Engineered gene transcriptional repression tool targeting vascular endothelial growth factor and use thereof

Abstract

The present application relates to an engineered gene transcriptional repression tool targeting a vascular endothelial growth factor and a use thereof, and in particular to a complex and a fusion. The complex comprises a first fusion and a second fusion, wherein: 1) one of the first fusion and the second fusion comprises a DNA methylation domain and at least one recruitment domain A, and the other fusion comprises a transcriptional repressor domain and at least one recruitment domain A'; and 2) the first fusion or the second fusion comprises a nucleic acid binding domain; moreover, the recruitment domain A and the recruitment domain A' can interact such that one of the first fusion and the second fusion or a portion thereof can be recruited to the vicinity of the other fusion; and the nucleic acid binding domain can specifically bind to a target nucleotide sequence on a VEGFA gene and / or a regulatory element thereof.

Description

Engineered gene transcription repression tools targeting vascular endothelial growth factor and uses thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a complex for regulating vascular endothelial growth factor (VEGFA) gene expression and uses thereof. BACKGROUND

[0002] Currently, wet age-related macular degeneration (wet-AMD) is an eye disease that affects central vision, mainly due to abnormal blood vessel growth in the central area of the retina (macula), leading to subretinal space fluid accumulation, hemorrhage or scarring. This lesion often leads to rapid decline in central vision, thus significantly affecting daily activities such as reading and facial recognition. According to data from the World Health Organization, millions of people worldwide are affected by AMD, with wet macular degeneration being particularly severe, posing a serious threat to the vision of the elderly. With the intensification of global population aging, the number of patients with wet macular degeneration is expected to continue to increase. It is estimated that by 2050, the number of people with AMD could double as the global elderly population increases. Globally, the prevalence of wet AMD is gradually increasing, which poses a significant burden to the public health system.

[0003] The pathological basis of wet AMD is related to the neovascularization under the retina, which is prone to leakage and hemorrhage, leading to impaired function in the macular area. Studies have shown that the formation of this lesion is closely related to the expression level of VEGF, a factor that promotes angiogenesis, which is abnormally increased in the eyes of patients with wet AMD. Current treatments for wet AMD mainly include anti-VEGF therapy, which can effectively inhibit the growth and leakage of abnormal blood vessels and slow down the progression of the disease. However, this treatment cannot cure wet AMD, and long-term periodic injections of drugs are required, and once treatment is stopped, abnormal blood vessels may quickly recur. Therefore, developing a safe and long-term effective treatment strategy has become a market pain point that needs to be addressed.

[0004] One of the current research focuses is to find therapeutic strategies that can precisely regulate the expression of VEGF and other related factors. Epigenetic regulation, such as modulating the expression of specific genes by modifying their epigenetic markers, provides an innovative approach to treating wet-AMD. It regulates the expression of key factors such as VEGF by modifying the epigenetic markers of genes, inhibiting the formation of new blood vessels, without involving DNA cleavage, thereby reducing the risk of genomic damage. The reversibility, low immunogenicity, and high targeting of this method make it a promising treatment option. Although this technology is still in development, its potential to combine with traditional treatments and flexible adjustment of treatment strategies demonstrates its great potential in future medical practice. SUMMARY

[0005] In one aspect, the present application provides a complex comprising a first fusion and a second fusion, wherein: 1) one of the fusions of the first fusion and the second fusion comprises a DNA methylation domain and at least one recruiting domain A, and wherein the other fusion comprises a transcription repressor domain and at least one recruiting domain A'; and 2) the first fusion or the second fusion comprises a nucleic acid binding domain; and the recruiting domain A and the recruiting domain A' are capable of interacting to enable the one of the fusions of the first fusion and the second fusion or a portion thereof to be recruited to the vicinity of the other fusion; the nucleic acid binding domain is capable of specifically binding to a target nucleotide sequence on the VEGFA gene and / or a regulatory element of the VEGFA gene.

[0006] In some embodiments, the nucleic acid binding domain is a DNA binding domain.

[0007] In some embodiments, the DNA binding domain is selected from the group consisting of: a TALE domain, a zinc finger domain, a tetR domain, a meganuclease, a Cas protein, an Argonaute (Ago) protein, and homologs, modified forms, or variants thereof.

[0008] In some embodiments, the DNA binding domain is capable of binding to the target nucleotide sequence.

[0009] In some embodiments, the DNA binding domain is capable of binding to a guide RNA.

[0010] In some embodiments, the guide RNA is capable of specifically recognizing and hybridizing to the target nucleotide sequence.

[0011] In some embodiments, the guide RNA is capable of specifically recognizing and hybridizing to the target nucleotide sequence within a region from about 500 bp, about 400 bp, about 300 bp, about 200 bp, about 100 bp, about 50 bp upstream of the transcription start site of the VEGFA gene to about 500 bp, about 400 bp, about 300 bp, about 200 bp, about 100 bp, about 50 bp downstream of the transcription start site of the VEGFA gene.

[0012] In some embodiments, the guide RNA is capable of specifically recognizing and hybridizing to the target nucleotide sequence within a region from about 500 bp, about 400 bp, about 300 bp, about 200 bp, about 100 bp, about 50 bp upstream of the transcription start site of the VEGFA gene to about 4000 bp, about 3500 bp, about 3000 bp, about 2500 bp, about 2000 bp, about 1500 bp, about 1000 bp, about 500 bp, about 250 bp, about 100 bp, about 50 bp downstream of the transcription start site of the VEGFA gene.

[0013] In some embodiments, the guide RNA is capable of specifically recognizing and hybridizing to the target nucleotide sequence within a region from about 500 bp, about 400 bp, about 300 bp, about 200 bp, about 100 bp, about 50 bp upstream of the transcription start site of the VEGFA gene to about 3500 bp, about 3000 bp, about 2500 bp, about 2000 bp, about 1500 bp, about 1000 bp, about 500 bp, about 250 bp, about 100 bp, about 50 bp downstream of the transcription start site of the VEGFA gene.

[0014] In some embodiments, the guide RNA is capable of specifically recognizing and hybridizing to the target nucleotide sequence within a region from about 250 bp upstream of the transcription start site of the VEGFA gene to about 250 bp downstream of the transcription start site of the VEGFA gene.

[0015] In some embodiments, the guide RNA comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 337-368, 382-404, 406-421, 423-425, 427, 429, 430-455, 457-460, 462, 463, and 465.

[0016] In some embodiments, the guide RNA comprises a partial sequence of a nucleotide sequence as set forth in any one of SEQ ID NOs: 337-368, 382-404, 406-421, 423-425, 427, 429, 430-455, 457-460, 462, 463, and 465, the partial sequence having a length of 15-20 base pairs.

[0017] In some embodiments, the DNA binding domain is a Cas protein, and the Cas protein is a Class II Cas nuclease.

[0018] In some embodiments, the Cas protein is selected from the group consisting of a Class II Type II Cas nuclease and a Class II Type V Cas nuclease.

[0019] In some embodiments, the Cas protein is a Cas9 or Cas12 protein.

[0020] In some embodiments, the Cas protein is a deactivated Cas9 (dCas9) protein or a deactivated Cas12 (dCas12) protein.

[0021] In some embodiments, the DNA-binding domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-9.

[0022] In some embodiments, the first fusion comprises a DNA methylation domain, a nucleic acid binding domain, and at least one recruitment domain A, and the second fusion comprises a transcription repressor domain and at least one recruitment domain A’.

[0023] In some embodiments, the first fusion comprises a DNA methylation domain, a nucleic acid binding domain, and at least one recruitment domain A, and the second fusion comprises a transcription repressor domain and at least one recruitment domain A’.

[0024] In some embodiments, the second fusion comprises a transcription repressor domain and at least one recruitment domain A’ in N-terminal to C-terminal order, or a recruitment domain A’ and a transcription repressor domain in N-terminal to C-terminal order.

[0025] In some embodiments, the first fusion comprises a transcription repressor domain, a nucleic acid binding domain, and at least one recruitment domain A, and the second fusion comprises a DNA methylation domain and at least one recruitment domain A’.

[0026] In some embodiments, the first fusion comprises a transcription repressor domain, a nucleic acid binding domain, and at least one recruitment domain A, and the second fusion comprises a DNA methylation domain and at least one recruitment domain A’.

[0027] In some embodiments, the second fusion comprises a DNA methylation domain and at least one recruitment domain A’ in N-terminal to C-terminal order, or a recruitment domain A’ and a DNA methylation domain in N-terminal to C-terminal order.

[0028] In some embodiments, the complex is characterized in that: 1) the first fusion comprises, in order from N- to C-terminus, a DNA methylation domain, a nucleic acid binding domain, and a recruitment domain A, and the second fusion comprises, in order from N- to C-terminus, a transcription repressor domain and a recruitment domain A'; or 2) the first fusion comprises, in order from N- to C-terminus, a DNA methylation domain, a nucleic acid binding domain, and a recruitment domain A, and the second fusion comprises, in order from N- to C-terminus, a recruitment domain A' and a transcription repressor domain; or 3) the first fusion comprises, in order from N- to C-terminus, a recruitment domain A, a nucleic acid binding domain, and a transcription repressor domain, and the second fusion comprises, in order from N- to C-terminus, a DNA methylation domain and a recruitment domain A'; or 4) the first fusion comprises, in order from N- to C-terminus, a recruitment domain A, a nucleic acid binding domain, and a transcription repressor domain, and the second fusion comprises, in order from N- to C-terminus, a recruitment domain A' and a DNA methylation domain.

[0029] In some embodiments, the recruitment domain A is selected from any one of one of the following two groups of domains, and the recruitment domain A' is selected from any one of the other of the following two groups of domains: 1) a general control non-derepressible protein 4 (GCN4), a GFP11 fragment derived from split green fluorescent protein (GFP), or a GVKESLV polypeptide; and 2) a single-chain antibody (scFv), a GFP1-10 fragment derived from split green fluorescent protein (GFP), or a PDZ protein domain.

[0030] In some embodiments, wherein: 1) the domain of one of the recruitment domain A and the recruitment domain A' is GCN4, and the domain of the other is a scFv; or 2) the domain of one of the recruitment domain A and the recruitment domain A' is a GFP11 fragment, and the domain of the other is a GFP1-10; or 3) the domain of one of the recruitment domain A and the recruitment domain A' is GVKESLV, and the domain of the other is a PDZ protein domain.

[0031] In some embodiments, the DNA methylation domain comprises at least one DNA methyltransferase or a functionally active fragment thereof.

[0032] In some embodiments, the DNA methyltransferase is selected from the group consisting of DNMT3A, DNMT3B, DNMT3C, DNMT1, DNMT2, and DNMT3L.

[0033] In some embodiments, the DNA methylation domain comprises at least one DNMT3A and at least one DNMT3L.

[0034] In some embodiments, the DNA methyltransferase comprises an amino acid sequence set forth in any one of SEQ ID NOs: 19-24.

[0035] In some embodiments, the DNA methylation domain comprises a DNMT3A-DNMT3L domain or a DNMT3L-DNMT3A domain; wherein - indicates that the domains at both ends thereof are directly or indirectly connected in order from the N-terminus to the C-terminus.

[0036] In some embodiments, the transcriptional repressor is selected from one or more of the following domains: KRAB, ZIM3KRAB, ZNF680, ZNF554, ZNF264, ZNF582, ZNF324, ZNF669, ZNF354A, ZNF82, ZNF595, ZNF419, ZNF566, ZIM2, EHMT2, SUV39H1, ZFPM1, TRIM28, EZH2, MXD1, SID, LSD1, HP1a, HDAC3, HDAC1, PRMT1, SETDB1, hSIRT1, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF41, ZNF189, ZNF528, ZNF543, ZNF140, ZNF610, ZNF350, ZNF8, ZNF30, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP82, ZNF224, ZNF33A, ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, CBX1, SCMH1, MPP8, SUMO3, HERC2, BIN1, PCGF2, TOX, FOXA1, FOXA2, IRF2BP1, IRF2BP2, IRF2BPL IRF-2BP1_2 N-terminal domain, HOXA13, HOXB13, HOXC13, HOXA11, HOXC11, HOXC10, HOXA10, HOXB9, HOXA9, ZFP28, ZN334, ZN568, ZN37A, ZN181, ZN510, ZN862, ZN140, ZN208, ZN248, ZN571, ZN699, ZN726, ZIK1, ZNF2, Z705F, ZNF14, ZN471, ZN624, ZNF84, ZNF7, ZN891, ZN337, Z705G, ZN529, ZN729, ZN419, Z705A, ZN302, ZN486, ZN621, ZN688, ZN33A, ZN554, ZN878, ZN772, ZN224, ZN184, ZN544, ZNF57, ZN283, ZN549, ZN211, ZN615, ZN253, ZN226, ZN730, Z585A, ZN732, ZN681,ZN667, ZN649, ZN470, ZN484, ZN431, ZN382, ZN254, ZN124, ZN607, ZN317, ZN620, ZN141, ZN584, ZN540, ZN75D, ZN555, ZN658, ZN684, RBAK, ZN829, ZN582, ZN112, ZN716, HKR1, ZN350, ZN480, ZN416, ZNF92, ZN100, ZN736, ZNF74, ZN443, ZN195, ZN530, ZN782, ZN791, ZN331, Z354C, ZN157, ZN727, ZN550, ZN793, ZN235, ZN724, ZN573, ZN577, ZN789, ZN718, ZN300, ZN383, ZN429, ZN677, ZN850, ZN454, ZN257, ZN264, ZN485, ZN737, ZNF44, ZN596, ZN565, ZN543, ZFP69, SUMO1, ZNF12, ZN169, ZN433, ZN175, ZN347, ZNF25, ZN519, Z585B, ZN517, ZN846, ZN230, ZNF66, ZN713, ZN816, ZN426, ZN674, ZN627, ZNF20, Z587B, ZN316, ZN233, ZN611, ZN556, ZN234, ZN560, ZNF77, ZN682, ZN614, ZN785, ZN445, ZFP30, ZN225, ZN551, ZN610, ZN528, ZN284, ZN418, ZN490, ZN805, Z780B, ZN763, ZN285, ZNF85, ZN223, ZNF90, ZN557, ZN425, ZN229, ZN606, ZN155, ZN222, ZN442, ZNF91, ZN135, ZN778, ZN534, ZN586, ZN567, ZN440, ZN583, ZN441, ZNF43, ZN589, ZN563, ZN561, ZN136, ZN630, ZN527, ZN333, Z324B, ZN786, ZN709, ZN792, ZN599, ZN613, ZF69B, ZN799, ZN569, ZN564, ZN546, ZFP92, ZN723, ZN439, ZFP57, ZNF19, ZN404, ZN274, CBX3, ZN250, ZN570, ZN675, ZN695, ZN548, ZN132, ZN738, ZN420, ZN626, ZN559, ZN460, ZN268, ZN304, ZN605, ZN844,SUMO5, ZN101, ZN783, ZN417, ZN182, ZN823, ZN177, ZN197, ZN717, ZN669, ZN256, ZN251, CBX4, CDY2, CDYL2, ZN562, ZN461, Z324A, ZN766, ID2, ZN214, CBX7, ID1, CREM, SCX, ASCL1, ZN764, SCML2, TWST1, CREB1, TERF1, ID3, CBX8, GSX1, NKX22, ATF1, TWST2, ZNF17, TOX3, TOX4, ZMYM3, I2BP1, RHXF1, SSX2, I2BPL, ZN680, TRI68, HXA13, PHC3, TCF24, HXB13, HEY1, PHC2, ZNF81, FIGLA, SAM11, KMT2B, HEY2, JDP2, HXC13, ASCL4, HHEX, GSX2, ETV7, ASCL3, PHC1, OTP, I2BP2, VGLL2, HXA11, PDLI4, ASCL2, CDX4, ZN860, LMBL4, PDIP3, NKX25, CEBPB, ISL1, CDX2, PROP1, SIN3B, SMBT1, HXC11, HXC10, PRS6A, VSX1, NKX23, MTG16, HMX3, HMX1, KIF22, CSTF2, CEBPE, DLX2, PPARG, PRIC1, UNC4, BARX2, ALX3, TCF15, TERA, VSX2, HXD12, CDX1, TCF23, ALX1, HXA10, RX, CXXC5, SCML1, NFIL3, DLX6, MTG8, CEBPD, SEC13, FIP1, ALX4, LHX3, PRIC2, MAGI3, NELL1, PRRX1, MTG8R, RAX2, DLX3, DLX1, NKX26, NAB1, SAMD7, PITX3, WDR5, MEOX2, NAB2, DHX8, CBX6, EMX2, CPSF6, HXC12, KDM4B, LMBL3, PHX2A, EMX1, NC2B, DLX4, SRY, ZN777, ZN398, GATA3, BSH, SF3B4, TEAD1, TEAD3, RGAP1, PHF1, GATA2, FOXO3, ZN212, IRX4, ZBED6, LHX4, SIN3A, RBBP7, NKX61, R51A1, MB3L1, DLX5, NOTC1, TERF2, ZN282, RGS12, ZN840, SPI2B, PAX7, NKX62, ASXL2, FOXO1, GATA1,ZMYM5, LRP1, MIXL1, SGT1, LMCD1, CEBPA, SOX14, WTIP, PRP19, NKX11, RBBP4, DMRT2, SMCA2, and functionally active fragments thereof.

[0037] In some embodiments, the transcription repressor domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 25-50.

[0038] In some embodiments, wherein: 1) one of the first fusion and the second fusion comprises a DNA methylation domain-dCas9 or dCas12-n x GCN4, and the other comprises a transcription repressor domain-scFv; or 2) one of the first fusion and the second fusion comprises a DNA methylation domain-dCas9 or dCas12-scFv, and the other comprises a transcription repressor domain-GCN4; or 3) one of the first fusion and the second fusion comprises a DNA methylation domain-dCas9 or dCas12-n x GFP11, and the other comprises a transcription repressor domain-GFP1-10; or 4) one of the first fusion and the second fusion comprises a DNA methylation domain-dCas9 or dCas12-GFP1-10, and the other comprises a transcription repressor domain-GFP11; or 5) one of the first fusion and the second fusion comprises a DNA methylation domain-dCas9 or dCas12-n x GCN4, and the other comprises a scFv-transcription repressor domain; or 6) one of the first fusion and the second fusion comprises a DNA methylation domain-dCas9 or dCas12-scFv, and the other comprises a GCN4-transcription repressor domain; or 7) one of the first fusion and the second fusion comprises a DNA methylation domain-dCas9 or dCas12-n x GFP11, and the other comprises a GFP1-10-transcription repressor domain; or 8) one of the first fusion and the second fusion comprises a DNA methylation domain-dCas9 or dCas12-GFP1-10, and the other comprises a GFP11-transcription repressor domain; wherein - indicates that the domains at its two ends are directly or indirectly connected in order from N-terminus to C-terminus; n x GCN4 or n x GFP11 indicates n copies of GCN4 or n copies of GFP11, respectively, connected by a linker sequence, n being selected from any integer from 1 to 20.

[0039] In some embodiments, the first fusion and / or the second fusion comprises the amino acid sequence set forth in any one of SEQ ID NOs: 51-76, 78-82, 85-93, 103-105, 110-115, 123, and 124.

[0040] In some embodiments, the complex comprises the amino acid sequence set forth in any one of SEQ ID NOs: 133-142, 153, 154, 158-163, and 168.

