associated viruses. In some embodiments, the hybrid adenovirus vector may contain one or more features from spumaretroviral or filamentous viruses (FVs). See, for example, Ehrhardt et al., 2007, Mol. Ther. 15:146-156; Liu et al., 2007, Mol. Ther. 15:1834-1841. The techniques and vectors described in the above literature can also be used in the engineered AAV capsid system of the present invention. The advantages of incorporating one or more features from FVs into the hybrid adenovirus vector or its system include: the resulting viral particles can infect a broad range of cells; their packaging capacity is greater than that of other retroviruses; and they can persist persistently in quiescent (non-dividing) cells.

[0073] Adeno-associated virus vector In some embodiments, the engineered vector or system may be an adeno-associated virus vector (AAV vector). See, for example, 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).

[0074] Although adeno-associated viruses are similar to adenovirus vectors in some respects, they have certain defects in replication and / or pathogenicity, and may therefore be safer than adenovirus vectors.

[0075] In some embodiments, the adeno-associated virus can integrate into a specific site on human chromosome 19, and the formulation can be used for treatment without significant side effects.

[0076] In some embodiments, the loading capacity of the AAV vector, its system, and / or AAV particles is approximately 4.7 kb. The AAV vector or its system may comprise one or more engineered capsid polynucleotides described herein.

[0077] The AAV vector or system thereof may include one or more regulatory molecules. In some embodiments, the regulatory molecules may be promoters, enhancers, repressors, etc., which are described in more detail elsewhere in this document.

[0078] In some embodiments, the AAV vector or its system may contain one or more polynucleotides encoding one or more regulatory proteins.

[0079] In some embodiments, the one or more regulatory proteins may be selected from Rep78, Rep68, Rep52, Rep40, their variants, combinations, etc. In some embodiments, the promoter may be one of the tissue-specific promoters discussed above.

[0080] In some embodiments, the tissue-specific promoter can drive the expression of the engineered AAV capsid polynucleotide described herein.

[0081] The AAV vector or its system may contain one or more polynucleotides encoding one or more capsid proteins, such as the engineered AAV capsid proteins described herein. These engineered capsid proteins are capable of assembling to form the protein coat of AAV viral particles.

[0082] This engineered capsid can achieve tropism towards specific cells, tissues, and / or organs.

[0083] In some embodiments, the AAV vector or its system may also contain one or more adenovirus helper factors or polynucleotides encoding these factors.

[0084] The adenovirus helper factors may include, but are not limited to, E1A, E1B, E2A, E4 ORF6, and VA RNAs.

[0085] In some embodiments, the host cell line that produces AAV particles expresses one or more adenovirus helper factors.

[0086] In some embodiments, the AAV vector or system thereof may be designed to generate AAV particles of a specific serotype.

[0087] In some embodiments, the serotype may be AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, AAV-9, or any combination of these serotypes.

[0088] In some embodiments, the AAV can be AAV1, AAV-2, AAV-5, AAV-9, or any combination thereof.

[0089] Technicians can select specific AAV serotypes based on the cell type to be targeted. For example, for targeting brain and / or neuronal cells, AAV serotypes 1, 2, 5, and 9, or any combination of hybrid capsid AAV1, AAV2, AAV5, and AAV9, can be selected. For targeting myocardial tissue, AAV4 can be selected; For targeting the liver, AAV8 can be selected.

[0090] Therefore, in some embodiments, an AAV carrier and system thereof capable of generating AAV particles that target brain and / or neuronal cells can be designed to generate AAV particles having AAV1, AAV2, AAV5 or hybrid capsid particles thereof.

[0091] In some embodiments, an AAV carrier system capable of generating AAV particles that target myocardial tissue can be designed to generate AAV4 type AAV particles.

[0092] In some embodiments, the AAV vector system capable of generating AAV particles targeting the liver can be designed to generate AAV8 type AAV particles. See also Srivastava (2017, Curr. Opin. Virol. 21:75–80).

[0093] It should be understood that although different serotypes can provide cell, tissue and / or organ specificity to some extent, each serotype is still multi-tropic, so using a serotype to target tissues with low transduction efficiency may lead to tissue toxicity.

