CN117070540A - Polynucleotide expression cassette, plasmid and recombinant adeno-associated virus for encoding VEGF protein - Google Patents
Polynucleotide expression cassette, plasmid and recombinant adeno-associated virus for encoding VEGF protein Download PDFInfo
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- CN117070540A CN117070540A CN202311103435.4A CN202311103435A CN117070540A CN 117070540 A CN117070540 A CN 117070540A CN 202311103435 A CN202311103435 A CN 202311103435A CN 117070540 A CN117070540 A CN 117070540A
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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Abstract
The present invention relates to polynucleotide expression cassettes encoding VEGF antagonists, plasmids containing the polynucleotide expression cassettes, host cells, recombinant viruses, and compositions and uses thereof.
Description
Technical Field
The invention relates to the field of genetic engineering/biological medicine, in particular to a polynucleotide expression cassette for encoding anti-VEGF protein, and plasmids, host cells, recombinant viruses, compositions, uses and treatment methods containing the polynucleotide expression cassette.
Background
The formation of new blood vessels is a key place for the development and spread of many diseases. Many diseases of the eye involve angiogenesis, including age-related macular degeneration (AMD), retinal Vein Occlusion (RVO), diabetic Retinopathy (DR), and pathological myopia, among others. VEGF is a highly specific vascular endothelial cell growth factor and has the effects of promoting vascular permeability increase, extracellular matrix denaturation, vascular endothelial cell migration, proliferation, angiogenesis and the like. VEGF exerts its biological effects after binding to 3 tyrosine kinase receptors, namely vascular endothelial growth factor receptors (VEGFR-1, VEGFR-2, VEGFR-3). The extra-membrane portion of VEGFR has approximately 750 amino acid residues and consists of 7 Ig domains that resemble immunoglobulin structures. When the receptor is activated by VEGF, intracellular tyrosine kinase groups phosphorylate and cause a series of signalling, ultimately triggering angiogenesis. Studies have shown that the crystal structure of the VEGFR-1 extracellular region shows that the second Ig domain is the region that binds to the ligand. The third Ig domain of VEGFR-2 plays a role in the specificity of ligand binding.
VEGF is widely distributed in various tissues in humans and animals, and normal ocular retinal pigment epithelial cells, vascular endothelial cells and pericytes produce lower levels of VEGF. Many studies have demonstrated that pathological neovascular ocular disease can be induced if VEGF is overexpressed. Due to the importance of VEGF signaling on angiogenesis, blocking VEGF or VEGF receptors to inhibit angiogenesis has important therapeutic effects on angiogenesis-related diseases including cancer, retinal vascular disease, etc. Over the past decade, some anti-VEGF drugs for the treatment of ocular neovascular eye diseases have been born, such as pipadatinib (Macugen), bevacizumab (Avastin), ranibizumab (Lucentis), albesiex (Eylea), and the like. Although these drugs are clinically effective, they require repeated intravitreal administration due to their short half-life, which increases the burden on both the physician and patient, and thus gene therapy is a desirable therapeutic regimen.
Gene therapy achieves therapeutic effects by introducing exogenous anti-VEGF genes into target cells via appropriate vectors (e.g., adenoviruses) and by effectively expressing the corresponding agents. It aims to achieve long-term maintenance of drug efficacy through sustained expression of drugs to significantly improve physiological and psychological burden of patients. However, a significant challenge of gene therapy is how to make exogenous genes sufficiently highly express functional proteins or antibodies in target cells, which becomes critical to the success or failure of gene therapy.
Disclosure of Invention
Based on the above objects, an aspect of the present invention is to provide a polynucleotide expression cassette comprising: 1) An expression regulatory element;
2) A coding sequence encoding an anti-VEGF protein.
The polynucleotide expression cassette of the invention includes a coding sequence that encodes an anti-VEGF protein. In certain embodiments, the anti-VEGF protein comprises extracellular domain 2 of VEGFR-1.
The anti-VEGF proteins of the invention are human VEGF receptors or portions of receptors. Which is capable of recognizing and binding to VEGF ligand but does not activate the receptor complex, acts as an inhibitor, binds to ligand and prevents ligand binding to conventional receptor. For example, the anti-VEGF protein of the invention is any one of the extracellular domains 1-7 of human VEGF receptor 1, or a combination of multiple domains. For another example, an anti-VEGF protein of the invention is any one of the extracellular domains 1-7 of human VEGF receptor 2, or a combination of multiple domains. For another example, the anti-VEGF protein of the invention is any one of the extracellular domains 1-7 of human VEGF receptor 3, or a combination of multiple domains.
In some embodiments, the anti-VEGF protein of the invention is human VEGF receptor 1, and/or VEGF receptor 2, and/or a combination of one or more extracellular domains of 1-7 domains in VEGF receptor 3.