[0041] In some embodiments, wherein: 1) one of the first and second fusions comprises a fusion of n x GCN4-dCas9 or dCasl2-transcription repressor domain, and the other comprises a DNA methylation domain-scFv; or 2) one of the first and second fusions comprises a fusion of scFv-dCas9 or dCasl2-transcription repressor domain, and the other comprises a DNA methylation domain-GCN4; or 3) one of the first and second fusions comprises a fusion of n x GFPn-dCas9 or dCasl2-transcription repressor domain, and the other comprises a DNA methylation domain-GFPi-io; or 4) one of the first and second fusions comprises a fusion of GFPi-io-dCas9 or dCasl2-transcription repressor domain, and the other comprises a DNA methylation domain-GFPn; or 5) one of the first and second fusions comprises a fusion of n x GCN4-dCas9 or dCasl2-transcription repressor domain, and the other comprises a scFv-DNA methylation domain; or 6) one of the first and second fusions comprises a fusion of scFv-dCas9 or dCasl2-transcription repressor domain, and the other comprises a GCN4-DNA methylation domain; or 7) one of the first and second fusions comprises a fusion of n x GFPn-dCas9 or dCasl2-transcription repressor domain, and the other comprises a GFPi-io-DNA methylation domain; or 8) one of the first and second fusions comprises a fusion of GFPi-io-dCas9 or dCasl2-transcription repressor domain, and the other comprises a GFPn-DNA methylation domain; wherein - indicates that the domains at its two ends are connected in order from N-terminus to C-terminus, directly or indirectly; n x GCN4 or n x GFPn indicates n copies of GCN4 or n copies of GFPn connected by a linker sequence, respectively, n is selected from any integer from 1 to 20.

[0042] In some embodiments, the first and / or second fusion comprises an amino acid sequence set forth in any one of SEQ ID NOs: 83, 84, 94-102, 106-109, and 116-122.

[0043] In some embodiments, the complex comprises an amino acid sequence set forth in any one of SEQ ID NOs: 143-152, 155-157, and 164-167.

[0044] In some embodiments, the complex further comprises a nuclear localization signal and / or a marker domain.

[0045] In some embodiments, the complex is capable of providing modification of at least one nucleotide within a regulatory element of the VEGFA gene.

[0046] In some embodiments, the complex is capable of providing modification of at least one nucleotide within a region from about 500 bp, about 400 bp, about 300 bp, about 200 bp, about 100 bp, about 50 bp upstream of the transcription start site of the VEGFA gene.

[0047] In some embodiments, the complex is capable of providing modification of at least one nucleotide within a region from about 4000 bp, about 3500 bp, about 3000 bp, about 2500 bp, about 2000 bp, about 1500 bp, about 1000 bp, about 500 bp, about 250 bp, about 100 bp, about 50 bp downstream of the transcription start site of the VEGFA gene.

[0048] In some embodiments, the complex is capable of providing modification of at least one nucleotide within a region from about 500 bp upstream to about 3500 bp downstream, from about 500 bp upstream to about 3000 bp downstream, from about 500 bp upstream to about 2500 bp downstream, from about 500 bp upstream to about 2000 bp downstream, from about 500 bp upstream to about 1500 bp downstream, from about 500 bp upstream to about 1000 bp downstream, from about 500 bp upstream to about 500 bp downstream of the transcription start site of the VEGFA gene.

[0049] In some embodiments, the complex is capable of providing modification of at least one nucleotide within a region from about 250 bp upstream to about 250 bp downstream of the transcription start site of the VEGFA gene.

[0050] In another aspect, the present application provides a fusion comprising a nucleic acid binding domain, and one or more effector domains; the effector domains comprise one or more of an epigenetic modification domain and / or a transcriptional regulation domain, and the fusion comprises no less than two of the epigenetic modification domain and / or the transcriptional regulation domain in total, the nucleic acid binding domain is capable of specifically binding to a target nucleotide sequence on a VEGFA gene and / or a regulatory element of a VEGFA gene, the regulatory element of a VEGFA gene comprises a transcription start site, a core promoter, a promoter, an enhancer, a silencer, an insulator element, a boundary element, and / or a locus control region, the nucleic acid binding domain is capable of specifically binding to a target nucleotide sequence within a region from 500 bp upstream to 4000 bp downstream of a transcription start site of a VEGFA gene.

[0051] In some embodiments, the nucleic acid binding domain is a DNA binding domain.

[0052] In some embodiments, the DNA binding domain is selected from the group consisting of: a TALE domain, a zinc finger domain, a tetR domain, a meganuclease, a Cas protein, an Argonaute (Ago) protein, and a homolog or a modified form thereof.

[0053] In some embodiments, the DNA binding domain is capable of binding to a target sequence of a target locus.

[0054] In some embodiments, the DNA binding domain is capable of binding to a guide RNA.

[0055] In some embodiments, the guide RNA is capable of specifically recognizing and hybridizing to a target sequence of the target locus.

[0056] In some embodiments, the guide RNA is capable of specifically recognizing and hybridizing to a target nucleotide sequence within a region from a transcription start site of a VEGFA gene to about 500 bp, about 400 bp, about 300 bp, about 200 bp, about 100 bp, about 50 bp upstream thereof.

[0057] In some embodiments, the guide RNA is capable of specifically recognizing and hybridizing to a target nucleotide sequence within a region from a transcription start site of a VEGFA gene to about 4000 bp, about 3500 bp, about 3000 bp, about 2500 bp, about 2000 bp, about 1500 bp, about 1000 bp, about 500 bp, about 250 bp, about 100 bp, about 50 bp downstream thereof.

[0058] In some embodiments, the guide RNA is capable of specifically recognizing and hybridizing to the target nucleotide sequence within a region from about 500 bp upstream to about 3500 bp downstream, from about 500 bp upstream to about 3000 bp downstream, from about 500 bp upstream to about 2500 bp downstream, from about 500 bp upstream to about 2000 bp downstream, from about 500 bp upstream to about 1500 bp downstream, from about 500 bp upstream to about 1000 bp downstream, or from about 500 bp upstream to about 500 bp downstream of the transcription start site of the VEGFA gene.

[0059] In some embodiments, the guide RNA is capable of specifically recognizing and hybridizing to the target nucleotide sequence within a region from about 250 bp upstream to about 250 bp downstream of the transcription start site of the VEGFA gene.

[0060] In some embodiments, the guide RNA comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 337-368, 382-404, 406-421, 423-425, 427, 429, 430-455, 457-460, 462, 463, and 465.

[0061] In some embodiments, the guide RNA comprises a partial sequence of the nucleotide sequence as set forth in any one of SEQ ID NOs: 337-368, 382-404, 406-421, 423-425, 427, 429, 430-455, 457-460, 462, 463, and 465, the partial sequence being 15-20 base pairs in length.

[0062] In some embodiments, the DNA-binding domain is a Cas protein, and the Cas protein is a Class II Cas nuclease.

[0063] In some embodiments, the Cas protein is selected from the group consisting of a Class II Type II Cas nuclease and a Class II Type V Cas nuclease.

[0064] In some embodiments, the DNA-binding domain is a Cas9 protein.

[0065] In some embodiments, the Cas9 protein is a deactivated Cas9 protein (dCas9).

[0066] In some embodiments, the nucleic acid-binding domain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-9.

[0067] In some embodiments, the epigenetic modification domain is selected from the group consisting of DNA deaminase activity, DNA methyltransferase activity, DNA demethylase activity, DNA aminase activity, DNA oxidizing activity, DNA helicase activity, histone acetyltransferase activity, histone deacetylase activity, histone methyltransferase activity, histone demethylase activity, histone kinase activity, histone phosphatase activity, histone ubiquitin ligase activity, and histone deubiquitinating activity.

[0068] In some embodiments, the epigenetic modification domain comprises a DNA methyltransferase and / or a functionally active fragment thereof.

[0069] In some embodiments, the DNA methyltransferase is selected from the group consisting of DNMT3A, DNMT3B, DNMT3C, DNMT1, DNMT2, and DNMT3L.

[0070] In some embodiments, the epigenetic modification domain comprises a plurality of DNA methyltransferases and / or functionally active fragments thereof, and the plurality of DNA methyltransferases and / or functionally active fragments thereof are linked by a linker sequence.

[0071] In some embodiments, the epigenetic modification domain comprises at least one DNMT3A and at least one DNMT3L.

[0072] In some embodiments, the DNA methyltransferase comprises an amino acid sequence set forth in any one of SEQ ID NOs: 19-24.

[0073] In some embodiments, the transcriptional regulation domain is a transcriptional activation domain or a transcriptional repressor domain.

[0074] In some embodiments, the transcription repressor domain is selected from the group consisting of: KRAB, ZIM3, ZNF680, ZNF554, ZNF264, ZNF582, ZNF324, ZNF669, ZNF354A, ZNF82, ZNF595, ZNF419, ZNF566, ZIM2, EHMT2, SUV39H1, ZFPM1, TRIM28, EZH2, MXD1, SID, LSD1, HP1a, HDAC3, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF41, ZNF189, ZNF528, ZNF543, ZNF140, ZNF610, ZNF350, ZNF8, ZNF30, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP82, ZNF224, ZNF33A, ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, CBX1, SCMH1, MPP8, SUMO3, HERC2, BIN1, PCGF2, TOX, FOXA1, FOXA2, IRF2BP1, IRF2BP2, IRF2BPL IRF-2BP1_2 N-terminal domain, HOXA13, HOXB13, HOXC13, HOXA11, HOXC11, HOXC10, HOXA10, HOXB9, HOXA9, ZFP28, ZN334, ZN568, ZN37A, ZN181, ZN510, ZN862, ZN140, ZN208, ZN248, ZN571, ZN699, ZN726, ZIK1, ZNF2, Z705F, ZNF14, ZN471, ZN624, ZNF84, ZNF7, ZN891, ZN337, Z705G, ZN529, ZN729, ZN419, Z705A, ZN302, ZN486, ZN621, ZN688, ZN33A, ZN554, ZN878, ZN772, ZN224, ZN184, ZN544, ZNF57, ZN283, ZN549, ZN211, ZN615, ZN253, ZN226, ZN730, Z585A, ZN732, ZN681, ZN667, ZN649, ZN470, ZN484, ZN431, ZN382, ZN254,ZN124, ZN607, ZN317, ZN620, ZN141, ZN584, ZN540, ZN75D, ZN555, ZN658, ZN684, RBAK, ZN829, ZN582, ZN112, ZN716, HKR1, ZN350, ZN480, ZN416, ZNF92, ZN100, ZN736, ZNF74, ZN443, ZN195, ZN530, ZN782, ZN791, ZN331, Z354C, ZN157, ZN727, ZN550, ZN793, ZN235, ZN724, ZN573, ZN577, ZN789, ZN718, ZN300, ZN383, ZN429, ZN677, ZN850, ZN454, ZN257, ZN264, ZN485, ZN737, ZNF44, ZN596, ZN565, ZN543, ZFP69, SUMO1, ZNF12, ZN169, ZN433, ZN175, ZN347, ZNF25, ZN519, Z585B, ZN517, ZN846, ZN230, ZNF66, ZN713, ZN816, ZN426, ZN674, ZN627, ZNF20, Z587B, ZN316, ZN233, ZN611, ZN556, ZN234, ZN560, ZNF77, ZN682, ZN614, ZN785, ZN445, ZFP30, ZN225, ZN551, ZN610, ZN528, ZN284, ZN418, ZN490, ZN805, Z780B, ZN763, ZN285, ZNF85, ZN223, ZNF90, ZN557, ZN425, ZN229, ZN606, ZN155, ZN222, ZN442, ZNF91, ZN135, ZN778, ZN534, ZN586, ZN567, ZN440, ZN583, ZN441, ZNF43, ZN589, ZN563, ZN561, ZN136, ZN630, ZN527, ZN333, Z324B, ZN786, ZN709, ZN792, ZN599, ZN613, ZF69B, ZN799, ZN569, ZN564, ZN546, ZFP92, ZN723, ZN439, ZFP57, ZNF19, ZN404, ZN274, CBX3, ZN250, ZN570, ZN675, ZN695, ZN548, ZN132, ZN738, ZN420, ZN626, ZN559, ZN460, ZN268, ZN304, ZN605, ZN844, SUMO5, ZN101, ZN783, ZN417, ZN182, ZN823, ZN177,ZN197, ZN717, ZN669, ZN256, ZN251, CBX4, CDY2, CDYL2, ZN562, ZN461, Z324A, ZN766, ID2, ZN214, CBX7, ID1, CREM, SCX, ASCL1, ZN764, SCML2, TWST1, CREB1, TERF1, ID3, CBX8, GSX1, NKX22, ATF1, TWST2, ZNF17, TOX3, TOX4, ZMYM3, I2BP1, RHXF1, SSX2, I2BPL, ZN680, TRI68, HXA13, PHC3, TCF24, HXB13, HEY1, PHC2, ZNF81, FIGLA, SAM11, KMT2B, HEY2, JDP2, HXC13, ASCL4, HHEX, GSX2, ETV7, ASCL3, PHC1, OTP, I2BP2, VGLL2, HXA11, PDLI4, ASCL2, CDX4, ZN860, LMBL4, PDIP3, NKX25, CEBPB, ISL1, CDX2, PROP1, SIN3B, SMBT1, HXC11, HXC10, PRS6A, VSX1, NKX23, MTG16, HMX3, HMX1, KIF22, CSTF2, CEBPE, DLX2, PPARG, PRIC1, UNC4, BARX2, ALX3, TCF15, TERA, VSX2, HXD12, CDX1, TCF23, ALX1, HXA10, RX, CXXC5, SCML1, NFIL3, DLX6, MTG8, CEBPD, SEC13, FIP1, ALX4, LHX3, PRIC2, MAGI3, NELL1, PRRX1, MTG8R, RAX2, DLX3, DLX1, NKX26, NAB1, SAMD7, PITX3, WDR5, MEOX2, NAB2, DHX8, CBX6, EMX2, CPSF6, HXC12, KDM4B, LMBL3, PHX2A, EMX1, NC2B, DLX4, SRY, ZN777, ZN398, GATA3, BSH, SF3B4, TEAD1, TEAD3, RGAP1, PHF1, GATA2, FOXO3, ZN212, IRX4, ZBED6, LHX4, SIN3A, RBBP7, NKX61, R51A1, MB3L1, DLX5, NOTC1, TERF2, ZN282, RGS12, ZN840, SPI2B, PAX7, NKX62, ASXL2, FOXO1, GATA1, ZMYM5, LRP1, MIXL1, SGT1, LMCD1, CEBPA, SOX14, WTIP,PRP19, NKX11, RBBP4, DMRT2, SMCA2, and functionally active fragments thereof.

[0075] In some embodiments, the transcription repressor domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 25-50.

[0076] In some embodiments, one or more of the nucleic acid binding domain, the epigenetic modification domain, and the transcription regulation domain are connected by a linker sequence.

[0077] In some embodiments, the linker sequence comprises at least 16 amino acids.

[0078] In some embodiments, the linker sequence comprises an XTEN linker sequence.

[0079] In some embodiments, the linker sequence comprises a GS linker peptide comprising the sequence: (GS) a (GGS) b (GGGS) c (GGGGS) d wherein G represents a glycine residue (Gly), S represents a serine residue (Ser), and a, b, c, and d represent integers greater than or equal to 0.

[0080] In some embodiments, the linker sequence comprises an amino acid sequence selected from one or more of SEQ ID NOs: 125-132.

[0081] In some embodiments, the fusion comprises more than one effector domain, which is located N-terminal and C-terminal to the nucleic acid binding domain.

[0082] In some embodiments, the more than one effector domain comprises at least one epigenetic modification domain.

[0083] In some embodiments, the more than one effector domain comprises at least one transcription regulation domain.

[0084] In some embodiments, the effector domain C-terminal to the nucleic acid binding domain comprises at least one transcription regulation domain.

[0085] In some embodiments, the effector domain C-terminal to the nucleic acid binding domain comprises at least one transcription repressor domain.

[0086] In some embodiments, the effector domain N-terminal to the nucleic acid binding domain comprises an epigenetic modification domain.

[0087] In some embodiments, the effector domain N-terminal to the nucleic acid binding domain comprises an epigenetic modification domain that provides DNA modification.

[0088] In some embodiments, the fusion has, in order from N- to C-terminus: (1) an epigenetic modification domain that provides histone modification, the nucleic acid binding domain, and an epigenetic modification domain that provides histone modification; (2) a transcription repressor domain, the nucleic acid binding domain, and an epigenetic modification domain that provides histone modification; (3) an epigenetic modification domain that provides DNA modification, the nucleic acid binding domain, and an epigenetic modification domain that provides histone modification; (4) a transcription repressor domain, an epigenetic modification domain that provides DNA modification, the nucleic acid binding domain, and an epigenetic modification domain that provides histone modification; or (5) an epigenetic modification domain that provides DNA modification, a transcription repressor domain, the nucleic acid binding domain, and an epigenetic modification domain that provides histone modification; or (6) an epigenetic modification domain that provides DNA modification, the nucleic acid binding domain, and a transcription repressor domain.

[0089] In some embodiments, the epigenetic modification domain that provides histone modification is selected from the group consisting of: EZH2, HDAC3, HDAC1, EHMT2, PRMT1, PRMT5, SETDB1, hSIRT1, HP1a, LSD1, and functionally active fragments thereof.

[0090] In some embodiments, the epigenetic modification domain that provides DNA modification is selected from the group consisting of: DNMT3A, DNMT3L, a combination of DNMT3A and DNMT3L, or functionally active fragments thereof.

[0091] In some embodiments, the fusion comprises the following domains: DNMT3A-DNMT3L-dCas9-KRAB, DNMT3A-DNMT3L-dCas9-ZIM3, HDAC3-dCas9-EZH2, KRAB-dCas9-EZH2, KRAB-DNMT3A-dCas9-EZH2, KRAB-DNMT3A-dCas9-HDAC3, DNMT3A-DNMT3L-dCas9-(EZH2) n＝1-11DNMT3A-DNMT3L-dCas9-HDAC3, DNMT3A-DNMT3L-dCas9-EHMT2, DNMT3A- DNMT3L-dCas9-HDAC1, DNMT3A-DNMT3L-dCas9-PRMT1, DNMT3A-DNMT3L- dCas9-SETDB1, DNMT3A-DNMT3L-dCas9-hSIRT1, DNMT3A-DNMT3L-dCas9- PRMT5, DNMT3A-DNMT3L-dCas9-HPla, DNMT3A-DNMT3L-dCas9-LSD1, or DNMT3A-DNMT3L-TALE-KRAB; wherein - indicates that the respective domains of the fusion are directly and / or indirectly linked, and the respective domains are in order from N-terminus to C-terminus.

[0092] In some embodiments, the fusion comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1478-1480 and 1482.

[0093] In some embodiments, the fusion further comprises a nuclear localization signal and / or a marker domain.

[0094] In some embodiments, the fusion is capable of providing modification of at least one nucleotide within about 500 bp, about 400 bp, about 300 bp, about 200 bp, about 100 bp, about 50 bp of the transcription start site of the VEGFA gene.

[0095] In some embodiments, the fusion is capable of providing modification of at least one nucleotide within about 500 bp, about 400 bp, about 300 bp, about 200 bp, about 100 bp, about 50 bp of the transcription start site of the VEGFA gene.