[0094] Therefore, in addition to achieving a certain tissue targeting capability by selecting specific AAV serotypes, the tropism of AAV serotypes can also be modulated through the engineered AAV capsid described in this invention. As described elsewhere herein, wild-type AAV variants of any serotype can be generated using the methods described herein and determined to have specific cell tropisms, which may be the same as or different from the wild-type tropism of that serotype.

[0095] In some embodiments, the tropism of wild-type serotypes to specific cell types can be enhanced (e.g., making them more selective or specific to specific cells). For example, wild-type AAV6 in humans is biased towards muscle and liver. By including a variant of the capsid protein comprising the engineered AAV capsid and / or wild-type AAV6 described herein, tropism towards the liver can be reduced or eliminated, while tropism towards muscle can be increased, thereby improving muscle specificity compared to wild-type AAV6.

[0096] As previously mentioned, engineered capsid and / or capsid protein variants of the wild-type AAV serotype are included, and their tropism may differ from that of the wild-type reference serotype.

[0097] In some embodiments, the AAV vector is a hybrid AAV vector or a system thereof.

[0098] Hybrid AAV refers to a partial genome extracted from one AAV serotype and packaged into an AAV with 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 (glandular helper plasmids) will be consistent with those discussed in rAAV2 production; however, the second plasmid pRepCap will be different, referred to as pRep2 / Cap5, where the Rep gene is derived from AAV2 and the Cap gene is derived from AAV5.

[0099] The production protocol is consistent with the AAV2 production method described above. The resulting rAAV is referred to as rAAV2 / 5, where the genome is based on rAAV2 and the capsid is based on AAV5. It is expected that the cellular or tissue tropism exhibited by this AAV2 / 5 hybrid virus should be the same as that of AAV5. It should be understood that this type of wild-type hybrid AAV virus may also exhibit specificity issues similar to those of the aforementioned non-hybrid wild-type serotypes.

[0100] The advantages of the wild-type hybrid AAV system can be achieved by combining the enhanced and customizable cell specificity provided by the engineered AAV capsid described herein, through the generation of hybrid AAV vectors incorporating the engineered AAV capsid.

[0101] It should be understood that hybrid AAVs may also contain engineered AAV capsids, in which the genome contains elements of different serotypes (i.e., the engineered AAV capsid is a variant of a reference wild-type serotype).

[0102] For example, a hybrid AAV can be produced containing an engineered AAV capsid that is a variant of AAV9 serum and is used to package the rep element of AAV2 serum. As previously mentioned, the tropism of the hybrid AAV particles generated from the engineered AAV capsid will depend on the properties of the engineered capsid itself.

[0103] The beneficial effects of this invention are as follows: This invention relates to an AAV capsid protein mutant, in which the insertion of amino acid sequences significantly increases the transduction efficiency of adeno-associated virus (AAV) into peripheral blood macrophages, bone marrow macrophages, and microglia. This AAV capsid protein mutant is the first to target human macrophages, achieving highly efficient and specific delivery of nucleic acids into macrophages, exhibiting excellent cross-species transduction characteristics, unrestricted by species, and possessing broad prospects for basic and clinical applications. This provides a more flexible and efficient approach for basic and clinical translational research related to macrophages. This invention also develops an AAV vector with an engineered capsid through directed evolution, achieving highly efficient gene delivery to macrophages and microglia. Attached Figure Description

[0104] Figure 1 This is the first screening process for the AAV variant library; Figure 2 This is the second screening process for AAV capsid; Figure 3 morphological observation of CD14-positive cells after 7 days of culture; Figure 4 Flow cytometry identification of human PBMC CD14+ cells; Figure 5 In the table, A represents the percentage of the top 100 sequences by count, and B represents the feature analysis of the top 200 inserted segments by count. Figure 6 In the figure, A represents the ratio of the barcode sequence in the parent library and macrophages, respectively, and B represents the transduction capacity of the AAV variant relative to wild-type AAV6. Figure 7 Observation of EGFP fluorescence in human macrophages transduced with four AAV6 variants and controls AAV9, AAV6, AAV9-HGTAASH, and AAV9-WPPKTTS; Figure 8 Flow cytometry analysis of EGFP was performed for each group; Figure 9 Observation of EGFP fluorescence in mouse bone marrow-derived macrophages transduced with four AAV6 variants and controls AAV9, AAV6, AAV9-HGTAASH, and AAV9-WPPKTTS; Figure 10 Flow cytometry analysis was performed on EGFP for each group. Detailed Implementation