In some embodiments, the anti-VEGF proteins of the invention comprise a fusion of any of the extracellular domains of a human VEGF receptor to an immunoglobulin Fc. Preferably, the immunoglobulin Fc is a human immunoglobulin Fc fragment. Preferably, the human immunoglobulin Fc fragment is selected from one of the following fragments: igG1Fc, igG2Fc, igG3Fc, and IgG4Fc.
In certain embodiments the anti-VEGF proteins of the invention comprise extracellular domain 2 of VEGFR-1 and extracellular domain 3 of VEGFR-2.
In other embodiments, the anti-VEGF proteins of the invention comprise extracellular domain 2 of VEGFR-1, extracellular domain 3 of VEGFR-2, and an immunoglobulin Fc fragment.
In certain embodiments, the anti-VEGF proteins of the invention are aflibercept or an analog thereof (e.g., one or more amino acid mutations, substitutions, insertions, etc., based on the structure of al Bai Xi). In some specific embodiments, the anti-VEGF proteins of the invention have the amino acid sequence set forth in SEQ ID NO:1, and a polypeptide comprising the amino acid sequence of 1.
The present invention includes coding sequences encoding the above anti-VEGF proteins, which are operably linked to expression regulatory elements. In other embodiments, the coding sequence encoding an anti-VEGF protein comprises SEQ ID NO:2, and a nucleotide sequence as described in 2. In other embodiments, the coding sequence of the anti-VEGF protein of the invention is codon optimized, e.g., the coding sequence is modified by methods known in the art, e.g., using codons that are expressed at high frequencies in the target cell instead of codons that are expressed at low frequencies, so that the coding sequence of the invention has increased expression in the target cell relative to the original codon, and the expression level of the encoded product in the target cell can be increased.
The polynucleotide expression cassettes of the invention also include expression control elements, e.g., the expression control elements are promoters operably linked to coding sequences. Promoters include promoter sequences or functional fragments thereof. The promoter is specific for eukaryotic cells or mammalian cells. The promoter is selected from the group consisting of promoters of: beta-actin promoter (CBA), cytomegalovirus promoter (CMV), elongation factor 1 alpha promoter (EF 1 alpha), MNT promoter, UB6 promoter, CAG promoter, RPE65 promoter, opsin promoter, artificial splice promoter (CASS), pMNTC promoter. Preferably, the promoter comprises a CBA promoter, a CMV promoter, a CAG promoter or an EF1 a promoter. In certain embodiments.
The expression regulatory element of the present invention may further include a polyadenylation signal (polyA). Polyadenylation signals protect the mRNA from exonuclease attack and are important for transcription termination, export of mRNA from the nucleus and translation. Polyadenylation signals comprise a plurality of consecutive adenosine monophosphates, typically containing AAUAAA repeats. The polyadenylation signal of the present invention is located downstream of the coding sequence encoding the anti-VEGF protein. In some embodiments, the polyadenylation signal comprises monkey vacuolated virus 40 (SV 40), human Growth Hormone (HGH), bovine Growth Hormone (BGH), or β -globin. .
In certain embodiments, the polynucleotide expression cassettes of the invention flank functional adenovirus Inverted Terminal Repeats (ITRs) at the 5 'and 3' ends. Functional adenovirus Inverted Terminal Repeats (ITRs) refer to ITR sequences as used for integration, replication and packaging of AAV virions. The inverted terminal repeat is an adeno-associated viral ITR of serotype selected from the group consisting of AAV1ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, AAV9 ITR, AAV10 ITR, AAV11 ITR, and AAV12 ITR.
In some embodiments, the polynucleotide expression cassette comprises an RNA export signal downstream of the coding sequence encoding the anti-VEGF protein and upstream of the polyadenylation region. The RNA output signal is illustratively selected from the group consisting of a human hepatitis b virus posttranscriptional element (HPRE) sequence and a woodchuck hepatitis virus posttranscriptional element (WPRE) sequence. Preferably, the RNA export signal is selected from woodchuck hepatitis virus post-transcriptional elements (WPREs).
In some embodiments, the polynucleotide expression cassette of the invention comprises, in order from 5 'to 3':
1)AAV 5'ITR；
2) A CMV promoter sequence;
3) A coding sequence encoding an anti-VEGF protein operably linked to a promoter sequence;
4) HGH polyadenylation signal sequence;
5)AAV 3'ITR。
in some preferred embodiments, the polynucleotide expression cassette of the invention comprises SEQ ID NO:3, and a nucleotide sequence as described in 3.