[0096] In some embodiments, the fusion is capable of providing modification of at least one nucleotide within about 500 bp, about 400 bp, about 300 bp, about 200 bp, about 100 bp, about 50 bp of the transcription start site of the VEGFA gene.

[0097] In some embodiments, the fusion is capable of providing modification of at least one nucleotide within a region from about 500 bp upstream to about 3500 bp downstream, from about 500 bp upstream to about 3000 bp downstream, from about 500 bp upstream to about 2500 bp downstream, from about 500 bp upstream to about 2000 bp downstream, from about 500 bp upstream to about 1500 bp downstream, from about 500 bp upstream to about 1000 bp downstream, from about 500 bp upstream to about 500 bp downstream of the transcription start site of the VEGFA gene.

[0098] In some embodiments, the fusion is capable of providing modification of at least one nucleotide within a region from about 250 bp upstream to about 250 bp downstream of the transcription start site of the VEGFA gene.

[0099] In another aspect, the present application provides a nucleic acid encoding the complex described herein and / or encoding the fusion described herein.

[0100] In some embodiments, the nucleic acid is a recombinant vector.

[0101] In some embodiments, the recombinant vector further comprises a non-coding region.

[0102] In some embodiments, the non-coding region is selected from the group consisting of an intron, a regulatory element, a promoter, an enhancer, a termination sequence, and a 5' and 3' untranslated region.

[0103] In some embodiments, the nucleic acid comprises a first nucleic acid segment encoding the first fusion, and a second nucleic acid segment encoding the second fusion.

[0104] In some embodiments, the first nucleic acid segment and the second nucleic acid segment are connected by a nucleic acid segment encoding a cleavable peptide.

[0105] In some embodiments, the cleavable peptide is a 2A peptide and / or an IRES.

[0106] In some embodiments, the 2A peptide is selected from the group consisting of P2A, T2A, E2A, and F2A.

[0107] In some embodiments, the nucleic acid comprises a nucleic acid sequence as set forth in any one of SEQ ID NOs: 169-335, 1483-1487.

[0108] In another aspect, the present application provides a delivery vehicle comprising the complex described herein, the fusion described herein, and / or the nucleic acid described herein, and optionally comprising a liposome and / or a lipid nanoparticle.

[0109] In another aspect, the present application provides a composition comprising the complex described herein, the fusion described herein, the nucleic acid described herein, and / or the delivery vehicle described herein.

[0110] In another aspect, the present application provides a cell comprising the complex described herein, the fusion described herein, the nucleic acid described herein, the delivery vehicle described herein, and / or the composition described herein.

[0111] In another aspect, the present application provides a kit comprising the complex described herein, the fusion described herein, the nucleic acid described herein, the delivery vehicle described herein, the composition described herein, and / or the cell described herein.

[0112] In another aspect, the present application provides a method of modulating expression of a VEGFA gene product, the method comprising administering the complex described herein, the fusion described herein, the nucleic acid described herein, the delivery vehicle described herein, the composition described herein, the cell described herein and / or the kit described herein.

[0113] In some embodiments, the method comprises introducing the complex, the fusion, the nucleic acid, the delivery vehicle, the composition, the cell, and / or the kit into a cell containing the VEGFA gene.

[0114] In some embodiments, the method comprises contacting the complex, the fusion, the nucleic acid, the delivery vehicle, and / or the composition with the VEGFA gene and / or a regulatory element of the VEGFA gene.

[0115] In some embodiments, the regulatory element comprises a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a boundary element, and / or a locus control region.

[0116] In another aspect, the present application provides a method of treating or ameliorating a disease or a condition thereof associated with abnormal expression of a VEGFA gene and / or abnormal activity of a VEGFA gene, the method comprising administering to a subject in need thereof an effective amount of the complex described herein, the fusion described herein, the nucleic acid described herein, the delivery vehicle described herein, the composition described herein, the cell described herein and / or the kit described herein.

[0117] In another aspect, the present application provides use of the complex described in the present application, the fusion described in the present application, the nucleic acid described in the present application, the delivery vector described in the present application, the composition described in the present application, the cell described in the present application and / or the kit described in the present application for the preparation of a medicament for treating or alleviating a disease or a condition thereof associated with abnormal expression and / or abnormal activity of VEGFA gene.

[0118] In some embodiments, the disease or the condition thereof associated with abnormal expression and / or abnormal activity of VEGFA gene comprises wet macular degeneration, diabetic retinopathy, age-related macular degeneration, and other corneal or choroidal neovascularization-related eye diseases.

[0119] The complex, fusion and its encoding nucleic acid, vector, composition, cell and the like products provided in the present application have at least one of the following advantages: significant VEGFA gene transcription regulation efficiency (up to ~96%), rich VEGFA gene regulation range, flexible and diverse connection mode between various regulation elements, and significantly improved recruitment effect of the complex peptide based on SunTag recruitment strategy. BRIEF DESCRIPTION OF DRAWINGS

[0120] The specific features of the invention involved in the present application are shown in the appended claims. The features and advantages of the invention involved in the present application can be better understood by referring to the exemplary embodiments and the accompanying drawings described in detail below. A brief description of the drawings is as follows:

[0121] Figure 1 shows the inhibitory effect of the complex described in the present application on the expression of VEGFA gene in mouse liver cell line N2a.

[0122] Figure 2 shows the inhibitory effect of the complex described in the present application on the expression of VEGFA gene in human retinal pigment epithelial cells ARPE19.

[0123] Figure 3 shows the inhibitory effect of the epigenetic editing tool containing different histone modification elements described in the present application on the expression of VEGFA gene in human retinal pigment epithelial cells ARPE19.

[0124] Figure 4 shows the inhibitory effect of different epigenetic editing tools described in the present application on the expression of VEGFA gene in human retinal pigment epithelial cells ARPE19.

[0125] Figure 5 shows the inhibitory effect of different candidate sgRNAs described in the present application on the expression of VEGFA gene in human retinal pigment epithelial cells ARPE19. DETAILED DESCRIPTION

[0126] The following illustrates the embodiments of the present application by specific examples, and other advantages and effects of the present application can be easily understood by those skilled in the art from the disclosure of the present application.

[0127] Definitions of terms

[0128] [Corrected according to Rule 91 on 18.07.2025] In the present application, the term "recruitment" generally refers to the recruitment effect between protein molecules, which specifically refers to the recruitment of other molecules by proteins to perform specific biological functions. This recruitment effect mainly depends on the affinity of intermolecular interaction, and its affinity is generally considered to be related to the spatial structure of the protein molecule, which is more complex. The interaction mechanism may, for example, include but is not limited to hydrogen bonds, ionic interactions, hydrophobic interactions, van der Waals forces, and other non-covalent bond interactions. For example, some proteins can recruit enzymes to catalyze chemical reactions, or recruit other proteins to form complexes. These recruitment effects are crucial for many cellular processes, such as signal transduction, DNA replication, and gene expression.

[0129] In the present application, the term "nucleic acid binding domain" generally refers to a part of a polypeptide or composition that can bind to a specific nucleic acid, which can include a region that contacts nucleic acid, nucleic acid and / or protein matter. Examples of nucleic acid binding domains can include, but are not limited to, helix-turn-helix domains, zinc finger domains, leucine zipper (bZIP) domains, winged helix domains, winged helix-turn-helix domains, helix-loop-helix domains, HMG-box domains, Wor3 domains, immunoglobulin domains, B3 domains, TALE domains, and / or CRISPR / CasX protein domains, etc.

[0130] In this application, the term "DNA-binding domain" generally refers to a folded protein domain containing at least one motif that recognizes double-stranded or single-stranded DNA. For example, the DNA-binding domain may recognize a specific DNA sequence (recognition or regulatory sequence) or have general affinity for DNA. In some cases, other domains of the DNA-binding domain typically regulate the activity of the DNA-binding domain; the DNA-binding function may be structural or include transcriptional regulation, and sometimes these two functions overlap. In some embodiments of the methods and gene expression regulatory molecules provided in this application, the DNA-binding domain may comprise a (DNA) nuclease, such as a nuclease capable of targeting DNA in a sequence-specific manner or capable of being directed or instructed to target DNA in a sequence-specific manner, such as the CRISPR-Cas system, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or a broad range of nucleases. In some embodiments, the DNA-binding domain is a DNA nuclease derived from the CRISPR-Cas system. For example, the CRISPR-Cas-derived DNA nuclease is a Cas protein.

[0131] In this application, the term "TALE DNA-binding domain" or "TALE" refers to a polypeptide containing one or more TALE repeating domains / units. Naturally occurring TALEs, or "wild-type TALEs," are nucleic acid-binding proteins secreted by numerous species of Proteobacteria. TALE polypeptides contain a nucleic acid-binding domain consisting of tandem repeats of highly conserved monomeric polypeptides, said monomeric polypeptides being primarily 33, 34, or 35 amino acids in length and differing primarily from each other at amino acid positions 12 and 13. In a preferred embodiment, the nucleic acid is DNA. As used herein, a polypeptide monomer of TALE is used to refer to a highly conserved repeating polypeptide sequence within the TALE nucleic acid-binding domain, and the term "repeated variable diresidue" or "RVD" is used to refer to a highly variable amino acid at positions 12 and 13 of the polypeptide monomer. A general representation of a TALE monomer contained within a DNA-binding domain is X. 1-11 -(X 12 X 13 )-X 14-33或34或35 The subscript indicates the position of the amino acid, and X represents any amino acid. 12 X 13 Indicating RVD. In some TALE polypeptide monomers, the variable amino acid at position 13 is missing or absent, and in such monomers, RVD consists of a single amino acid. In such cases, RVD can alternatively be represented as X*, where X represents X. 12 And (*) indicates X 13 No. The DNA-binding domain contains several repeats of the TALE monomer, and this can be represented as (X 1-11- (X 12 X 13 - (X 14-33或34或35 ) z wherein in preferred embodiments, z is at least 5-40. In further preferred embodiments, z is at least 10-26.

[0132] TALE monomers have nucleotide binding affinities that are determined by the type of amino acid within their RVD. For example, polypeptide monomers with RVDs of NI preferentially bind to adenine (A), polypeptide monomers with RVDs of NG preferentially bind to thymine (T), polypeptide monomers with RVDs of HD preferentially bind to cytosine (C), and monomers with RVDs of NN preferentially bind to both adenine (A) and guanine (G). In additional embodiments, monomers with RVDs of IG preferentially bind to T. Thus, the number and order of polypeptide monomer repeats in the nucleic acid binding domain of a TALE determines its nucleic acid target specificity. In further embodiments of the application, monomers with RVDs of NS recognize all four base pairs and can bind to A, T, G, or C. The structure and function of TALEs are further described, for example, in Moscou et al., Science 326: 1501 (2009); Boch et al., Science 326: 1509-1512 (2009); and Zhang et al., Nature Biotechnology 29: 149-153 (2011), each of which is incorporated by reference in its entirety. The repeat domains of TALEs are involved in the binding of TALEs to their cognate target DNA sequences. These repeat units (or "repeat sequences") exhibit at least some sequence homology to other TALE repeat sequences within naturally occurring TALE proteins. See, e.g., U.S. Patent Publication No. 20110301073. The TALE binding domains involved in the present application can be "engineered" to bind to a predetermined nucleotide sequence, for example, via engineering (altering one or more amino acids) of the recognition helix region of naturally occurring TALE proteins. Thus, engineered DNA binding proteins (TALEs) are non-naturally occurring proteins. Non-limiting examples of methods for engineering DNA binding proteins are design and selection. Designed DNA binding proteins are non-naturally occurring proteins whose design and / or composition is derived primarily from rational criteria. Rational design criteria include the application of substitution rules and computational algorithms for processing information in information databases that store existing TALE designs and binding data. See, e.g., U.S. Patents 6,140,081; 6,453,242; and 6,534,261; see also WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536 and WO 03 / 016496 and U.S. Publication No. 20110301073.

[0133] In the present application, "Cas enzyme" is used interchangeably with "Cas protein", "CRISPR protein", "CRISPR enzyme", "CRISPR-Cas protein", "CRISPR-Cas enzyme", "Cas", "CRISPR effector", or "Cas effector protein", which generally refers to a class of enzymes that are complementary to CRISPR sequences, capable of using CRISPR sequences as guides to recognize and cut specific DNA strands. Non-limiting examples of Cas proteins include: Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csf1, Csf2, Csf3, Csf4, and / or their homologues, or modified versions thereof. These proteins are known, for example, the amino acid sequence of the S. pyogenes Cas9 protein can be found under SwissProt database accession number Q99ZW2.

[0134] In the present application, the term "class II Cas nuclease" generally refers to a class of Cas proteins that function in a single protein to recognize and / or cleave according to the updated classification scheme of CRISPR / Cas loci (Makarova et al. (2015) Nat Rev Microbiol 13(11):722-36; Shmakov et al. (2015) Mol Cell 60:385-397).

[0135] In the present application, the term "class II type II Cas nuclease and class II type V Cas nuclease" generally refers to a single-protein, RNA-guided endonuclease in class II Cas nucleases. Among them, the Cas nucleases in type II and type V-B type Cas nucleases require the cooperation of tracrRNA (trans-activating CRISPR RNA) and crRNA (CRISPR RNA) to function normally, and the crRNA and tracrRNA can be artificially combined into a single guide RNA (sgRNA); the Cas nucleases in type V-A type Cas nucleases require crRNA alone to function as a guide. Non-limiting examples of class II type II Cas nucleases include Cas9 and its family-related nucleases, and non-limiting examples of class II type V Cas nucleases include Cas12a (also known as Cpf1), Cas12b (also known as C2c1), Cas12c (also known as C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c1, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b, Cas14c nucleases and / or TnpB.

[0136] In the present application, the term "dCas" can refer to a dCas protein or a fragment thereof. For example, as used herein, "dCas9" can refer to a dCas9 protein or a fragment thereof. As used herein, the terms "iCas" and "dCas" are used interchangeably to refer to a CRISPR-associated protein without catalytic activity. In one embodiment, the dCas protein comprises one or more mutations in the DNA cleavage domain. In one embodiment, the dCas protein comprises one or more mutations in the RuvC or domain. In one embodiment, the dCas molecule comprises one or more mutations in both the RuvC and HNH domains. In one embodiment, the dCas protein is a fragment of a wild-type Cas protein. In one embodiment, the dCas protein comprises a functional domain from a wild-type Cas protein, wherein the functional domain is selected from a Reel domain, a bridge helix domain, or a PAM interaction domain. In one embodiment, the dCas has at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% less nuclease activity compared to the nuclease activity of the corresponding wild-type Cas protein.

[0137] In the present application, the term "capable of binding" can be used interchangeably with "binds to", "specifically recognizes", "targets", and the like, and generally refers to the ability of a binding molecule (e.g., a gene expression modulating molecule of the present application) to interact with a nucleotide on a target gene or target site, or the sufficient affinity of the binding molecule (e.g., a gene expression modulating molecule of the present application) to a target gene or target site, such interaction can be through conjugation, coupling, attachment, providing complementarity, providing covalent forces or providing non-covalent forces, increasing binding stability, and the like.

[0138] In the present application, the terms "guide RNA", "guide DNA", and "gRNA" are used interchangeably, and generally refer to a DNA molecule capable of directing a nuclease (e.g., Argonaute, or Ago) to bind and / or cleave a target gene. In some preferred embodiments, a guide DNA can comprise: a single-stranded DNA molecule (ssDNA), a 5' end phosphorylated single-stranded DNA molecule, a 5' end hydroxylated single-stranded DNA molecule, a base fragment capable of complementarity to a target gene, and / or a length of 8-35 nt. In some embodiments of the present application, the term "guide RNA" refers to an RNA comprising: (1) an "activating" nucleotide sequence that binds to and activates an RNA-guided endonuclease (e.g., a Class II Cas nuclease, such as a Type II, Type V, or Type VI Cas endonuclease); and (2) a "target" nucleotide sequence comprising a nucleotide sequence that hybridizes to a target nucleic acid. The "activating" nucleotide sequence and the "target" nucleotide sequence can be on separate RNA molecules (e.g., "dual guide RNA"); or can be on the same RNA molecule ("single guide RNA", also referred to as sgRNA).

[0139] In the present application, the term "DNA methyltransferase" generally refers to an enzyme that catalyzes the transfer of a methyl group to DNA. Non-limiting examples of DNA methyltransferases include DNMT1, DNMT 3A, DNMT 3B, and DNMT 3L. For example, by DNA methylation, DNA methyltransferases can modify the activity (e.g., regulate gene expression) of a DNA fragment without altering the DNA sequence. As described herein, a gene expression modulating molecule can include one or more (e.g., two) DNA methyltransferases. When a DNA methyltransferase is included as part of a gene expression modulating molecule, the DNA methyltransferase can be referred to as a "DNA methyltransferase domain." In aspects, a DNA methyltransferase domain comprises a variant or homolog of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to DNMT 3A. In aspects, a DNA methyltransferase domain comprises a variant or homolog of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to DNMT 3L.

[0140] In the present application, the term "functionally active fragment" generally refers to a fragment that has a partial region of a full-length protein or nucleic acid, but retains or partially retains the biological activity or function of the full-length protein or nucleic acid. For example, a functionally active fragment can retain or partially retain the ability of a full-length protein to bind to another molecule. For example, a functionally active fragment of a DNA methyltransferase can retain or partially retain the biological active function of a full-length DNA methyltransferase to catalyze the transfer of a methyl group to DNA.

[0141] In the present application, the term "transcriptional repressor" generally refers to a substance and / or agent, such as a protein (e.g., a transcription factor or fragment thereof), that binds to a target nucleic acid sequence and results in a decrease in the expression level of a gene product associated with the target nucleic acid sequence. For example, the gene product can be an RNA (e.g., mRNA) transcribed from a gene or a polypeptide translated from an mRNA transcribed from a gene. Generally, an increase or decrease in mRNA level results in an increase or decrease in the level of a polypeptide translated therefrom. Expression level can be determined using standard techniques for measuring mRNA or protein. Non-limiting examples of transcriptional repressors include: mSin3 interaction domain (SID) protein, methyl-CpG-binding domain 2 (MBD2), MBD3, DNA methyltransferase (DNMT) 1 (DNMT1), DNMT2A, DNMT3A, DNMT3B, DNMT3L, retinoblastoma protein (Rb), methyl CpG binding protein 2 (Mecp2), GATA-1 and its cofactor Fog1, MAT2 regulator (ROM2), Arabidopsis thaliana HD2A protein (AtHD2A), lysine-specific demethylase 1 (LSD1), and / or Krüppel-associated box (KRAB).

[0142] In the present application, the term "KRAB" also referred to as "Krüppel-associated box domain" or "Krüppel-associated box domain" generally refers to a transcriptional repression domain of about 45 to about 75 amino acid residues present in the transcription factor of human zinc finger protein. In aspects, the KRAB domain can include a variant or homolog of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a ZIM3 KRAB domain or a KOX1 KRAB domain.

[0143] In the present application, the term "split green fluorescent protein" generally refers to a polypeptide that is capable of splitting and upon recombination immediately forms an active green fluorescent protein.

[0144] In the present application, the term "GCN4" is a transcription factor in S. cerevisiae, a "master regulator" in the yeast genome, regulating nearly one-tenth of the yeast genome, is a highly conserved protein, whose homolog in mammals is Activating Transcription factor-4 (ATF4).