[0105] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0106] AAV screening process: Starting with the wild-type AAV6 or AAV9 capsid, we generated an AAV6 or AAV9 capsid library in which each variant had a random heptapeptide sequence inserted between amino acids 588 and 589 of the AAV6 or AAV9 VP1 protein. This insertion site is located at a protrusion along the triple symmetry axis of the capsid, which facilitates the interaction of the inserted peptide with molecules on the target cell membrane. We transduced the rAAV from the capsid library into cultured human monocyte-derived macrophages and recovered successfully transduced capsid variants. Using next-generation sequencing, we identified capsid variants that were highly enriched after screening, such as... Figure 1 As shown.

[0107] Next, the first round of highly enriched capsid variants were packaged with tagged EGFP to prepare barcode libraries. These libraries were then transduced again into human monocyte-derived macrophages and subjected to next-generation DNA sequencing to ultimately identify the truly highly enriched capsid variants, such as... Figure 2 As shown.

[0108] Finally, we packaged the EGFP reporter gene vector into rAAVs containing candidate capsid variants and used these AAV variants to transduce cultured human monocyte-derived macrophages, mouse bone marrow-derived macrophages, and microglia, respectively. Their transduction capabilities were evaluated and compared with parental AAV9, AAV6, AAV9-HGTAASH, and AAV9-WPPKTTS, the two AAV9 variants of which have been reported to efficiently transduce cultured mouse microglia (Lin et al. Nat Methods. 2022, 19(8): 976-985).

[0109] Screening of AAV6 variants that efficiently target macrophages 1. Induction and identification of human macrophages To obtain human macrophages suitable for AAV capsid selection, human PBMC CD14+ cells were isolated and induced to differentiate. The entire experimental procedure is as follows: CD14+ cell isolation → M-CSF-induced differentiation → cell identification Specifically: (1) Experimental methods: A. Commercially available PBMC cells were resuscitated; B. CD14 positive cells were isolated using Easy Human CD14 positive selection kit II; C. Cells were cultured in a cell culture medium containing M-CSF for 7 days to induce differentiation into macrophages; D. Flow cytometry was used to identify anti-CD11b and anti-CD45.

[0110] (2) Experimental results: Microscopic observation showed that the cells cultured for 7 days exhibited a spindle shape or a pancake shape, such as... Figure 3 As shown. Flow cytometry analysis revealed that the percentage of CD11b+CD45+ cells was 96.1%, as indicated. Figure 4 As shown, the vast majority of cells are human macrophages.

[0111] 2. AAV6-7NNK Library Filtering This is the first screening of a library of AAV6 variants targeting macrophages. The screening process is as follows: Figure 1 As shown.

[0112] (1) Experimental methods: A novel AAV library construction strategy was adopted to construct an AAV variant library by inserting a random heptapeptide sequence between positions 588 and 589 of the VP1 protein. Since the insertion site is located in the common region of VP1, VP2 and VP3 proteins, the inserted peptide will also be present in VP2 and VP3 proteins. The AAV6 library was transduced into CD14+ induced macrophages according to 1E+5. RNA samples were collected after 48 h, reverse transcribed into cDNA, and then the variable region of Capsid was amplified by PCR. Finally, the PCR product was subjected to NGS sequencing.

[0113] Bioinformatics analysis was used to sort AAV variants from high to low based on copy number, and candidate AAV variants were obtained for a second screening.

[0114] (2) Experimental Results: The AAV6 variant library constructed using the novel strategy minimizes the problem of cross-packaging between different AAV capsids. NGS sequencing analysis revealed approximately 1E+6 unique nucleotide sequences in the AAV6 parent library, consistent with the sequence numbers obtained in the first round of screening of most known libraries. NGS sequencing analysis of human macrophages infected with the AAV6-7NNK library revealed approximately 1E+5 unique nucleotide sequences in this sample.