In some embodiments, the polynucleotide expression cassette of the invention comprises, in order from 5 'to 3':
1)AAV 5'ITR；
2) A CMV promoter sequence;
3) A coding sequence encoding an anti-VEGF protein operably linked to a promoter sequence;
4) An SV40 polyadenylation signal sequence;
5)AAV 3'ITR。
in some preferred embodiments, the polynucleotide expression cassette of the invention comprises SEQ ID NO:4, and a nucleotide sequence as described in (a).
In some embodiments, the polynucleotide expression cassette of the invention comprises, in order from 5 'to 3':
1)AAV 5'ITR；
2) A CMV promoter sequence;
3) A coding sequence encoding an anti-VEGF protein operably linked to a promoter sequence;
4) A rabbit beta globin polyadenylation signal sequence;
5)AAV 3'ITR。
in some preferred embodiments, the polynucleotide expression cassette of the invention comprises SEQ ID NO:5, and a nucleotide sequence described in seq id no.
In some embodiments, the polynucleotide expression cassette of the invention comprises, in order from 5 'to 3':
1)AAV 5'ITR；
2) A CBA promoter sequence;
3) A coding sequence encoding an anti-VEGF protein operably linked to a promoter sequence;
4) BGH polyadenylation signal sequence;
5)AAV 3'ITR。
in some preferred embodiments, the polynucleotide expression cassette of the invention comprises SEQ ID NO:6, and a nucleotide sequence described in the following.
In some embodiments, the polynucleotide expression cassette of the invention comprises, in order from 5 'to 3':
1)AAV 5'ITR；
2) MNTC promoter sequence;
3) A coding sequence encoding an anti-VEGF protein operably linked to a promoter sequence;
4) BGH polyadenylation signal sequence;
5)AAV 3'ITR。
in some embodiments, the polynucleotide expression cassette of the invention comprises, in order from 5 'to 3':
1)AAV 5'ITR；
2) MNTC promoter sequence;
3) A coding sequence encoding an anti-VEGF protein operably linked to a promoter sequence;
4) HGH polyadenylation signal sequence;
5)AAV 3'ITR。
in some embodiments, the polynucleotide expression cassette of the invention comprises, in order from 5 'to 3':
1)AAV 5'ITR；
2) MNTC promoter sequence;
3) A coding sequence encoding an anti-VEGF protein operably linked to a promoter sequence;
4) A rabbit beta globin polyadenylation signal sequence;
5)AAV 3'ITR。
in some embodiments, the polynucleotide expression cassette of the invention comprises, in order from 5 'to 3':
1)AAV 5'ITR；
2) MNTC promoter sequence;
3) A coding sequence encoding an anti-VEGF protein operably linked to a promoter sequence;
4) An SV40 polyadenylation signal sequence;
5)AAV 3'ITR。
in some preferred embodiments, the anti-VEGF proteins of the invention have the amino acid sequence set forth in SEQ ID NO:1, and a polypeptide comprising the amino acid sequence of 1.
In some embodiments, the polynucleotide expression cassettes of the invention may also include Kozak sequences. The Kozak sequence is located between the promoter and the coding sequence for the anti-VEGF protein.
In some embodiments, the polynucleotide expression cassette of the invention comprises an RNA export signal downstream of the coding sequence encoding the anti-VEGF protein and upstream of the polyadenylation region. The RNA output signal is illustratively selected from the group consisting of a human hepatitis b virus posttranscriptional element (HPRE) sequence and a woodchuck hepatitis virus posttranscriptional element (WPRE) sequence.
It is another object of the present invention to provide a plasmid comprising the polynucleotide expression cassette of the present invention.
It is another object of the present invention to provide a host cell transfected with a polynucleotide expression cassette or plasmid of the present invention. In some embodiments, the host cell is a mammalian cell, a yeast cell, a bacterial cell, or an insect cell.
In another aspect of the invention, there is provided a vector for gene delivery comprising the nucleotide cassette of the invention. In some embodiments, the gene delivery vector is a recombinant virus comprising: capsid proteins and polynucleotide expression cassettes according to the invention. In some embodiments, the recombinant virus is a recombinant adeno-associated virus (rAAV), wherein the recombinant adeno-associated virus comprises an AAV capsid protein and a polynucleotide cassette of the invention. In some embodiments, the capsid protein is a wild-type AAV capsid protein, illustratively, the AAV serotype capsid protein is selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12. In other embodiments, the AAV capsid protein is a variant AAV capsid protein. In one embodiment, the AAV capsid variant is a variant of AAV 2.
In another aspect, the invention provides a pharmaceutical composition. The pharmaceutical composition comprises the polynucleotide expression cassette, plasmid or recombinant virus of the invention, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a recombinant virus described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is prepared as a formulation for intravitreal injection, subretinal injection, suprachoroidal injection, intravenous injection, intratumoral injection, or intramuscular injection.