[0145] In the present application, the term "PDZ protein" generally refers to a naturally occurring protein containing a PDZ domain. Exemplary PDZ proteins include CASK, MPP1, DLG1, DLG2, PSD95, NeDLG, TIP-33, SYNla, TIP-43, LDP, LIM, LIMK1, LIMK2, MPP2, NOS 1, AF6, PTN_4, prIL16, 41.8kD, KIAA0559, RGS12, KIAA0316, DVL1, TIP-40, TIAMl, MINTl, MAGI-I, MAGI-2, MAGI-3, KIAA0303, CBP, MINT3, TIP-2, KIAA0561, and / or TIP-I.

[0146] In the present application, the term "single chain antibody" or "scFv (Single Chain Antibody)" generally refers to a single chain polypeptide containing one or more antigen binding sites. In addition, although the H and L chains of Fv fragments are encoded by different genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain (known as single chain antibody, sAb; Bird et al. 1988 Science 242:423-426; and Huston et al. 1988 PNAS 85:5879-5883). Such single chain antibodies are also encompassed within the term "antibody", can be used as binding determinants in the design and manufacture of multispecific binding molecules, and can be prepared by recombinant techniques or enzymatic or chemical cleavage of intact antibodies.

[0147] In the present application, the term "directly or indirectly bound" generally refers to either "directly bound" or "indirectly bound". "Directly bound" generally refers to a direct linkage. For example, the directly bound can be a case where there is no spacer component (e.g., an amino acid residue or a derivative thereof) between the linked substances (e.g., an amino acid sequence segment) and they are directly linked; for example, an amino acid sequence segment X is directly linked to another amino acid sequence segment Y through an amide bond formed between the C-terminal amino acid of the amino acid sequence segment X and the N-terminal amino acid of the amino acid sequence segment Y. "Indirectly bound" generally refers to a case where there is a spacer component (e.g., an amino acid residue or a derivative thereof) between the linked substances (e.g., an amino acid sequence segment) and they are indirectly linked. For example, the spacer component used in the present application can be a stretch of amino acid residues, the sequence of which is selected from the amino acid sequence shown in any one of SEQ ID NOs: 125-132 (SEQ ID NO: 126 is GSG).

[0148] In the present application, "nuclear localization sequence" or "NLS" generally refers to a peptide that directs a protein to the nucleus. In certain embodiments, the NLS comprises five basic, positively charged amino acids. The NLS can be located anywhere on the peptide chain. In certain embodiments, the NLS is an NLS derived from SV40. In certain embodiments, the NLS comprises a sequence set forth in any one of SEQ ID NOs: 379-381. In some embodiments, the NLS has an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identical to any one of SEQ ID NOs: 379-381.

[0149] In the present application, the term "marker" refers to a peptide that can be introduced into an expression vector that can be used to allow for the deletion and / or purification of the expression product of one or more vector inserts. Such markers are well known in the art and include radiolabeled amino acids or polypeptides to which biotin moieties can be attached that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent label or enzymatic activity that can be detected by optical or colorimetric methods). Affinity markers such as FLAG, glutathione-S-transferase, maltose binding protein, cellulose binding domain, thioredoxin, NusA, mistin, chitin binding domain, cutinase, AGT, GFP and other markers in widespread use such as those used in the ProBond® protein expression and purification system. Further non-limiting examples for polypeptides include, but are not limited to, the following: histidine tag, a radioisotope or radionuclide (e.g.,3H,14C,35S,90Y,99Tc,111In,125I,177Lu,166Ho or153Sm); a fluorescent marker (e.g., FITC, rhodamine, lanthanide phosphors), an enzymatic marker (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); a chemiluminescent marker; a biotin moiety; an overhanging polypeptide epitope recognized by a second reporter (e.g., leucine zipper pair sequence, binding site for a secondary antibody, metal binding domain, epitope tag); and a magnetic reagent such as a gadolinium chelate.

[0150] In the present application, the terms "nucleic acid" and "polynucleotide," "nucleotide," "nucleotide sequence," and "oligonucleotide" are used interchangeably and generally refer to a polymer of nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and its complements in single-, double-, or multi-stranded form. For example, a nucleotide can be a ribonucleotide, a deoxyribonucleotide, or a modified version thereof. For example, a nucleotide can be single- and double-stranded DNA, single- and double-stranded RNA, and hybrid molecules having a mixture of single- and double-stranded DNA and RNA. For example, a nucleotide can include, but is not limited to, any type of RNA, such as mRNA, siRNA, miRNA, sgRNA, and guide RNA, and any type of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragment thereof. The terms also encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring.

[0151] In the present application, the term "a sequence encoding for" or "a nucleic acid encoding for" generally refers to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence that encodes for a protein. The coding sequence can also include initiation and termination signals operably linked to regulatory elements comprising a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence can be codon optimized. In the present application, the term "intron" generally refers to a segment of DNA that is transcribed but removed from the RNA transcript by splicing together any of the ends of the sequences (exons). Introns are considered interfering sequences within the protein coding region of a gene and generally do not contain information represented by the protein produced by the gene.

[0152] In the present application, the term "recombinant vector" generally refers to a nucleic acid molecule that is capable of transporting another nucleic acid with it. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Alternatively, the vector can be linear. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell and thereby are replicated along with the host genome.

[0153] In the present application, the term "transcription start site" refers to the first base transcribed from the 5' end of a gene, which is the base on the DNA strand corresponding to the first nucleotide of the mRNA strand when transcribed, and can typically be a purine (e.g., A or G). The sequence before the transcription start site (i.e., 5' end) can be referred to as upstream, and the sequence after (i.e., 3' end) can be referred to as downstream. In the present application, the transcription start site is denoted by "0". In the present application, the upstream of the transcription start site is denoted by "-". For example, 250 bp upstream of the transcription start site is denoted by "-250 bp". In the present application, the downstream of the transcription start site is denoted by "+". For example, 250 bp downstream of the transcription start site can be denoted by "+250 bp" or simply "250 bp".

[0154] In the present application, the term "regulatory element" refers to a genetic element that is capable of controlling the expression of a nucleic acid sequence. For example, a splice signal, a promoter sequence, a polyadenylation signal, a transcription termination sequence, an upstream regulatory domain, an origin of replication, an internal ribosome entry site ("IRES"), an enhancer, and the like, which collectively provide for replication, transcription, and translation of a coding sequence in a recipient cell. Not all of these control sequences need to be present.

[0155] In the present application, the term "promoter" generally refers to a nucleotide sequence that controls or regulates the transcription of a nucleotide sequence (e.g., a coding sequence) operably associated with the promoter. The coding sequence controlled or regulated by the promoter can encode a polypeptide and / or a functional RNA. Typically, a "promoter" refers to a nucleotide sequence that includes a binding site for RNA polymerase II and directs the initiation of transcription. Typically, the promoter is located 5' or upstream relative to the start of the coding region of the corresponding coding sequence. The promoter can include other elements that act as regulators of gene expression; for example, a promoter region. In some embodiments, the promoter region can include at least one intron. The promoter can include, for example, constitutive, inducible, time-regulated, developmentally-regulated, chemically-regulated, tissue-preferred, and / or tissue-specific promoters, for making a recombinant nucleic acid molecule, e.g., a "synthetic nucleic acid construct" or a "protein-RNA complex". These different types of promoters are known in the art.

[0156] In the present application, the term "enhancer" generally refers to a regulatory DNA sequence, e.g., 50-1500 bp, that can be bound by proteins (activators) to stimulate or enhance transcription of a gene or several genes. These activators (also known as transcription factors) interact with mediator complexes and recruit polymerase II and general transcription factors, which then initiate transcription of the gene. Enhancers are generally cis-acting, but can be located upstream or downstream of the start site of the gene or genes they regulate. In addition, enhancers can be forward or backward oriented and do not need to be located near the transcription start site to affect transcription, as some enhancers have been found to be located hundreds of thousands of base pairs upstream or downstream of the start site. Enhancers can also be found in introns.

[0157] In the present application, the term "cleavage peptide" refers to a class of polypeptides that are capable of effecting cleavage of a protein. For example, the cleavage peptide can effect cleavage of a protein via ribosomal skipping rather than protease hydrolysis. For example, the cleavage peptide can be a cleavage 2A peptide, which can include T2A, F2A, P2A, and / or E2A.

[0158] In the present application, the term "delivery vehicle" generally refers to a transfer vehicle that is capable of delivering an agent (e.g., a nucleic acid molecule) to a target cell. The delivery vehicle can deliver the agent to a specific subset of cells. For example, the delivery vehicle can be targeted to certain types of cells by virtue of the inherent characteristics of the delivery vehicle or by virtue of moieties coupled to the vehicle, contained within (or bound to the vehicle such that the moiety and the delivery vehicle remain together, and such that the moiety is sufficient to target the delivery vehicle). The delivery vehicle can also increase the in vivo half-life of the agent to be delivered and / or the bioavailability of the agent to be delivered. The delivery vehicle can include a viral vector, a virus-like particle, a polycationic vector, a peptide vector, a liposome, and / or a hybrid vector. For example, if the target cell is a hepatocyte, the properties of the delivery vehicle (e.g., size, charge, and / or pH) can be effective to deliver the delivery vehicle and / or the molecule encapsulated therein to the target cell, reduce immune clearance, and / or promote residence in the target cell.

[0159] In the present application, the term "liposome" generally refers to a vesicle that has an internal space isolated from the external medium by one or more bilayered membranes. In some embodiments, the bilayered membrane can be formed by amphiphilic molecules, such as synthetic or naturally-occurring lipids comprising a spatially segregated hydrophilic and hydrophobic domain; in other embodiments, the bilayered membrane can be formed by amphiphilic polymers and surfactants. In some embodiments, the liposome is a spherical vesicular structure consisting of a single or multiple lipid bilayers surrounding an internal aqueous compartment, and an outer, relatively impermeable, lipophilic phospholipid bilayer. In some embodiments, liposomes are biocompatible, non-toxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood-brain barrier (BBB). Liposomes can be made from several different types of lipids, e.g., phospholipids. Liposomes can comprise natural phospholipids and lipids such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), sphingomyelin, egg phosphatidylcholine, monosialic ganglioside, or any combination thereof. To alter the structure and properties of the liposome, several other additives can be added to the liposome. For example, the liposome can also comprise cholesterol, sphingomyelin, and / or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), e.g., to increase stability and / or prevent leakage of the internal cargo of the liposome.

[0160] The term "lipid nanoparticle (LNP)" generally refers to a particle comprising a plurality (i.e., more than one) of lipid molecules physically associated (e.g., covalently or non-covalently) with one another by intermolecular forces. The LNP can be, for example, a microsphere (including unilamellar and multilamellar vesicles, e.g., liposomes), a dispersed phase in an emulsion, a micelle, or an internal phase in a suspension. The LNP can encapsulate a nucleic acid within a cationic lipid particle (e.g., a liposome) and can be relatively easily delivered to cells. In some examples, the lipid nanoparticle does not contain any viral components, which helps to minimize safety and immunogenicity issues. The lipid particles can be used for delivery in vitro, ex vivo, and in vivo. The lipid particles can also be used for cell populations of various scales. The LNP of the present application can be readily prepared by various methods known in the art, for example, by mixing an organic phase with an aqueous phase. The mixing of the two phases can be achieved by microfluidic devices and impinging stream reactors. The more thoroughly the organic and aqueous phases are mixed, the better the entrapment efficiency and particle size distribution of the LNP obtained. Preferably, the particle size of the LNP can be adjusted by varying the speed of mixing of the organic and aqueous phases. The faster the speed of mixing, the smaller the particle size of the LNP prepared. The entrapment efficiency can be optimized by adjusting the N / P (ionizable lipid / nucleic acid) ratio of the LNP system. In some examples, the LNP can be used to deliver DNA molecules and / or RNA molecules (e.g., mRNA of Cas, sgRNA). In certain instances, the LNP can be used to deliver RNP complexes of Cas / gRNA. In some embodiments, the LNP is used to deliver mRNA and gRNA.

[0161] In the present application, the term "subject" generally refers to an animal, typically a mammal, such as a human, a non-human primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), and a laboratory animal (mice, rats, rabbits, guinea pigs). Human subjects include fetal, neonatal, infant, juvenile, and adult subjects. Subjects include animal disease models, for example, mice and other animal models of blood coagulation diseases (such as HemA), and other animal models known to those skilled in the art.

[0162] In the present application, the term "comprising" generally means including the recited elements but not excluding other elements.

[0163] In the present application, the term "consisting of generally means including only the recited elements and no other elements.

[0164] In the present application, the term "about" generally refers to a variation within a range of 0.5-10% above or below the specified numerical value, such as within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified numerical value.

[0165] DETAILED DESCRIPTION

[0166] In one aspect, the present application provides a complex comprising a first fusion and a second fusion, wherein: 1) one of the first fusion and the second fusion comprises a DNA methylation domain and at least one recruiting domain A, and wherein the other fusion comprises a transcriptional repressor domain and at least one recruiting domain A'; and 2) the first fusion or the second fusion comprises a nucleic acid binding domain; and the recruiting domain A and the recruiting domain A' are capable of interacting to enable the one of the first fusion and the second fusion or a portion thereof to be recruited in proximity to the other fusion; the nucleic acid binding domain is capable of specifically binding to a target nucleotide sequence on the VEGFA gene and / or a regulatory element of the VEGFA gene. For example, the nucleic acid binding domain is capable of specifically binding to a target nucleotide sequence within a region from 500 bp upstream to 4000 bp downstream of the transcription start site of the VEGFA gene. For example, the target nucleotide sequence is located within a region from the transcription start site of the VEGFA gene to about 500 bp, about 450 bp, about 400 bp, about 350 bp, about 300 bp, about 250 bp, about 200 bp, about 150 bp, about 100 bp, about 50 bp upstream thereof. For example, the target nucleotide sequence is located within a region from the transcription start site of the VEGFA gene to about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1500 bp, about 2000 bp, about 2500 bp, about 3000 bp, about 3500 bp, about 4000 bp downstream thereof.

[0167] For example, the target nucleotide sequence is located within a region from about 500 bp upstream to about 3500 bp downstream, from about 500 bp upstream to about 3000 bp downstream, from about 500 bp upstream to about 2500 bp downstream, from about 500 bp upstream to about 2000 bp downstream, from about 500 bp upstream to about 1500 bp downstream, from about 500 bp upstream to about 1000 bp downstream, from about 500 bp upstream to about 500 bp downstream, from about 500 bp upstream to about 250 bp downstream, from about 500 bp upstream to about 200 bp downstream, from about 500 bp upstream to about 100 bp downstream, from about 400 bp upstream to about 3500 bp downstream, from about 400 bp upstream to about 3000 bp downstream, from about 400 bp upstream to about 2500 bp downstream, from about 400 bp upstream to about 2000 bp downstream, from about 400 bp upstream to about 1500 bp downstream, from about 400 bp upstream to about 1000 bp downstream, from about 400 bp upstream to about 500 bp downstream, from about 400 bp upstream to about 250 bp downstream, from about 400 bp upstream to about 200 bp downstream, from about 400 bp upstream to about 100 bp downstream, from about 300 bp upstream to about 3500 bp downstream, from about 300 bp upstream to about 3000 bp downstream, from about 300 bp upstream to about 2500 bp downstream, from about 300 bp upstream to about 2000 bp downstream, from about 300 bp upstream to about 1500 bp downstream, from about 300 bp upstream to about 1000 bp downstream, from about 300 bp upstream to about 500 bp downstream, from about 300 bp upstream to about 250 bp downstream, from about 300 bp upstream to about 200 bp downstream, from about 300 bp upstream to about 100 bp downstream, from about 250 bp upstream to about 3500 bp downstream, from about 250 bp upstream to about 3000 bp downstream, from about 250 bp upstream to about 2500 bp downstream, from about 250 bp upstream to about 2000 bp downstream, from about 250 bp upstream to about 1500 bp downstream, from about 250 bp upstream to about 1000 bp downstream, from about 250 bp upstream to about 500 bp downstream, from about 250 bp upstream to about 250 bp downstream, from about 250 bp upstream to about 200 bp downstream, from about 250 bp upstream to about 100 bp downstream, from about 200 bp upstream to about 3500 bp downstream, from about 200 bp upstream to about 3000 bp downstream, from about 200 bp upstream to about 2500 bp downstream, from about 200 bp upstream to about 2000 bp downstream, from about 200 bp upstream to about 1500 bp downstream, from about 200 bp upstream to about 1000 bp downstream, from about 200 bp upstream to about 500 bp downstream, from about 200 bp upstream to about 250 bp downstream, from about 200 bp upstream to about 200 bp downstream, from about 200 bp upstream to about 100 bp downstream, from about 100 bp upstream to about 3500 bp downstream, from about 100 bp upstream to about 3000 bp downstream, from about 100 bp upstream to about 2500 bp downstream, from about 100 bp upstream to about 2000 bp downstream, from about 100 bp upstream to about 1500 bp downstream, from about 100 bp upstream to about 1000 bp downstream, from about 100 bp upstream to about 500 bp downstream, from about 100 bp upstream to about 250 bp downstream, from about 100 bp upstream to about 200 bp downstream, or from about 100 bp upstream to about 100 bp downstream of the VEGFA transcription start site.about 1000 bp downstream, about 50 bp upstream to about 500 bp downstream, about 50 bp upstream to about 250 bp downstream, about 50 bp upstream to about 200 bp downstream, about 50 bp upstream to about 100 bp downstream. In another aspect, the present application provides a nucleic acid encoding the complex described herein. For example, the nucleic acid comprises DNA and / or mRNA. For example, the nucleic acid can be used to treat or alleviate a disease or a condition thereof associated with abnormal expression of a target gene and / or abnormal activity of a target gene. In some embodiments, the nucleic acid is mRNA; one or more modification techniques can be used to generate more stable mRNA. Known mRNA modification techniques can be broadly divided into three categories: synthesizing mRNA with artificially synthesized non-natural ribonucleic acid instead of natural ribonucleic acid; adding 5' caps, 3' poly(A) "tails" and UTR (untranslated region) sequences; and using special new formulation technology to effectively protect mRNA. Among them, the preferred mRNA modification technique can be to synthesize mRNA with artificially synthesized non-natural ribonucleic acid instead of natural ribonucleic acid. Chemical modifications on eukaryotic mRNA can be broadly divided into three categories: methylation, pseudouridine (Ψ) and hypoxanthine. For example, the chemical modification can be selected from the group consisting of: pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine and 2'-O-methyluridine. For example, the nucleic acid is a recombinant vector comprising a nucleic acid encoding the complex described herein. For example, the recombinant vector can refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. The recombinant vector can include single-stranded, double-stranded,double-stranded or partially double-stranded nucleic acid molecules; nucleic acid molecules comprising one or more free ends, without free ends (e.g., circular); nucleic acid molecules comprising DNA, RNA, or both; and other classes of polynucleotides known in the art. For example, viral vectors can be used. Viral vectors can comprise viral-derived DNA or RNA sequences for packaging into a virus (e.g., retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus AAV). Viruses and viral vectors can be used for delivery in vitro, ex vivo, and / or in vivo.