[0115] Comparison with the AAV6 parent library revealed significant enrichment of over 27,700 DNA sequences. The top 200 sequences by count accounted for 16.7% of the total count, and the top 20 sequences accounted for 3.8%. The highest count reached over 38,000, exceeding 0.4% of the total count. Figure 5 As shown in Figure A.

[0116] At amino acids positions 588 to 595 of VP1, the top 200 sequences by count exhibit certain DNxxxxG characteristics, such as... Figure 5 As shown in Figure B. Considering that the copy number of AAV detected in macrophages is closely related to the production of AAV, the top 20 copies with the highest count will be selected for further screening.

[0117] 3. AAV6-barcode document filtering This study selected the top 20 in terms of count from the first screening for a second screening. The screening process is as follows: Figure 2 As shown.

[0118] (1) Experimental methods: In this study, barcode-bearing EGFP was used as the GOI. AAV6 variants and controls AAV9, AAV6, AAV9-HGTAASH, and AAV9-WPPKTTS were packaged using a three-plasmid method. CD14+ cells were 4.2E6 cells. Barcode-bearing AAV was added at an MOI of 1.9E3. RNA samples were collected after 48 hours, reverse transcribed into cDNA, and then the barcode region was amplified by PCR. Finally, NGS sequencing was performed. Bioinformatics analysis was used to determine the proportion of each barcode in the total count.

[0119] (2) Experimental results: NGS analysis of the AAV6 barcode parent library showed that the parent library contained 23 barcodes. Except for 3 barcodes whose distribution in the parent library was either too high or too low, the other barcodes showed a relatively uniform distribution.

[0120] NGS analysis of the experimental group revealed that the counts of two barcodes accounted for the vast majority, such as Figure 6 As shown in Figure A.

[0121] Compared with the control AAV6, all 20 AAV variants showed enhanced transduction capabilities, with DC-11 (corresponding to the heptapeptide sequence QNDIKNG), DC-16 (corresponding to the heptapeptide sequence GNDLRPT), DC-25 (corresponding to the heptapeptide sequence NGNAIVG), and DD-2 (corresponding to the heptapeptide sequence DNNLAKL) exhibiting the best performance. Their transduction capabilities were 1215-fold, 499-fold, 820-fold, and 524-fold, respectively, compared to the parent AAV6. Figure 6 As shown in B. Subsequent verification experiments will also focus on these four AAV6 variants. The amino acid sequences of the four AAV6 variants VP1 are shown in SEQ ID NO. 2-5, and the gene sequences of the four AAV6 variants VP1 are shown in SEQ ID NO. 6-9.

[0122] Validation of AAV variants' macrophage transduction capabilities 1. Transduction of AAV variants in human macrophages Through secondary screening, a series of AAV variants with better transduction capacity than the parent were obtained, and the four better ones were selected for further verification.

[0123] (1) Experimental methods: EGFP with barcode was used as GOI. Four AAV6 variants and controls AAV9, AAV6, AAV9-HGTAASH, and AAV9-WPPKTTS were obtained by packaging and screening using the three-plasmid method. CD14+ cells were seeded in 12-well plates at 4E5 / well and transduction was performed at MOI 5E4. After 48 h, cell fluorescence and flow cytometry were used to detect EGFP expression.

[0124] (2) Experimental results: Microscopic observation revealed that, except for wild-type AAV9, all other AAV groups had EGFP-positive macrophages, such as Figure 7 As shown in the figure. Flow cytometry analysis revealed that the positivity rate of AAV6 variants was significantly higher than that of wild-type AAV6 (24.6% for wild-type AAV6), with the variant AAV6-QNDIKNG exhibiting the highest positivity rate at 51.8%. Figure 8 As shown.

[0125] 2. Transduction of AAV variants in mouse bone marrow-derived macrophages Through secondary screening, a series of AAV variants with better transduction capabilities than the parent were obtained, and four of the better ones were selected for further verification.