[0168] In another aspect, the present application provides a delivery vehicle comprising the complex described herein and / or the nucleic acid described herein, and optionally comprising a liposome and / or a lipid nanoparticle. For example, the delivery vehicle can be introduced into a cell by a physical delivery method. Examples of physical methods include microinjection, electroporation, and hydrodynamic delivery. For example, LNPs can encapsulate nucleic acids in cationic lipid particles (e.g., liposomes), and can be relatively easy to deliver to cells. In some examples, the lipid nanoparticles are free of any viral components, which helps to minimize safety and immunogenicity concerns. Lipid particles can be used for delivery in vitro, ex vivo, and in vivo. The components of the LNP can include a cationic lipid, an ionizable lipid, a PEGylated lipid, and / or a supporting lipid, and optionally a cholesterol component.

[0169] In another aspect, the present application provides a composition comprising the complex described herein, the nucleic acid described herein, and / or the delivery vehicle described herein. For example, the complex, the nucleic acid (or recombinant vector) encoding the complex, and the delivery vehicle in the composition can be included in one composition, or separately included in different compositions. For example, the complex, the nucleic acid (or recombinant vector) encoding the complex, and / or the delivery vehicle in the composition can be used simultaneously, or separately.

[0170] In another aspect, the present application provides a cell comprising the complex described herein, the nucleic acid described herein, the delivery vehicle described herein, and / or the composition described herein.

[0171] In another aspect, the present application provides a kit comprising a complex described herein, a nucleic acid described herein, a delivery vehicle described herein, a composition described herein, and / or a cell described herein. For example, the kit further comprises at least one container holding the components described above. For example, the kit comprises more than one of the components described above, which further comprises a second, third, and / or other container(s) in addition to the container(s), which can hold the more than one of the components described above separately. For example, the kit can hold various combinations of the components described above in the container(s). For example, the kit further comprises a buffer reagent, a device for mixing, a device for measuring, a device for sorting, and / or a device for labeling. For example, the kit further comprises a package for holding the various containers. For example, the kit further comprises instructions for using the components of the kit. For example, the instructions comprise a paper physical form and / or a machine-readable electronic form.

[0172] In another aspect, the present application provides a method of modulating expression of a VEGFA gene product, the method comprising administering the complex described herein, the nucleic acid described herein, the delivery vehicle described herein, the composition described herein, the cell described herein, and / or the kit described herein. For example, the method of inhibiting expression of a target gene is introducing the complex, the nucleic acid, the delivery vehicle, the composition, the cell, and / or the kit into a cell containing a VEGFA gene. For example, the introducing into a cell can be introducing into a cell using a non-viral or viral-based transfection method. For example, the non-viral transfection method includes any suitable method of introducing into a cell without using viral DNA or viral particles as a delivery system, non-limiting examples of non-viral transfection methods include nanoparticle encapsulation of nucleic acid encoding the complex (e.g., lipid nanoparticle, gold nanoparticle, etc.), calcium phosphate transfection, liposome transfection, nucleofection, sonoporation, transfection by heat shock, magnetic transfection, and electroporation. For example, the viral-based transfection method includes any viral vector suitable for use in the method described herein, non-limiting examples of which include, but are not limited to, retroviral, adenoviral, lentiviral, and / or adeno-associated viral vectors. For example, the method of inhibiting expression of a VEGFA gene further comprises introducing the complex, the nucleic acid, the delivery vehicle, the composition, the cell, and / or the kit into a cell from an external environment. For another example, the method of inhibiting expression of a VEGFA gene comprises contacting the complex, the nucleic acid, the delivery vehicle, and / or the composition with a VEGFA gene and / or a transcriptional regulatory element of the VEGFA gene. For example, the contacting refers to contacting the first fusion, the second fusion, and the guide RNA described herein with a VEGFA gene and / or a transcriptional regulatory element of the VEGFA gene, and the guide RNA forms a complex with the fusion comprising a DNA-binding domain, the complex specifically recognizes and hybridizes to a specific region in the VEGFA gene, while the first fusion and the second fusion are recruited to the vicinity of the DNA-binding domain through direct or indirect interaction of their recruitment domain A and recruitment domain A’, thereby modulating expression of the target nucleic acid. For example, the method comprises bringing the first fusion, the second fusion, and the guide RNA described herein to exist in the form of a complex (e.g., an assembled ribonucleoprotein complex), and bringing the complex into contact with a VEGFA gene and / or a transcriptional regulatory element of the VEGFA gene.

[0173] In another aspect, the present application provides a method of treating or ameliorating a disease or a condition associated with abnormal VEGFA gene expression and / or abnormal VEGFA gene activity, comprising administering to a subject in need thereof an effective amount of the complex described herein, the nucleic acid described herein, the delivery vehicle described herein, the composition described herein, the cell described herein, and / or the kit described herein. For example, the method of treatment comprises mixing the complex, the nucleic acid, the delivery vehicle, the composition, the cell, and / or the kit with a therapeutic agent and performing systemic delivery to a subject in need thereof such that it is widely exposed to a substantial portion of the body, which can be performed by any means known in the art, including but not limited to intravenous, intra-arterial, subcutaneous, intracavitary, and intraperitoneal delivery. For example, the method of treatment comprises mixing the complex, the nucleic acid, the delivery vehicle, the composition, the cell, and / or the kit with a therapeutic agent and performing local delivery to a subject in need thereof such that it is directed to a target site within the organism, which can be performed by, for example, direct injection into a disease site (e.g., a tumor or an inflammation site) or a target organ (e.g., liver, heart, pancreas, kidney, etc.). For example, the local delivery includes local administration or local injection techniques, including but not limited to intramuscular, subcutaneous, or intradermal injection. For example, the local delivery does not exclude systemic pharmacological effects.

[0174] In another aspect, the present application provides use of the complex described herein, the nucleic acid described herein, the delivery vehicle described herein, the composition described herein, the cell described herein, and / or the kit described herein for the manufacture of a medicament for treating or ameliorating a disease or a condition associated with abnormal VEGFA gene expression and / or abnormal VEGFA gene activity.

[0175] In another aspect, the present application provides the complex described herein, the nucleic acid described herein, the delivery vehicle described herein, the composition described herein, the cell described herein, or the kit described herein for use in treating or ameliorating a disease or a condition associated with abnormal VEGFA gene expression and / or abnormal VEGFA gene activity.

[0176] the first fusion or the second fusion

[0177] In some embodiments, the first fusion and the second fusion of the complex of the present application can be generally classified into two scenarios: (1) one of the two fusions comprises a nucleic acid binding domain, a DNA methylation domain, and a recruitment domain A, and the other fusion comprises a transcriptional repressor domain and a recruitment domain A’, or (2) one of the two fusions comprises a nucleic acid binding domain, a transcriptional repressor, and a recruitment domain A, and the other fusion comprises a DNA methylation domain and a recruitment domain A’.

[0178] In particular, in some embodiments of the above (1), one of the two fusions can comprise, in order from N- to C-terminus, a DNA methylation domain, a nucleic acid binding domain, and a recruitment domain A. For example, in some embodiments of the above (2), one of the two fusions can comprise, in order from N- to C-terminus, a recruitment domain A, a nucleic acid binding domain, and a transcription repressor domain. For example, in some embodiments of the above (1), the other of the two fusions can comprise, in order from N- to C-terminus, a transcription repressor domain and a recruitment domain A’, or a recruitment domain A’ and a transcription repressor domain, i.e., the transcription repressor domain and the recruitment domain A’ can be connected in an order that is interchangeable. For example, in some embodiments of the above (2), the other of the two fusions can comprise, in order from N- to C-terminus, a DNA methylation domain and a recruitment domain A’, or a recruitment domain A’ and a DNA methylation domain, i.e., the DNA methylation domain and the recruitment domain A’ can be connected in an order that is interchangeable.

[0179] In some more particular embodiments, the nucleic acid binding domain is a DNA binding domain. For example, the DNA binding domain can be selected from the group consisting of: a TALE domain, a zinc finger domain, a tetR domain, a meganuclease, a Cas protein, an Argonaute (Ago) protein, and a homolog, modified version, or variant thereof. For example, the DNA binding domain can be a Cas protein, and the Cas protein is a Class II Cas nuclease. Further, the Cas protein can be selected from the group consisting of a Class II Type II Cas nuclease and a Class II Type V Cas nuclease; for example, the Cas protein can be a Cas9 or Cas12 protein. In certain embodiments, the Cas protein can be a deactivated Cas9 (dCas9) protein or a deactivated Cas12 (dCas12) protein. For example, the DNA binding domain described herein can comprise, but is not limited to, an amino acid sequence set forth in any one of SEQ ID NOs: 1-9. For example, the DNA binding domain can be capable of binding to a guide RNA. For example, the guide RNA can comprise a nucleotide sequence set forth in any one of 337-368, 382-1477.

[0180] In some more specific embodiments, the transcriptional repressor is selected from one or more of the following domains: KRAB, ZIM3 KRAB, ZNF680, ZNF554, ZNF264, ZNF582, ZNF324, ZNF669, ZNF354A, ZNF82, ZNF595, ZNF419, ZNF566, ZIM2, EHMT2, SUV39H1, ZFPM1, TRIM28, EZH2, MXD1, SID, LSD1, HP1a, HDAC3, HDAC1, PRMT1, SETDB1, hSIRT1, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF41, ZNF189, ZNF528, ZNF543, ZNF140, ZNF610, ZNF350, ZNF8, ZNF30, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP82, ZNF224, ZNF33A, ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, CBX1, SCMH1, MPP8, SUMO3, HERC2, BIN1, PCGF2, TOX, FOXA1, FOXA2, IRF2BP1, IRF2BP2, IRF2BPL IRF-2BP1_2 N-terminal domain, HOXA13, HOXB13, HOXC13, HOXA11, HOXC11, HOXC10, HOXA10, HOXB9, HOXA9, ZFP28, ZN334, ZN568, ZN37A, ZN181, ZN510, ZN862, ZN140, ZN208, ZN248, ZN571, ZN699, ZN726, ZIK1, ZNF2, Z705F, ZNF14, ZN471, ZN624, ZNF84, ZNF7, ZN891, ZN337, Z705G, ZN529, ZN729, ZN419, Z705A, ZN302, ZN486, ZN621, ZN688, ZN33A, ZN554, ZN878, ZN772, ZN224, ZN184, ZN544, ZNF57, ZN283, ZN549, ZN211, ZN615, ZN253, ZN226, ZN730, Z585A, ZN732,ZN681, ZN667, ZN649, ZN470, ZN484, ZN431, ZN382, ZN254, ZN124, ZN607, ZN317, ZN620, ZN141, ZN584, ZN540, ZN75D, ZN555, ZN658, ZN684, RBAK, ZN829, ZN582, ZN112, ZN716, HKR1, ZN350, ZN480, ZN416, ZNF92, ZN100, ZN736, ZNF74, ZN443, ZN195, ZN530, ZN782, ZN791, ZN331, Z354C, ZN157, ZN727, ZN550, ZN793, ZN235, ZN724, ZN573, ZN577, ZN789, ZN718, ZN300, ZN383, ZN429, ZN677, ZN850, ZN454, ZN257, ZN264, ZN485, ZN737, ZNF44, ZN596, ZN565, ZN543, ZFP69, SUMO1, ZNF12, ZN169, ZN433, ZN175, ZN347, ZNF25, ZN519, Z585B, ZN517, ZN846, ZN230, ZNF66, ZN713, ZN816, ZN426, ZN674, ZN627, ZNF20, Z587B, ZN316, ZN233, ZN611, ZN556, ZN234, ZN560, ZNF77, ZN682, ZN614, ZN785, ZN445, ZFP30, ZN225, ZN551, ZN610, ZN528, ZN284, ZN418, ZN490, ZN805, Z780B, ZN763, ZN285, ZNF85, ZN223, ZNF90, ZN557, ZN425, ZN229, ZN606, ZN155, ZN222, ZN442, ZNF91, ZN135, ZN778, ZN534, ZN586, ZN567, ZN440, ZN583, ZN441, ZNF43, ZN589, ZN563, ZN561, ZN136, ZN630, ZN527, ZN333, Z324B, ZN786, ZN709, ZN792, ZN599, ZN613, ZF69B, ZN799, ZN569, ZN564, ZN546, ZFP92, ZN723, ZN439, ZFP57, ZNF19, ZN404, ZN274, CBX3, ZN250, ZN570, ZN675, ZN695, ZN548, ZN132, ZN738, ZN420, ZN626, ZN559, ZN460, ZN268, ZN304, ZN605,ZN844, SUMO5, ZN101, ZN783, ZN417, ZN182, ZN823, ZN177, ZN197, ZN717, ZN669, ZN256, ZN251, CBX4, CDY2, CDYL2, ZN562, ZN461, Z324A, ZN766, ID2, ZN214, CBX7, ID1, CREM, SCX, ASCL1, ZN764, SCML2, TWST1, CREB1, TERF1, ID3, CBX8, GSX1, NKX22, ATF1, TWST2, ZNF17, TOX3, TOX4, ZMYM3, I2BP1, RHXF1, SSX2, I2BPL, ZN680, TRI68, HXA13, PHC3, TCF24, HXB13, HEY1, PHC2, ZNF81, FIGLA, SAM11, KMT2B, HEY2, JDP2, HXC13, ASCL4, HHEX, GSX2, ETV7, ASCL3, PHC1, OTP, I2BP2, VGLL2, HXA11, PDLI4, ASCL2, CDX4, ZN860, LMBL4, PDIP3, NKX25, CEBPB, ISL1, CDX2, PROP1, SIN3B, SMBT1, HXC11, HXC10, PRS6A, VSX1, NKX23, MTG16, HMX3, HMX1, KIF22, CSTF2, CEBPE, DLX2, PPARG, PRIC1, UNC4, BARX2, ALX3, TCF15, TERA, VSX2, HXD12, CDX1, TCF23, ALX1, HXA10, RX, CXXC5, SCML1, NFIL3, DLX6, MTG8, CEBPD, SEC13, FIP1, ALX4, LHX3, PRIC2, MAGI3, NELL1, PRRX1, MTG8R, RAX2, DLX3, DLX1, NKX26, NAB1, SAMD7, PITX3, WDR5, MEOX2, NAB2, DHX8, CBX6, EMX2, CPSF6, HXC12, KDM4B, LMBL3, PHX2A, EMX1, NC2B, DLX4, SRY, ZN777, ZN398, GATA3, BSH, SF3B4, TEAD1, TEAD3, RGAP1, PHF1, GATA2, FOXO3, ZN212, IRX4, ZBED6, LHX4, SIN3A, RBBP7, NKX61, R51A1, MB3L1, DLX5, NOTC1, TERF2, ZN282, RGS12, ZN840, SPI2B, PAX7, NKX62, ASXL2, FOXO1,GATA1, ZMYM5, LRP1, MIXL1, SGT1, LMCD1, CEBPA, SOX14, WTIP, PRP19, NKX11, RBBP4, DMRT2, SMCA2, and functionally active fragments thereof.

[0181] In some more specific embodiments, the DNA methylation domain comprises at least one DNA methyltransferase or a functionally active fragment thereof. For example, the DNA methyltransferase is selected from the group consisting of DNMT3A, DNMT3B, DNMT3c, DNMT1, DNMT2, and DNMT3L. For example, the DNA methylation domain comprises at least one DNMT3A and at least one DNMT3L. For example, the at least one DNMT3A and the at least one DNMT3L are linked in an interchangeable order. For example, the DNA methylation domain comprises one DNMT3A and one DNMT3L, and they are linked in an interchangeable order. For example, the DNA methyltransferase comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 19-24.

[0182] The first fusion and the second fusion of the present complexes are formed by the interaction between the recruiting domains comprised in each, thus forming an aggregated complex. Accordingly, the present application provides non-limiting examples of combinations of recruiting domain A and recruiting domain A' : (1) one of the domains of the recruiting domain A and the recruiting domain A' is GCN4, and the other domain is scFv; or (2) one of the domains of the recruiting domain A and the recruiting domain A' is GFP11 fragment, and the other domain is GFP1-10; or (3) one of the domains of the recruiting domain A and the recruiting domain A' is GVKESLV, and the other domain is PDZ protein domain. The same applies to the case where GFP11 and GFP1-10 are derived from split GFP (SEQ ID NO: 15) to form the recruiting domain A and the recruiting domain A', respectively, which is also applicable to other categories of fluorescent proteins, such as mCherry (SEQ ID NO: 16), eYFP (SEQ ID NO: 18), eCFP (SEQ ID NO: 17), etc., i.e., different sets of recruiting domain A and recruiting domain A' can be obtained by splitting mCherry, splitting eYFP, or splitting eCFP, respectively, for use in the present complexes. In some embodiments, one of the first fusion and the second fusion of the present complexes can comprise two or more recruiting domains, and they are linked by a linker sequence. Exemplary amino acid sequences of recruiting domains can comprise any one of SEQ ID NOs: 10-14.

[0183] In view of the above, the present application can provide the amino acid sequence of the first fusion or the second fusion as shown below:

[0184] Table 1 Exemplary fusions of the present application

[0185] Fusions of the present application

[0186] In another aspect, the present application provides a fusion comprising a nucleic acid binding domain, and one or more effector domains; the effector domains comprise one or more of an epigenetic modification domain and / or a transcriptional regulation domain, and the fusion comprises no less than two of the epigenetic modification domain and / or the transcriptional regulation domain in total, the nucleic acid binding domain is capable of specifically binding to a target nucleotide sequence on a VEGFA gene and / or a regulatory element of a VEGFA gene, the regulatory element of a VEGFA gene comprises a transcription start site, a core promoter, a promoter, an enhancer, a silencer, an insulator element, a boundary element, and / or a locus control region, the nucleic acid binding domain is capable of specifically binding to a target nucleotide sequence within a region from 500 bp upstream to 4000 bp downstream of a transcription start site of a VEGFA gene. For example, the target nucleotide sequence is located within a region from the transcription start site of the VEGFA gene to about 500 bp, about 450 bp, about 400 bp, about 350 bp, about 300 bp, about 250 bp, about 200 bp, about 150 bp, about 100 bp, about 50 bp upstream thereof. For example, the target nucleotide sequence is located within a region from the transcription start site of the VEGFA gene to about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1500 bp, about 2000 bp, about 2500 bp, about 3000 bp, about 3500 bp, about 4000 bp downstream thereof.

[0187] For example, the target nucleotide sequence is located within a region from about 500 bp upstream to about 3500 bp downstream, from about 500 bp upstream to about 3000 bp downstream, from about 500 bp upstream to about 2500 bp downstream, from about 500 bp upstream to about 2000 bp downstream, from about 500 bp upstream to about 1500 bp downstream, from about 500 bp upstream to about 1000 bp downstream, from about 500 bp upstream to about 500 bp downstream, from about 500 bp upstream to about 250 bp downstream, from about 500 bp upstream to about 200 bp downstream, from about 500 bp upstream to about 100 bp downstream, from about 400 bp upstream to about 3500 bp downstream, from about 400 bp upstream to about 3000 bp downstream, from about 400 bp upstream to about 2500 bp downstream, from about 400 bp upstream to about 2000 bp downstream, from about 400 bp upstream to about 1500 bp downstream, from about 400 bp upstream to about 1000 bp downstream, from about 400 bp upstream to about 500 bp downstream, from about 400 bp upstream to about 250 bp downstream, from about 400 bp upstream to about 200 bp downstream, from about 400 bp upstream to about 100 bp downstream, from about 300 bp upstream to about 3500 bp downstream, from about 300 bp upstream to about 3000 bp downstream, from about 300 bp upstream to about 2500 bp downstream, from about 300 bp upstream to about 2000 bp downstream, from about 300 bp upstream to about 1500 bp downstream, from about 300 bp upstream to about 1000 bp downstream, from about 300 bp upstream to about 500 bp downstream, from about 300 bp upstream to about 250 bp downstream, from about 300 bp upstream to about 200 bp downstream, from about 300 bp upstream to about 100 bp downstream, from about 250 bp upstream to about 3500 bp downstream, from about 250 bp upstream to about 3000 bp downstream, from about 250 bp upstream to about 2500 bp downstream, from about 250 bp upstream to about 2000 bp downstream, from about 250 bp upstream to about 1500 bp downstream, from about 250 bp upstream to about 1000 bp downstream, from about 250 bp upstream to about 500 bp downstream, from about 250 bp upstream to about 250 bp downstream, from about 250 bp upstream to about 200 bp downstream, from about 250 bp upstream to about 100 bp downstream, from about 200 bp upstream to about 3500 bp downstream, from about 200 bp upstream to about 3000 bp downstream, from about 200 bp upstream to about 2500 bp downstream, from about 200 bp upstream to about 2000 bp downstream, from about 200 bp upstream to about 1500 bp downstream, from about 200 bp upstream to about 1000 bp downstream, from about 200 bp upstream to about 500 bp downstream, from about 200 bp upstream to about 250 bp downstream, from about 200 bp upstream to about 200 bp downstream, from about 200 bp upstream to about 100 bp downstream, from about 100 bp upstream to about 3500 bp downstream, from about 100 bp upstream to about 3000 bp downstream, from about 100 bp upstream to about 2500 bp downstream, from about 100 bp upstream to about 2000 bp downstream, from about 100 bp upstream to about 1500 bp downstream, from about 100 bp upstream to about 1000 bp downstream, from about 100 bp upstream to about 500 bp downstream, from about 100 bp upstream to about 250 bp downstream, from about 100 bp upstream to about 200 bp downstream, or from about 100 bp upstream to about 100 bp downstream of the VEGFA transcription start site.for example, the effector domain N-terminal to the nucleic acid binding domain can comprise an epigenetic modification domain. Specifically, the effector domain N-terminal to the nucleic acid binding domain can comprise an epigenetic modification domain that provides DNA modification. More specifically, the epigenetic modification domain that provides DNA modification can be a DNA methylation domain. More specifically, the DNA methylation domain can comprise at least one DNA methyltransferase or a functionally active fragment thereof. More specifically, the DNA methyltransferase can be selected from the group consisting of DNMT3A, DNMT3B, DNMT3C, DNMT1, DNMT2, and DNMT3L. More specifically, the DNA methylation domain can comprise at least one DNMT3A and at least one DNMT3L.

[0188] In some embodiments, the fusion described herein can comprise more than one effector domain N-terminal and C-terminal to the nucleic acid binding domain.

[0189] For example, the effector domain C-terminal to the nucleic acid binding domain can comprise at least one transcriptional modulation domain. Specifically, the effector domain C-terminal to the nucleic acid binding domain can comprise at least one transcriptional repressor domain. More specifically, the transcriptional repressor domain can be KRAB, ZIM3, and / or a functionally active fragment thereof.

[0190] More specifically, the effector domain C-terminal to the nucleic acid binding domain can comprise an epigenetic modification domain that provides histone modification. More specifically, the epigenetic modification domain that provides histone modification can comprise HP1a.

[0191] For example, the effector domain N-terminal to the nucleic acid binding domain can comprise an epigenetic modification domain. Specifically, the effector domain N-terminal to the nucleic acid binding domain can comprise an epigenetic modification domain that provides DNA modification. More specifically, the epigenetic modification domain that provides DNA modification can be a DNA methylation domain. More specifically, the DNA methylation domain can comprise at least one DNA methyltransferase or a functionally active fragment thereof. More specifically, the DNA methyltransferase can be selected from the group consisting of DNMT3A, DNMT3B, DNMT3C, DNMT1, DNMT2, and DNMT3L. More specifically, the DNA methylation domain can comprise at least one DNMT3A and at least one DNMT3L.

[0192] In some embodiments of the above cases, the fusion has, in order from N- to C-terminus, an epigenetic modification domain that provides DNA modification, the nucleic acid binding domain, and an epigenetic modification domain that provides histone modification. For example, the fusion comprises the following domains: DNMT3A-DNMT3L-dCas9-HP1a. For example, the fusion comprises the amino acid sequence of any one of SEQ ID NOs: 1480, 1482.

[0193] In some embodiments of the above cases, the fusion has, in order from N- to C-terminus, an epigenetic modification domain that provides DNA modification, the nucleic acid binding domain, and a transcriptional repressor domain. For example, the fusion comprises the following domains: DNMT3A-DNMT3L-dCas9-KRAB, DNMT3A-DNMT3L-dCas9-ZIM3. For example, the fusion comprises the amino acid sequence of any one of SEQ ID NOs: 1478, 1479.

[0194] Without wishing to be bound by any theory, the examples below are merely to illustrate the complexes, the methods of preparation, and the uses of the present application, and are not intended to limit the scope of the present application.

[0195] Examples

[0196] Example 1

[0197] Design and construction of plasmids containing the complexes of the present application

[0198] The amino acid sequences of the epigenetic modification recruitment system with HA epitope, P2A, and the recruited elements (including DNMT3A, DNMT3L, dSpCas9, KRAB) were optimized by Genscript for mammalian expression and synthesized, and then cloned into the pLV-CAG vector with CAG promoter and WPRE, to express the recruited elements and the self-cleaved recruitment system fusion protein from the CAG promoter.

[0199] In the optimization of different functional elements, different functional elements were optimized by Genscript for mammalian expression and synthesized. First, the vector except the element that needs to be replaced was amplified by PCR, and then the element that needs to be replaced was amplified from the sequence synthesized by the company, while introducing the homologous arm sequence, and finally the different elements were recombined into the vector by NEBuilder reagent to construct the final expression plasmid.

[0200] Example 2

[0201] Inhibition efficiency of the complexes of the present application on VEGFA gene expression in mouse hepatocyte cell line N2a

[0202] The mouse N2a cell line was used as a research model in this example. The guide RNA was designed according to the CpG island sequence (SEQ ID NO: 377) near the transcription start site of the mouse VEGFA genome, and the mRNA (SEQ ID NO: 288) of the epigenetic editing tool and different sgRNAs (SEQ ID NO: 337-351) were prepared by LNP embedding at a mass ratio of 1:1 to obtain LNP test samples targeting different positions (LNP reference: Musunuru, K., Chadwick, A. C., Mizoguchi, T. et al. In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates. Nature 593, 429-434 (2021)). The test cells used the mouse neuroblastoma cell line N2a, and 5 million cells / well were plated into a 24-well plate. After 12 hours, the LNP samples were added to the well plate at a dose of 2.5 ug / ml, and fresh medium was replaced after 4-6 hours. The cells were cultured, and the cells were collected after 3 days for mRNA extraction, reverse transcription into cDNA, and detection of the mRNA expression level of the target gene VEGFA by qPCR. By comparing with the Non-target group (mRNA and sgRNA without targeting site, SEQ ID NO: 336), the VEGFA gene knockdown efficiency corresponding to each sgRNA was obtained. The qPCR primer sequences are shown in SEQ ID NO: 369, 370, 373 and 374.

[0203] The results 3 days after administration (Figure 1) showed that basically all sgRNAs could achieve a certain degree of VEGFA gene inhibition, with the highest efficiency reaching 94% (sgRNA15, SEQ ID NO: 351), followed by sgRNA3 (SEQ ID NO: 339) with an efficiency of 92%, and sgRNA7 (SEQ ID NO: 343) with an efficiency of 91%.

[0204] Example 3

[0205] Inhibition efficiency of the complex of the present application on VEGFA gene expression in human retinal pigment epithelial cells ARPE19

[0206] In this embodiment, human ARPE-19 was used as the research model. Guide RNA was designed targeting the CpG island sequence (SEQ ID NO:378) near the transcription start site in the human VEGFA genome. The mRNA of the epigenetic editing tool (SEQ ID NO:288) and different sgRNAs (SEQ ID NO:352-368, 382) were embedded in LNP at a mass ratio of 1:1 to prepare LNP test samples targeting different locations (LNP reference: Musunuru, K., Chadwick, AC, Mizoguchi, T. et al. In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates. Nature 593,429–434(2021)). Human ARPE-19 cells were used for testing. Cells were seeded at 50,000 cells / well in 24-well plates. After 12 hours, LNP samples were added to the plates at a dosage of 2.5 μg / ml. The culture medium was replaced with fresh medium after 4-6 hours, and the cells were cultured for another 3 days. Cells were then collected for mRNA extraction, reverse transcribed into cDNA, and qPCR was used to detect the mRNA expression level of the target gene. The knockdown efficiency of each sgRNA was obtained by comparing it with the Non-target group (mRNA and sgRNA without a target site, SEQ ID NO: 336). The qPCR primer sequences are shown in SEQ ID NO: 371, 372, 375, and 376.

[0207] Results 3 days after administration (Figure 2) showed that almost all sgRNAs could achieve some degree of VEGFA gene inhibition, with the highest efficiency reaching 96% (sgRNA8, SEQ ID NO:359), followed by sgRNA5 (SEQ ID NO:356) with an efficiency of 92%, sgRNA9 (SEQ ID NO:360) with an efficiency of 93%, and sgRNA12 (SEQ ID NO:363) with an efficiency of 91%.

[0208] Example 4

[0209] The efficiency of different epigenetic editing tools in inhibiting VEGFA gene expression in human retinal pigment epithelial cells ARPE19, as described in this application.

[0210] In this embodiment, human ARPE-19 was used as a research model. Different epigenetic editing tools were combined with the same sgRNA (sgRNA9, SEQ ID NO:360) to screen the editing efficiency of different epigenetic editing tools against the VEGFA gene.

[0211] The epigenetic editing tools were designed for three different histone modification elements, and the structures are shown in Table 2.

[0212] Table 2: Epigenetic editing tools containing three different modification elements

[0213] The mRNA of the epigenetic editing tools of the three different histone modification elements and the same sgRNA (sgRNA9, SEQ ID NO: 360) were prepared by LNP embedding at a mass ratio of 1:1 to obtain LNP test samples of different tools (LNP is cited from the literature: Musunuru, K., Chadwick, A.C., Mizoguchi, T. et al. In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates. Nature 593, 429-434 (2021)). The test cells used human ARPE-19, and the cells were plated into 24-well plates at 50,000 cells per well. After 12 hours, the LNP samples were added to the well plates at a dose of 5 ug / ml, and fresh medium was replaced after 4-6 hours. The cells were continued to be cultured, and the mRNA extraction, reverse transcription into cDNA, and qPCR detection of the target gene mRNA expression level were performed after 3 days. By comparing with the Non-target group (mRNA, SEQ ID NO: 288 and sgRNA without targeting site, SEQ ID NO: 336), the corresponding knockdown efficiency of each group was obtained. Subsequently, the cells were continued to be cultured, and regular detection was performed to compare the persistence of each epigenetic editing tool. The qPCR primer sequences are shown in SEQ ID NO: 371, 372, 375, and 376.

[0214] [According to Rule 91 correction 18.07.2025] The efficacy results after administration were tracked to 83 days (Figure 3), which showed that the epigenetic editing tools containing three different histone modification elements could all significantly inhibit the expression of the VEGFA gene. Among them, H014 could reduce VEGFA to the lowest level, and the persistence was the best, indicating that the histone modification element HP1a had the highest efficiency for VEGFA gene inhibition.

[0215] According to the above results, the efficiency of HP1a as a histone modification element for VEGFA gene inhibition was higher than that of Zim3. Next, the Zim3 in the mRNA of the recruitment system fusion protein (SEQ ID NO: 288) was replaced with HP1a, and the differences between the two histone modification elements in this structure were compared. In addition, for the H014 structure, the differences in the efficiency of human and mouse DNMT3L for VEGFA gene inhibition were compared. According to the above two purposes, new epigenetic editing tools were designed, and the structures are shown in Table 3.

[0216] Table 3 Structures of different apparent editing tools

[0217] The same ARPE-19 cell line was used, and the same operation was used to compare the inhibition efficiency of different tools on VEGFA gene expression. The pharmacodynamic results (Figure 4) tracked to 42 days after administration showed that in the recruitment system fusion protein, E009-HP1a was superior to E009 in the effect of inhibiting VEGFA gene expression at each time point, indicating that the histone modification element HP1a was superior to Zim3 in the efficiency of VEGFA gene inhibition. After replacing the mouse DNMT3L in H014 with human DNMT3L, the effect and persistence of VEGFA gene inhibition were reduced, indicating that the mouse DNMT3L was superior to the human DNMT3L in the efficiency of VEGFA gene inhibition. Parallel comparison of the four tools showed that H014 was the best in terms of pharmacodynamic effect and persistence of VEGFA gene expression inhibition.

[0218] Example 5

[0219] Inhibition efficiency of different candidate VEGFA sgRNAs on VEGFA gene expression in human retinal pigment epithelial cells ARPE19

[0220] In this study, guide RNA was designed according to the CpG island sequence (SEQ ID NO: 378) near the transcription start site of the human VEGFA genome.

[0221] Human ARPE-19 was used as a research model in this example, and the epigenetic editing tool H014 was prepared by LNP embedding with different candidate sgRNAs at a mass ratio of 1:1 to obtain LNP test samples of different tools (LNP was derived from the literature: Musunuru, K., Chadwick, A.C., Mizoguchi, T. et al. In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates. Nature 593, 429-434 (2021)). The test cells used human ARPE-19, and 50,000 cells / well were plated into a 24-well plate. After 12 hours, the LNP samples were added to the well plate at a dose of 5 ug / ml, and fresh medium was replaced after 4-6 hours. The cells were cultured, and after 7 days, the cells were collected for mRNA extraction, reverse transcription into cDNA, and detection of the mRNA expression level of the target gene using qPCR. By comparing with the Non-target group (sgRNA without a target site, SEQ ID NO: 336), the knockdown efficiency of different sgRNAs was obtained. The qPCR primer sequences are shown in SEQ ID NOs: 371, 372, 375, and 376.

[0222] [According to Rule 91, corrected on 18.07.2025] The inhibition rate of different candidate sgRNAs on VEGFA expression is shown in the following Figure 5. The dashed area is -250bp to 250bp (genomic region: chromosome 6, 43769961 to 43770460, reference genome: GRCh38.p14 Primary Assembly). The 11 sgRNAs in this region all exhibit an inhibition efficiency of more than 80%, proving that this region has the best gene inhibition efficiency and drug development potential. Next, -500bp to +2000bp (genomic region: chromosome 6, 43769711 to 43772210, reference genome: GRCh38.p14 Primary Assembly) all exhibit good gene inhibition efficiency, and 63 of the 65 sgRNAs with more than 80% inhibition efficiency are in this region. The sequences and inhibition rates of the 65 different candidate sgRNAs with inhibition efficiency of more than 80% are shown in Table 4.

[0223] Table 4 Inhibition efficiency of different candidate sgRNAs on VEGFA gene expression in ARPE19

Claims

1. A complex comprising a first fusion and a second fusion, wherein: 1) one of the fusion of the first fusion and the second fusion comprises a DNA methylation domain and at least one recruiting domain A, and the other fusion comprises a transcription repressor domain and at least one recruiting domain A’; and 2) the first fusion or the second fusion comprises a nucleic acid binding domain; and the recruiting domain A and the recruiting domain A’ are capable of interacting to enable the fusion or a portion thereof of one of the first fusion and the second fusion to be recruited to the vicinity of the other fusion; the nucleic acid binding domain is capable of specifically binding to a target nucleotide sequence on a VEGFA gene and / or a regulatory element of a VEGFA gene, the regulatory element of a VEGFA gene comprising a transcription start site, a core promoter, a promoter, an enhancer, a silencer, an insulator element, a boundary element and / or a locus control region, the nucleic acid binding domain is capable of specifically binding to a target nucleotide sequence within a region from 500 bp upstream to 4000 bp downstream of a transcription start site of a VEGFA gene.

2. The complex of claim 1, the nucleic acid binding domain is a DNA binding domain.

3. The complex of claim 1 or 2, the DNA binding domain is selected from the group consisting of: a TALE domain, a zinc finger domain, a tetR domain, a meganuclease, a Cas protein, an Argonaute (Ago) protein, and a homolog, a modified version or a variant thereof.

4. The complex of claim 2 or 3, the DNA binding domain is capable of binding to the target nucleotide sequence.

5. The complex of any one of claims 2-4, the DNA binding domain is capable of binding to a guide RNA.

6. The complex of claim 5, the guide RNA is capable of specifically recognizing and hybridizing to the target nucleotide sequence.

7. The complex of any one of claims 5-6, the guide RNA is capable of specifically recognizing and hybridizing to the target nucleotide sequence within a region from the transcription start site of a VEGFA gene to about 500 bp, about 400 bp, about 300 bp, about 200 bp, about 100 bp, about 50 bp upstream thereof.

8. The complex of any one of claims 5-7, the guide RNA is capable of specifically recognizing and hybridizing to the target nucleotide sequence within a region from the transcription start site of a VEGFA gene to about 4000 bp, about 3500 bp, about 3000 bp, about 2500 bp, about 2000 bp, about 1500 bp, about 1000 bp, about 500 bp, about 250 bp, about 100 bp, about 50 bp downstream thereof.

9. The complex of any one of claims 5-8, wherein the guide RNA is capable of specifically recognizing and hybridizing to the target nucleotide sequence within a region from about 500 bp upstream to about 3500 bp downstream, from about 500 bp upstream to about 3000 bp downstream, from about 500 bp upstream to about 2500 bp downstream, from about 500 bp upstream to about 2000 bp downstream, from about 500 bp upstream to about 1500 bp downstream, from about 500 bp upstream to about 1000 bp downstream, or from about 500 bp upstream to about 500 bp downstream of the transcription start site of the VEGFA gene.

10. The complex of claim 9, wherein the guide RNA is capable of specifically recognizing and hybridizing to the target nucleotide sequence within a region from about 250 bp upstream to about 250 bp downstream of the transcription start site of the VEGFA gene.

11. The complex of any one of claims 5-10, wherein the guide RNA comprises a nucleotide sequence of any one of SEQ ID NOs: 337-368, 382-404, 406-421, 423-425, 427, 429, 430-455, 457-460, 462, 463, and 465.

12. The complex of any one of claims 5-11, wherein the guide RNA comprises a partial sequence of a nucleotide sequence of any one of SEQ ID NOs: 337-368, 382-404, 406-421, 423-425, 427, 429, 430-455, 457-460, 462, 463, and 465, the partial sequence having a length of 15-20 base pairs.

13. The complex of any one of claims 2-12, wherein the DNA-binding domain is a Cas protein, and the Cas protein is a Class II Cas nuclease.

14. The complex of claim 13, wherein the Cas protein is selected from the group consisting of a Class II Type II Cas nuclease and a Class II Type V Cas nuclease.