[0126] (1) Experimental method: EGFP with barcode was used as GOI. Four AAV6 variants and controls AAV9, AAV6, AAV9-HGTAASH and AAV9-WPPKTTS were obtained by packaging and screening using the three plasmid method. CD14+ cells were seeded in 12-well plates and transduction was performed according to MOI 1E4. After 48 h, cell fluorescence and flow cytometry were used to detect EGFP expression.

[0127] (2) Experimental results: Microscopic observation revealed that, except for wild-type AAV9, all other AAV groups had EGFP-positive macrophages, such as Figure 9 As shown in the figure. Flow cytometry analysis revealed that the positive rates of AAV6 variants were significantly higher than those of wild-type AAV6 (41.6% for wild-type AAV6), with the highest positive rate of variant AAV6-QNDIKNG at 87.9%. The positive rates of both wild-type and variant AAV9 were no higher than 13%. Figure 10 As shown.

[0128] As shown above, at the same dosage, the screened AAV variants can transduce macrophages more efficiently than wild-type AAV parents, and this transduction ability is applicable to macrophages from different species. These highly efficient AAV variants can not only promote basic research on macrophages, but also be applied to CAR-M cell therapy and the treatment of other macrophage-related diseases.

[0129] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes or modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions should also fall within the scope of the invention and should be defined by the claims.

Claims

1. An AAV capsid protein mutant, characterized in that, The capsid protein VP1 protein, or a protein having at least 80% sequence identity with the amino acid sequence of the above proteins, is a capsid protein of at least one of the wild-type or modified AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.74, and AAVrh.

10. A short peptide amino acid sequence of at least one of QNDIKNG, GNDLRPT, NGNAIVG, and DNNLAKL is inserted between any two amino acids in the surface variable region of the protein.

2. The AAV capsid protein mutant according to claim 1, characterized in that, The variable surface region of the capsid protein VP1 is at least one of VR-IV, VR-V, and VR-VIII.

3. An AAV capsid protein mutant according to claim 1 or 2, characterized in that, The variable surface region of the capsid protein VP1 includes at least one of the following positions: 262–269, 327–332, 382–386, 452–460, 488–505, 527–539, 545–558, 581–593, and 704–714.

4. The AAV capsid protein mutant according to claim 3, characterized in that, At least one of the amino acid sequences of the short peptide is inserted between amino acids 588 and 589 of the variable region on the surface of the capsid protein VP1.

5. The AAV capsid protein mutant according to claim 4, characterized in that, The AAV capsid protein VP1 is the wild-type AAV6 capsid protein VP1.

6. The AAV capsid protein mutant according to claim 5, characterized in that, Between amino acids 588 and 589 of the AAV6 capsid protein VP1, short peptide amino acids QNDIKNG, GNDLRPT, NGNAIVG, and DNNLAKL were inserted, respectively, to obtain four AAV6 capsid protein mutants, the amino acid sequences of which are shown in SEQ ID NO. 2-5.

7. A gene encoding the AAV capsid protein mutant of claim 5.

8. An expression carrier, characterized in that, It contains the gene encoding the AAV capsid protein mutant as described in claim 7.

9. A host cell, characterized in that, It comprises the gene encoding the AAV capsid protein mutant as described in claim 7 or the expression vector as described in claim 8.

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

11. 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 7 or the expression vector as described in claim 8; (2) GOI plasmids containing the target gene.

12. A pharmaceutical composition, characterized in that, It includes rAAV prepared by the preparation method of claim 11 and pharmaceutically acceptable carriers.

13. The gene encoding the AAV capsid protein mutant as described in claim 7, the expression vector as described in claim 8, or the rAAV prepared by the preparation method as described in claim 11, for use in the preparation of a medicament for delivering the gene product to the cells or tissues of a subject.

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

15. The virus particle according to claim 14, characterized in that, It also includes recombinant polynucleotides that encode genes of interest.

16. An engineered particle prepared from the virus particles according to claim 14, characterized in that, Its cargo carrier can be used to modulate macrophage function and treat or prevent macrophage-related diseases or disorders.

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