15. The complex of claim 13 or 14, wherein the Cas protein is a Cas9 or Cas12 protein.

16. The complex of any one of claims 13-15, wherein the Cas protein is a deactivated Cas9 (dCas9) protein or a deactivated Cas12 (dCas12) protein.

17. The complex of any one of claims 2-16, wherein the DNA-binding domain comprises an amino acid sequence of any one of SEQ ID NOs: 1-9.

18. The complex of any one of claims 1-17, wherein the first fusion comprises a DNA methylation domain, a nucleic acid binding domain, and at least one recruiting domain A, and the second fusion comprises a transcription repressor domain and at least one recruiting domain A’.

19. The complex of any one of claims 1-18, wherein the first fusion comprises, in order from N-terminus to C-terminus, a DNA methylation domain, a nucleic acid binding domain, and a recruiting domain A.

20. The complex of any one of claims 1-19, the second fusion comprising, in order from N- to C-terminus, a transcription repressor domain and a recruitment domain A’, or comprising, in order from N- to C-terminus, a recruitment domain A’ and a transcription repressor domain.

21. The complex of any one of claims 1-20, the first fusion comprising a transcription repressor domain, a nucleic acid binding domain, and at least one recruitment domain A, and the second fusion comprising a DNA methylation domain and at least one recruitment domain A’.

22. The complex of any one of claims 1-18, the first fusion comprising, in order from N- to C-terminus, a recruitment domain A, a nucleic acid binding domain, and a transcription repressor domain.

23. The complex of any one of claims 1-18 and 22, the second fusion comprising, in order from N- to C-terminus, a DNA methylation domain and a recruitment domain A’, or comprising, in order from N- to C-terminus, a recruitment domain A’ and a DNA methylation domain.

24. The complex of any one of claims 1-23, wherein: 1) the first fusion comprises, in order from N- to C-terminus, a DNA methylation domain, a nucleic acid binding domain, and a recruitment domain A, and the second fusion comprises, in order from N- to C-terminus, a transcription repressor domain and a recruitment domain A’; or 2) the first fusion comprises, in order from N- to C-terminus, a DNA methylation domain, a nucleic acid binding domain, and a recruitment domain A, and the second fusion comprises, in order from N- to C-terminus, a recruitment domain A’ and a transcription repressor domain; or 3) the first fusion comprises, in order from N- to C-terminus, a recruitment domain A, a nucleic acid binding domain, and a transcription repressor domain, and the second fusion comprises, in order from N- to C-terminus, a DNA methylation domain and a recruitment domain A’; or 4) the first fusion comprises, in order from N- to C-terminus, a recruitment domain A, a nucleic acid binding domain, and a transcription repressor domain, and the second fusion comprises, in order from N- to C-terminus, a recruitment domain A’ and a DNA methylation domain.

25. The complex of any one of claims 1-24, the recruitment domain A is selected from any one of one of the following two groups of domains, and the recruitment domain A’ is selected from any one of the other of the following two groups of domains: 1) a general control non-derepressible protein 4 (GCN4), a GFP 11 fragment derived from split green fluorescent protein (GFP), or a GVKESLV polypeptide; and 2) a single chain antibody (scFv), a GFP 1-10 fragment derived from split green fluorescent protein (GFP), or a PDZ protein domain.

26. The complex of any one of claims 1-25, wherein: 1) the domain of one of the recruitment domain A and the recruitment domain A’ is a GCN4, and the domain of the other is a scFv; or 2) one of the recruiting domain A and the recruiting domain A' is a domain of GFP11 fragment, and the other is GFP1-10; or 3) one of the recruiting domain A and the recruiting domain A' is GVKESLV, and the other is a PDZ protein domain.

27. The complex of any one of claims 1-26, the DNA methylation domain comprises at least one DNA methyltransferase or a functionally active fragment thereof.

28. The complex of claim 27, the DNA methyltransferase is selected from the group consisting of DNMT3A, DNMT3B, DNMT3C, DNMT1, DNMT2, and DNMT3L.

29. The complex of any one of claims 1-28, the DNA methylation domain comprises at least one DNMT3A and at least one DNMT3L.

30. The complex of claim 27 or 28, the DNA methyltransferase comprises an amino acid sequence set forth in any one of SEQ ID NOs: 19-24.

31. The complex of any one of claims 1-30, the DNA methylation domain comprising a DNMT3A-DNMT3L domain or a DNMT3L-DNMT3A domain; wherein, - indicates that the domains at its two ends are connected directly or indirectly in the order from N-terminus to C-terminus.

32. The complex of any one of claims 1-31, the transcriptional repressor is selected from one or more of the following domains: KRAB, ZIM3 KRAB, ZNF680, ZNF554, ZNF264, ZNF582, ZNF324, ZNF669, ZNF354A, ZNF82, ZNF595, ZNF419, ZNF566, ZIM2, EHMT2, SUV39H1, ZFPM1, TRIM28, EZH2, MXD1, SID, LSD1, HP1a, HDAC3, HDAC1, PRMT1, SETDB1, hSIRT1, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF41, ZNF189, ZNF528, ZNF543, ZNF140, ZNF610, ZNF350, ZNF8, ZNF30, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP82, ZNF224, ZNF33A, ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, CBX1, SCMH1, MPP8, SUMO3, HERC2, BIN1, PCGF2, TOX, FOXA1, FOXA2, IRF2BP1, IRF2BP2, IRF2BPL IRF-2BP1_2 N-terminal domain, HOXA13, HOXB13, HOXC13, HOXA11, HOXC11, HOXC10, HOXA10, HOXB9, HOXA9, ZFP28, ZN334, ZN568, ZN37A, ZN181, ZN510, ZN862, ZN140, ZN208, ZN248, ZN571, ZN699, ZN726, ZIK1, ZNF2, Z705F, ZNF14, ZN471, ZN624, ZNF84, ZNF7, ZN891, ZN337, Z705G, ZN529, ZN729, ZN419, Z705A, ZN302, ZN486, ZN621, ZN688, ZN33A, ZN554, ZN878, ZN772, ZN224, ZN184, ZN544, ZNF57, ZN283, ZN549, ZN211, ZN615, ZN253, ZN226, ZN730,Z585A, ZN732, ZN681, ZN667, ZN649, ZN470, ZN484, ZN431, ZN382, ZN254, ZN124, ZN607, ZN317, ZN620, ZN141, ZN584, ZN540, ZN75D, ZN555, ZN658, ZN684, RBAK, ZN829, ZN582, ZN112, ZN716, HKR1, ZN350, ZN480, ZN416, ZNF92, ZN100, ZN736, ZNF74, ZN443, ZN195, ZN530, ZN782, ZN791, ZN331, Z354C, ZN157, ZN727, ZN550, ZN793, ZN235, ZN724, ZN573, ZN577, ZN789, ZN718, ZN300, ZN383, ZN429, ZN677, ZN850, ZN454, ZN257, ZN264, ZN485, ZN737, ZNF44, ZN596, ZN565, ZN543, ZFP69, SUMO1, ZNF12, ZN169, ZN433, ZN175, ZN347, ZNF25, ZN519, Z585B, ZN517, ZN846, ZN230, ZNF66, ZN713, ZN816, ZN426, ZN674, ZN627, ZNF20, Z587B, ZN316, ZN233, ZN611, ZN556, ZN234, ZN560, ZNF77, ZN682, ZN614, ZN785, ZN445, ZFP30, ZN225, ZN551, ZN610, ZN528, ZN284, ZN418, ZN490, ZN805, Z780B, ZN763, ZN285, ZNF85, ZN223, ZNF90, ZN557, ZN425, ZN229, ZN606, ZN155, ZN222, ZN442, ZNF91, ZN135, ZN778, ZN534, ZN586, ZN567, ZN440, ZN583, ZN441, ZNF43, ZN589, ZN563, ZN561, ZN136, ZN630, ZN527, ZN333, Z324B, ZN786, ZN709, ZN792, ZN599, ZN613, ZF69B, ZN799, ZN569, ZN564, ZN546, ZFP92, ZN723, ZN439, ZFP57, ZNF19, ZN404, ZN274, CBX3, ZN250, ZN570, ZN675, ZN695, ZN548, ZN132, ZN738, ZN420, ZN626, ZN559, ZN460, ZN268,ZN304, ZN605, ZN844, SUMO5, ZN101, ZN783, ZN417, ZN182, ZN823, ZN177, ZN197, ZN717, ZN669, ZN256, ZN251, CBX4, CDY2, CDYL2, ZN562, ZN461, Z324A, ZN766, ID2, ZN214, CBX7, ID1, CREM, SCX, ASCL1, ZN764, SCML2, TWST1, CREB1, TERF1, ID3, CBX8, GSX1, NKX22, ATF1, TWST2, ZNF17, TOX3, TOX4, ZMYM3, I2BP1, RHXF1, SSX2, I2BPL, ZN680, TRI68, HXA13, PHC3, TCF24, HXB13, HEY1, PHC2, ZNF81, FIGLA, SAM11, KMT2B, HEY2, JDP2, HXC13, ASCL4, HHEX, GSX2, ETV7, ASCL3, PHC1, OTP, I2BP2, VGLL2, HXA11, PDLI4, ASCL2, CDX4, ZN860, LMBL4, PDIP3, NKX25, CEBPB, ISL1, CDX2, PROP1, SIN3B, SMBT1, HXC11, HXC10, PRS6A, VSX1, NKX23, MTG16, HMX3, HMX1, KIF22, CSTF2, CEBPE, DLX2, PPARG, PRIC1, UNC4, BARX2, ALX3, TCF15, TERA, VSX2, HXD12, CDX1, TCF23, ALX1, HXA10, RX, CXXC5, SCML1, NFIL3, DLX6, MTG8, CEBPD, SEC13, FIP1, ALX4, LHX3, PRIC2, MAGI3, NELL1, PRRX1, MTG8R, RAX2, DLX3, DLX1, NKX26, NAB1, SAMD7, PITX3, WDR5, MEOX2, NAB2, DHX8, CBX6, EMX2, CPSF6, HXC12, KDM4B, LMBL3, PHX2A, EMX1, NC2B, DLX4, SRY, ZN777, ZN398, GATA3, BSH, SF3B4, TEAD1, TEAD3, RGAP1, PHF1, GATA2, FOXO3, ZN212, IRX4, ZBED6, LHX4, SIN3A, RBBP7, NKX61, R51A1, MB3L1, DLX5, NOTC1, TERF2, ZN282, RGS12, ZN840, SPI2B, PAX7, NKX62,ASXL2, FOXOl, GATA1, ZMYM5, LRP1, MIXL1, SGT1, LMCD1, CEBPA, SOX14, WTIP, PRP19, NKX11, RBBP4, DMRT2, SMCA2, and functionally active fragments thereof.

33. The complex of any one of claims 1-32, the transcription repressor domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 25-50.

34. The complex of any one of claims 1-33, wherein: 1) one of the first fusion and the second fusion comprises a DNA methylation domain - dCas9 or dCas12-n x GCN4, and the other comprises a transcription repressor domain - scFv; or 2) one of the first fusion and the second fusion comprises a DNA methylation domain - dCas9 or dCas12-scFv, and the other comprises a transcription repressor domain - GCN4; or 3) one of the first fusion and the second fusion comprises a DNA methylation domain - dCas9 or dCas12-n x GFP11, and the other comprises a transcription repressor domain - GFP1-10; or 4) one of the first fusion and the second fusion comprises a DNA methylation domain - dCas9 or dCas12-GFP1-10, and the other comprises a transcription repressor domain - GFP11; or 5) one of the first fusion and the second fusion comprises a DNA methylation domain - dCas9 or dCas12-n x GCN4, and the other comprises a scFv - transcription repressor domain; or 6) one of the first fusion and the second fusion comprises a DNA methylation domain - dCas9 or dCas12-scFv, and the other comprises a GCN4 - transcription repressor domain; or 7) one of the first fusion and the second fusion comprises a DNA methylation domain-dCas9 or dCas12-n x GFP11, and the other comprises GFP1-10-transcription repressor domain; or 8) one of the first fusion and the second fusion comprises a DNA methylation domain-dCas9 or dCas12-GFP1-10, and the other comprises GFP11-transcription repressor domain; wherein - indicates that the domains at its two ends are connected directly or indirectly in the order from N-terminus to C-terminus; n x GCN4 or n x GFP11 indicates n copies of GCN4 or n copies of GFP11 connected by a linker sequence, respectively, and n is selected from any integer from 1 to 20.

35. The complex of any one of claims 1-33, the first fusion and / or the second fusion comprising the amino acid sequence of any one of SEQ ID NOs: 51-76, 78-82, 85-93, 103-105, 110-115, 123, and 124.

36. The complex of any one of claims 1-35, comprising the amino acid sequence of any one of SEQ ID NOs: 133-142, 153, 154, 158-163, 168, and 1481.

37. The complex of any one of claims 1-33, wherein: 1) one of the first fusion and the second fusion comprises n x GCN4-dCas9 or dCas12-transcription repressor domain, and the other comprises a DNA methylation domain-scFv; or 2) one of the first fusion and the second fusion comprises scFv-dCas9 or dCas12-transcription repressor domain, and the other comprises a DNA methylation domain-GCN4; or 3) one of the first fusion and the second fusion comprises n x GFP11-dCas9 or dCas12-transcription repressor domain, and the other comprises a DNA methylation domain-GFP1-10; or 4) one of the first fusion and the second fusion comprises GFP1-10-dCas9 or dCas12-transcription repressor domain, and the other comprises a DNA methylation domain-GFP11; or 5) one of the first fusion and the second fusion comprises n x GCN4-dCas9 or dCas12-transcription repressor domain, and the other comprises scFv-DNA methylation domain; or 6) one of the first fusion and the second fusion comprises scFv-dCas9 or dCas12-transcription repressor domain, and the other comprises GCN4-DNA methylation domain; or 7) the fusion of one of the first fusion and the second fusion comprises n x GFP11-dCas9 or dCas12-transcription repressor domain, and the other fusion comprises GFP1-10-DNA methylation domain; or 8) the fusion of one of the first fusion and the second fusion comprises GFP1-10-dCas9 or dCas12-transcription repressor domain, and the other fusion comprises GFP11-DNA methylation domain; wherein - indicates that the domains at its two ends are connected directly or indirectly in the order from N-terminus to C-terminus; n x GCN4 or n x GFP11 respectively indicates n copies of GCN4 or n copies of GFP11 connected by a linker sequence, n is selected from any integer from 1 to 20.

38. The complex of any one of claims 1-33 and 37, the first fusion and / or the second fusion comprises an amino acid sequence set forth in any one of SEQ ID NOs: 83, 84, 94-102, 106-109, and 116-122.

39. The complex of any one of claims 1-33, 37, and 38, comprising an amino acid sequence set forth in any one of SEQ ID NOs: 143-152, 155-157, and 164-167.

40. The complex of any one of claims 1-39, further comprising a nuclear localization signal and / or a marker domain.

41. The complex of any one of claims 1-40, capable of providing modification of at least one nucleotide in the vicinity of the VEGFA gene and / or within a regulatory element of the VEGFA gene.

42. The complex of any one of claims 1-41, capable of providing modification of at least one nucleotide within a region from the transcription start site of the VEGFA gene to about 500 bp, about 400 bp, about 300 bp, about 200 bp, about 100 bp, about 50 bp upstream thereof.

43. The complex of any one of claims 1-42, capable of providing modification of at least one nucleotide within a region from the transcription start site of the VEGFA gene to about 4000 bp, about 3500 bp, about 3000 bp, about 2500 bp, about 2000 bp, about 1500 bp, about 1000 bp, about 500 bp, about 250 bp, about 100 bp, about 50 bp downstream thereof.

44. The complex of any one of claims 1-43, which is capable of providing a modification of at least one nucleotide within a region from about 500 bp upstream to about 3500 bp downstream, from about 500 bp upstream to about 3000 bp downstream, from about 500 bp upstream to about 2500 bp downstream, from about 500 bp upstream to about 2000 bp downstream, from about 500 bp upstream to about 1500 bp downstream, from about 500 bp upstream to about 1000 bp downstream, from about 500 bp upstream to about 500 bp downstream of the transcription start site of the VEGFA gene.

45. The complex of claim 44, which is capable of providing a modification of at least one nucleotide within a region from about 250 bp upstream to about 250 bp downstream of the transcription start site of the VEGFA gene.

46. A fusion comprising a nucleic acid binding domain, and one or more effector domains; the effector domains comprise one or more of an epigenetic modification domain and / or a transcriptional regulation domain, and the epigenetic modification domain and / or the transcriptional regulation domain in the fusion is not less than two in total, the nucleic acid binding domain is capable of specifically binding to a target nucleotide sequence on the VEGFA gene and / or a regulatory element of the VEGFA gene, the regulatory element of the VEGFA gene comprises a transcription start site, a core promoter, a promoter, an enhancer, a silencer, an insulator element, a boundary element, and / or a locus control region, the nucleic acid binding domain is capable of specifically binding to a target nucleotide sequence within a region from 500 bp upstream to 4000 bp downstream of the transcription start site of the VEGFA gene.

47. The fusion of claim 46, the nucleic acid binding domain is a DNA binding domain.

48. The fusion of claim 47, the DNA binding domain is selected from the group consisting of: a TALE domain, a zinc finger domain, a tetR domain, a meganuclease, a Cas protein, an Argonaute (Ago) protein, and a homolog or a modified version thereof.

49. The fusion of any one of claims 47-48, the DNA binding domain is capable of binding to a target sequence of interest of a target locus.

50. The fusion of claim 49, the DNA binding domain is capable of binding to a guide RNA.

51. The fusion of claim 50, the guide RNA is capable of specifically recognizing and hybridizing to the target nucleotide sequence within a region from the transcription start site to about 500 bp, about 400 bp, about 300 bp, about 200 bp, about 100 bp, about 50 bp upstream thereof of the VEGFA gene.

52. The fusion of any one of claims 50-51, the guide RNA is capable of specifically recognizing and hybridizing to the target nucleotide sequence within a region from the transcription start site to about 500 bp, about 400 bp, about 300 bp, about 200 bp, about 100 bp, about 50 bp upstream thereof of the VEGFA gene.

53. The fusion of any one of claims 50-52, wherein the guide RNA is capable of specifically recognizing and hybridizing to the target nucleotide sequence within a region from about 4000 bp, about 3500 bp, about 3000 bp, about 2500 bp, about 2000 bp, about 1500 bp, about 1000 bp, about 500 bp, about 250 bp, about 100 bp, about 50 bp upstream of the transcription start site of the VEGFA gene to about 4000 bp, about 3500 bp, about 3000 bp, about 2500 bp, about 2000 bp, about 1500 bp, about 1000 bp, about 500 bp, about 250 bp, about 100 bp, about 50 bp downstream of the transcription start site of the VEGFA gene.

54. The fusion of any one of claims 50-53, wherein the guide RNA is capable of specifically recognizing and hybridizing to the target nucleotide sequence within a region from about 500 bp upstream of the transcription start site of the VEGFA gene to about 3500 bp downstream of the transcription start site of the VEGFA gene, from about 500 bp upstream of the transcription start site of the VEGFA gene to about 3000 bp downstream of the transcription start site of the VEGFA gene, from about 500 bp upstream of the transcription start site of the VEGFA gene to about 2500 bp downstream of the transcription start site of the VEGFA gene, from about 500 bp upstream of the transcription start site of the VEGFA gene to about 2000 bp downstream of the transcription start site of the VEGFA gene, from about 500 bp upstream of the transcription start site of the VEGFA gene to about 1500 bp downstream of the transcription start site of the VEGFA gene, from about 500 bp upstream of the transcription start site of the VEGFA gene to about 1000 bp downstream of the transcription start site of the VEGFA gene, or from about 500 bp upstream of the transcription start site of the VEGFA gene to about 500 bp downstream of the transcription start site of the VEGFA gene.

55. The fusion of claim 54, wherein the guide RNA is capable of specifically recognizing and hybridizing to the target nucleotide sequence within a region from about 250 bp upstream of the transcription start site of the VEGFA gene to about 250 bp downstream of the transcription start site of the VEGFA gene.

56. The fusion of any one of claims 50-55, wherein the guide RNA comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 337-368, 382-404, 406-421, 423-425, 427, 429, 430-455, 457-460, 462, 463, and 465.

57. The fusion of any one of claims 50-56, wherein the guide RNA comprises a partial sequence of the nucleotide sequence set forth in any one of SEQ ID NOs: 337-368, 382-404, 406-421, 423-425, 427, 429, 430-455, 457-460, 462, 463, and 465, the partial sequence being 15-20 base pairs in length.

58. The fusion of any one of claims 47-57, wherein the DNA-binding domain is a Cas protein, and the Cas protein is a Class II Cas nuclease.

59. The fusion of claim 58, wherein the Cas protein is selected from the group consisting of a Class II Type II Cas nuclease and a Class II Type V Cas nuclease.

60. The fusion of any one of claims 47-59, wherein the DNA-binding domain is a Cas9 protein.

61. The fusion of claim 60, wherein the Cas9 protein is a dead Cas9 protein (dCas9).

62. The fusion of any one of claims 46-61, wherein the nucleic acid-binding domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-9.

63. The fusion of any one of claims 46-62, wherein the epigenetic modification domain is selected from the group consisting of DNA deaminase activity, DNA methyltransferase activity, DNA demethylase activity, DNA aminase activity, DNA oxidizing activity, DNA helicase activity, histone acetyltransferase activity, histone deacetylase activity, histone methyltransferase activity, histone demethylase activity, histone kinase activity, histone phosphatase activity, histone ubiquitin ligase activity, and histone deubiquitination activity.

64. The fusion of any one of claims 46-63, wherein the epigenetic modification domain comprises a DNA methyltransferase and / or a functionally active fragment thereof.

65. The fusion of claim 64, wherein the DNA methyltransferase is selected from the group consisting of DNMT3A, DNMT3B, DNMT3C, DNMT1, DNMT2, and DNMT3L.

66. The fusion of any one of claims 46-65, wherein the epigenetic modification domain comprises a plurality of DNA methyltransferases and / or functionally active fragments thereof, and the plurality of DNA methyltransferases and / or functionally active fragments thereof are linked by a linker sequence.

67. The fusion of any one of claims 46-66, wherein the epigenetic modification domain comprises at least one DNMT3A and at least one DNMT3L.

68. The fusion of any one of claims 64-66, wherein the DNA methyltransferase comprises an amino acid sequence set forth in any one of SEQ ID NOs: 19-24.

69. The fusion of any one of claims 46-68, wherein the transcriptional regulation domain is a transcriptional activation domain or a transcriptional repressor domain.

70. The fusion of claim 69, the transcriptional repressor domain is selected from the group consisting of: KRAB, ZIM3, ZNF680, ZNF554, ZNF264, ZNF582, ZNF324, ZNF669, ZNF354A, ZNF82, ZNF595, ZNF419, ZNF566, ZIM2, EHMT2, SUV39H1, ZFPM1, TRIM28, EZH2, MXD1, SID, LSD1, HP1a, HDAC3, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF41, ZNF189, ZNF528, ZNF543, ZNF140, ZNF610, ZNF350, ZNF8, ZNF30, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP82, ZNF224, ZNF33A, ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, CBX1, SCMH1, MPP8, SUMO3, HERC2, BIN1, PCGF2, TOX, FOXA1, FOXA2, IRF2BP1, IRF2BP2, IRF2BPL IRF-2BP1_2 N-terminal domain, HOXA13, HOXB13, HOXC13, HOXA11, HOXC11, HOXC10, HOXA10, HOXB9, HOXA9, ZFP28, ZN334, ZN568, ZN37A, ZN181, ZN510, ZN862, ZN140, ZN208, ZN248, ZN571, ZN699, ZN726, ZIK1, ZNF2, Z705F, ZNF14, ZN471, ZN624, ZNF84, ZNF7, ZN891, ZN337, Z705G, ZN529, ZN729, ZN419, Z705A, ZN302, ZN486, ZN621, ZN688, ZN33A, ZN554, ZN878, ZN772, ZN224, ZN184, ZN544, ZNF57, ZN283, ZN549, ZN211, ZN615, ZN253, ZN226, ZN730, Z585A, ZN732, ZN681, ZN667, ZN649, ZN470, ZN484, ZN431,ZN382, ZN254, ZN124, ZN607, ZN317, ZN620, ZN141, ZN584, ZN540, ZN75D, ZN555, ZN658, ZN684, RBAK, ZN829, ZN582, ZN112, ZN716, HKR1, ZN350, ZN480, ZN416, ZNF92, ZN100, ZN736, ZNF74, ZN443, ZN195, ZN530, ZN782, ZN791, ZN331, Z354C, ZN157, ZN727, ZN550, ZN793, ZN235, ZN724, ZN573, ZN577, ZN789, ZN718, ZN300, ZN383, ZN429, ZN677, ZN850, ZN454, ZN257, ZN264, ZN485, ZN737, ZNF44, ZN596, ZN565, ZN543, ZFP69, SUMO1, ZNF12, ZN169, ZN433, ZN175, ZN347, ZNF25, ZN519, Z585B, ZN517, ZN846, ZN230, ZNF66, ZN713, ZN816, ZN426, ZN674, ZN627, ZNF20, Z587B, ZN316, ZN233, ZN611, ZN556, ZN234, ZN560, ZNF77, ZN682, ZN614, ZN785, ZN445, ZFP30, ZN225, ZN551, ZN610, ZN528, ZN284, ZN418, ZN490, ZN805, Z780B, ZN763, ZN285, ZNF85, ZN223, ZNF90, ZN557, ZN425, ZN229, ZN606, ZN155, ZN222, ZN442, ZNF91, ZN135, ZN778, ZN534, ZN586, ZN567, ZN440, ZN583, ZN441, ZNF43, ZN589, ZN563, ZN561, ZN136, ZN630, ZN527, ZN333, Z324B, ZN786, ZN709, ZN792, ZN599, ZN613, ZF69B, ZN799, ZN569, ZN564, ZN546, ZFP92, ZN723, ZN439, ZFP57, ZNF19, ZN404, ZN274, CBX3, ZN250, ZN570, ZN675, ZN695, ZN548, ZN132, ZN738, ZN420, ZN626, ZN559, ZN460, ZN268, ZN304, ZN605, ZN844, SUMO5, ZN101, ZN783, ZN417, ZN182,ZN823, ZN177, ZN197, ZN717, ZN669, ZN256, ZN251, CBX4, CDY2, CDYL2, ZN562, ZN461, Z324A, ZN766, ID2, ZN214, CBX7, ID1, CREM, SCX, ASCL1, ZN764, SCML2, TWST1, CREB1, TERF1, ID3, CBX8, GSX1, NKX22, ATF1, TWST2, ZNF17, TOX3, TOX4, ZMYM3, I2BP1, RHXF1, SSX2, I2BPL, ZN680, TRI68, HXA13, PHC3, TCF24, HXB13, HEY1, PHC2, ZNF81, FIGLA, SAM11, KMT2B, HEY2, JDP2, HXC13, ASCL4, HHEX, GSX2, ETV7, ASCL3, PHC1, OTP, I2BP2, VGLL2, HXA11, PDLI4, ASCL2, CDX4, ZN860, LMBL4, PDIP3, NKX25, CEBPB, ISL1, CDX2, PROP1, SIN3B, SMBT1, HXC11, HXC10, PRS6A, VSX1, NKX23, MTG16, HMX3, HMX1, KIF22, CSTF2, CEBPE, DLX2, PPARG, PRIC1, UNC4, BARX2, ALX3, TCF15, TERA, VSX2, HXD12, CDX1, TCF23, ALX1, HXA10, RX, CXXC5, SCML1, NFIL3, DLX6, MTG8, CEBPD, SEC13, FIP1, ALX4, LHX3, PRIC2, MAGI3, NELL1, PRRX1, MTG8R, RAX2, DLX3, DLX1, NKX26, NAB1, SAMD7, PITX3, WDR5, MEOX2, NAB2, DHX8, CBX6, EMX2, CPSF6, HXC12, KDM4B, LMBL3, PHX2A, EMX1, NC2B, DLX4, SRY, ZN777, ZN398, GATA3, BSH, SF3B4, TEAD1, TEAD3, RGAP1, PHF1, GATA2, FOXO3, ZN212, IRX4, ZBED6, LHX4, SIN3A, RBBP7, NKX61, R51A1, MB3L1, DLX5, NOTC1, TERF2, ZN282, RGS12, ZN840, SPI2B, PAX7, NKX62, ASXL2, FOXO1, GATA1, ZMYM5, LRP1, MIXL1, SGT1, LMCD1, CEBPA,SOX14, WTIP, PRP19, NKX11, RBBP4, DMRT2, SMCA2, and functionally active fragments thereof.

71. The fusion of claim 69 or 70, wherein the transcriptional repressor domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 25-50.

72. The fusion of any one of claims 46-71, wherein one or more of the nucleic acid binding domain, the epigenetic modification domain, and the transcriptional regulation domain are linked by a linker sequence.

73. The fusion of claim 72, wherein the linker sequence comprises at least 16 amino acids.

74. The fusion of claim 72 or 73, wherein the linker sequence comprises an XTEN linker sequence.

75. The fusion of any one of claims 72-74, said linker sequence comprising a GS linker peptide comprising the sequence: (GS) a (GGS) b (GGGS) c (GGGGS) d wherein G represents a glycine residue (Gly), S represents a serine residue (Ser), and a, b, c, and d represent integers greater than or equal to 0.

76. The fusion of any one of claims 72-75, wherein the linker sequence comprises an amino acid sequence set forth in one or more of SEQ ID NOs: 125-132.

77. The fusion of any one of claims 46-76, comprising one or more effector domains, wherein the effector domain is located N-terminal and C-terminal to the nucleic acid binding domain.

78. The fusion of claim 77, wherein the one or more effector domains comprise at least one epigenetic modification domain.

79. The fusion of any one of claims 77-78, wherein the one or more effector domains comprises at least one transcriptional modulator domain.

80. The fusion of any one of claims 77-79, wherein the effector domain C-terminal to the nucleic acid binding domain comprises at least one transcriptional modulator domain.

81. The fusion of any one of claims 77-80, wherein the effector domain C-terminal to the nucleic acid binding domain comprises at least one transcriptional repressor domain.

82. The fusion of any one of claims 77-81, wherein the effector domain N-terminal to the nucleic acid binding domain comprises an epigenetic modification domain.

83. The fusion of any one of claims 77-82, wherein the effector domain N-terminal to the nucleic acid binding domain comprises an epigenetic modification domain that provides DNA modification.

84. The fusion of any one of claims 77-83, wherein the fusion comprises, in order from N- to C-terminus: (1) an epigenetic modification domain that provides histone modification, the nucleic acid binding domain, and an epigenetic modification domain that provides histone modification; (2) a transcriptional repressor domain, the nucleic acid binding domain, and an epigenetic modification domain that provides histone modification; (3) an epigenetic modification domain that provides DNA modification, the nucleic acid binding domain, and an epigenetic modification domain that provides histone modification; (4) a transcriptional repressor domain, an epigenetic modification domain that provides DNA modification, the nucleic acid binding domain, and an epigenetic modification domain that provides histone modification; or (5) an epigenetic modification domain that provides DNA modification, a transcriptional repressor domain, the nucleic acid binding domain, and an epigenetic modification domain that provides histone modification; or (6) an epigenetic modification domain that provides DNA modification, the nucleic acid binding domain, and a transcriptional repressor domain.

85. The fusion of claim 84, wherein the epigenetic modification domain that provides histone modification is selected from the group consisting of EZH2, HDAC3, HDAC1, EHMT2, PRMT1, PRMT5, SETDB1, hSIRT1, HP1a, LSD1, and functionally active fragments thereof.

86. The fusion of any one of claims 83-85, wherein the epigenetic modification domain that provides DNA modification is selected from the group consisting of DNMT3A, DNMT3L, a combination of DNMT3A and DNMT3L, or functionally active fragments thereof.

87. The fusion of any one of claims 77-86, comprising the following domains: DNMT3A-DNMT3L-dCas9-KRAB, DNMT3A-DNMT3L-dCas9-ZIM3, HDAC3-dCas9-EZH2, KRAB-dCas9-EZH2, KRAB-DNMT3A-dCas9-EZH2, KRAB-DNMT3A-dCas9-HDAC3, DNMT3A-DNMT3L-dCas9-(EZH2) n＝1-11 , DNMT3A-DNMT3L-dCas9-HDAC3, DNMT3A-DNMT3L-dCas9-EHMT2, DNMT3A-DNMT3L-dCas9-HDAC1, DNMT3A-DNMT3L-dCas9-PRMT1, DNMT3A-DNMT3L-dCas9-SETDB1, DNMT3A-DNMT3L-dCas9-hSIRT1, DNMT3A-DNMT3L-dCas9-PRMT5, DNMT3A-DNMT3L-dCas9-HPla, DNMT3A-DNMT3L-dCas9-LSD1, or DNMT3A-DNMT3L-TALE-KRAB; wherein, - indicates that the domains of the respective portion of the fusion are directly and / or indirectly linked, and in order from N- to C-terminus.

88. The fusion of any one of claims 77-87, comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1478-1480 and 1482.

89. The fusion of any one of claims 77-88, further comprising a nuclear localization signal and / or a marker domain.

90. The fusion of any one of claims 46-89, which is capable of providing a modification of at least one nucleotide in the vicinity of the VEGFA gene and / or within a regulatory element of the VEGFA gene.

91. The fusion of any one of claims 46-90, which is capable of providing a modification of at least one nucleotide in the region from the transcription start site of the VEGFA gene to about 500 bp, about 400 bp, about 300 bp, about 200 bp, about 100 bp, about 50 bp upstream thereof.

92. The fusion of any one of claims 46-91, which is capable of providing a modification of at least one nucleotide in the region from the transcription start site of the VEGFA gene to about 4000 bp, about 3500 bp, about 3000 bp, about 2500 bp, about 2000 bp, about 1500 bp, about 1000 bp, about 500 bp, about 250 bp, about 100 bp, about 50 bp downstream thereof.

93. The fusion of any one of claims 46-92, which is capable of providing a modification of at least one nucleotide in the region from about 500 bp upstream of the transcription start site of the VEGFA gene to about 3500 bp downstream thereof, from about 500 bp upstream to about 3000 bp downstream, from about 500 bp upstream to about 2500 bp downstream, from about 500 bp upstream to about 2000 bp downstream, from about 500 bp upstream to about 1500 bp downstream, from about 500 bp upstream to about 1000 bp downstream, from about 500 bp upstream to about 500 bp downstream.

94. The fusion of claim 93, which is capable of providing a modification of at least one nucleotide in the region from about 250 bp upstream of the transcription start site of the VEGFA gene to about 250 bp downstream thereof.

95. A nucleic acid encoding the complex of any one of claims 1-45 and / or encoding the fusion of any one of claims 46-94.

96. The nucleic acid of claim 95, which is a recombinant vector.

97. The nucleic acid of claim 96, which further comprises a non-coding region.

98. The nucleic acid of claim 97, wherein the non-coding region is selected from the group consisting of an intron, a regulatory element, a promoter, an enhancer, a termination sequence, and a 5' and 3' untranslated region.

99. The nucleic acid of any one of claims 95-98, which comprises a first nucleic acid segment encoding the first fusion and a second nucleic acid segment encoding the second fusion.

100. The nucleic acid of claim 99, wherein the first nucleic acid segment and the second nucleic acid segment are linked by a nucleic acid segment encoding a cleavable peptide.

101. The nucleic acid of claim 100, wherein the cleavable peptide is a 2A peptide and / or an IRES.

102. The nucleic acid of claim 101, wherein the 2A peptide is selected from the group consisting of P2A, T2A, E2A, and F2A.

103. The nucleic acid of any one of claims 95-102, comprising a nucleic acid sequence set forth in any one of SEQ ID NOs: 169-335, 1483-1487.

104. A delivery vehicle comprising the complex of any one of claims 1-45, the fusion of any one of claims 46-94, and / or the nucleic acid of any one of claims 95-103, and optionally a liposome and / or a lipid nanoparticle.

105. A composition comprising the complex of any one of claims 1-45, the fusion of any one of claims 46-94, the nucleic acid of any one of claims 95-103, and / or the delivery vehicle of claim 104.

106. A cell comprising the complex of any one of claims 1-45, the fusion of any one of claims 46-94, the nucleic acid of any one of claims 95-103, the delivery vehicle of claim 104, and / or the composition of claim 105.

107. A kit comprising the complex of any one of claims 1-45, the fusion of any one of claims 46-94, the nucleic acid of any one of claims 95-103, the delivery vehicle of claim 104, the composition of claim 105, and / or the cell of claim 106.

108. A method of modulating expression of a VEGFA gene product, the method comprising administering the complex of any one of claims 1-45, the fusion of any one of claims 46-94, the nucleic acid of any one of claims 95-103, the delivery vehicle of claim 104, the composition of claim 105, the cell of claim 106, and / or the kit of claim 107.

109. The method of claim 108, comprising introducing the complex, the nucleic acid, the delivery vehicle, the composition, the cell, and / or the kit into a cell containing the VEGFA gene.

110. The method of claim 108, comprising contacting the complex, the nucleic acid, the delivery vehicle, and / or the composition with the VEGFA gene and / or a regulatory element of the VEGFA gene.

111. The method of claim 110, wherein the regulatory element comprises a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a boundary element, and / or a locus control region.

112. A method of treating or ameliorating a disease or a condition thereof associated with abnormal VEGFA gene expression and / or abnormal VEGFA gene activity, the method comprising administering to a subject in need thereof an effective amount of the complex of any one of claims 1-45, the fusion of any one of claims 46-94, the nucleic acid of any one of claims 95-103, the delivery vector of claim 104, the composition of claim 105, the cell of claim 106, and / or the kit of claim 107.

113. Use of the complex of any one of claims 1-45, the fusion of any one of claims 46-94, the nucleic acid of any one of claims 95-103, the delivery vector of claim 104, the composition of claim 105, the cell of claim 106, and / or the kit of claim 107 for the manufacture of a medicament for treating or ameliorating a disease or a condition thereof associated with abnormal VEGFA gene expression and / or abnormal VEGFA gene activity.

114. The method of claim 113, wherein the disease or disorder associated with abnormal expression of VEGFA gene and / or abnormal activity of VEGFA gene comprises: wet macular degeneration, diabetic retinopathy, age-related macular degeneration, and other ocular diseases associated with corneal or choroidal neovascularization.

Images