In another aspect, the invention provides the use of a polynucleotide expression cassette, plasmid or recombinant virus according to the invention in the manufacture of a medicament for the treatment of a disease associated with VEGF. In certain embodiments, the VEGF-related disease is ocular neovascular disease. In certain embodiments, the ocular neovascular disorder is selected from age-related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion, macular edema due to branch retinal vein occlusion, macular edema secondary to retinal vein occlusion, polypoidal choroidal vasculopathy, wet age-related macular degeneration of very low vision, choroidal neovascularization secondary to pathological myopia.
Another aspect of the invention is to provide a method of treating a VEGF-related disease in a mammalian subject, the method comprising delivering to the eye a therapeutically effective amount of a recombinant virus or pharmaceutical composition comprising a polynucleotide expression cassette of the invention. In certain embodiments, the VEGF-related disease is ocular neovascular disease. In certain embodiments, the ocular neovascular disorder is selected from age-related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion, macular edema due to branch retinal vein occlusion, macular edema secondary to retinal vein occlusion, polypoidal choroidal vasculopathy, wet age-related macular degeneration of very low vision, choroidal neovascularization secondary to pathological myopia. In some embodiments, the recombinant viruses or pharmaceutical compositions of the invention are administered by intravitreal injection, subretinal injection, suprachoroidal injection, intravenous injection, intratumoral injection, or intramuscular injection. In some embodiments, the method of treatment comprises administering 10 6 To 10 16 vg, preferably 10 8 To 10 13 vg, more preferably 10 9 To 10 12 vg to the eye.
Drawings
FIG. 1 provides a schematic representation of a recombinant plasmid in which an exemplary polynucleotide expression cassette is positioned between Inverted Terminal Repeats (ITRs) of adeno-associated virus serotype 2 (AAV 2)
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. And the following examples are not representative of all or only of the following experiments in which the present invention was developed.
Example 1: construction of plasmids
Construction using standard recombinant DNA cloning techniques or general molecular biology techniques produced a series of polynucleotide expression cassettes containing various combinations of regulatory elements and coding sequences (table 1). Recombinant plasmids comprising the polynucleotide expression cassettes described in table 1 were then constructed and cloned in e.coli using conventional DNA recombination and cloning techniques for subsequent preparation of recombinant AAV viruses. FIG. 1 shows a schematic representation of a recombinant plasmid expression cassette of the invention, wherein an exemplary polynucleotide expression cassette is positioned between Inverted Terminal Repeats (ITRs) of adeno-associated virus serotype 2 (AAV 2).
As represented in the schematic representation of the recombinant plasmid of FIG. 1, each cassette is positioned between the Inverted Terminal Repeats (ITRs) of adeno-associated virus serotype 2 (AAV 2), and cassettes 1-4 comprise, in order from 5 'to 3', a 5'ITR, a promoter, a coding sequence encoding Abelmoschus, a polyadenylation signal sequence, and a 3' ITR. The main composition of each polynucleotide expression cassette is shown in table 1.
TABLE 1 Polynucleotide expression cassettes
| Box numbering | Enhancer/promoter/intron | Target gene | Poly A |
| 1 | CMV | Coding sequence | HGH |
| 2 | CMV | Coding sequence | SV40 |
| 3 | CMV | Coding sequence | Rabbit beta globin |
| 4 | CBA | Coding sequence | BGH |
Then, plasmids based on the above polynucleotide expression cassettes 1 to 4 were constructed, respectively designated as No.1, no.2, no.3, no.4.
Example 2: protein expression in 293 cells transiently transfected with plasmids
To assess the expression profile of each polynucleotide expression cassette or plasmid in vitro, each recombinant construct was transfected into HEK293F cells in suspension using transfection reagents. The specific method comprises the following steps: selecting the appropriate cell number (no more than P15 generation), and carefully adding 10ml DMEM (10% fbs) medium 2h before plasmid transfection without disturbing the cell monolayer; then pass through Lipofectamine TM 2000, specifically comprising: 2. Mu.g of the plasmid containing the target gene (with endotoxin removed) was added to 1mL of serum-free medium, and 5. Mu.L of LiP2000 was added to 1mL of serum-free medium; standing for 5min respectively; then lightly mixing and standing for 15min; adding the mixed solution obtained in the previous step into a cell culture dish, uniformly distributing the mixed solution at each position when adding, slightly and uniformly mixing the whole culture solution after finishing, and culturing at 37 ℃ and 5% CO 2; after 6h of transfection, the medium was carefully aspirated and replaced with fresh medium (10% FBS,1% P/S),after culturing for 48-72 h, collecting cells and cell supernatant; cell lysates containing expressed proteins were prepared by repeating freeze thawing 3 times, and the protein content was measured by ELISA, and the results are shown in table 2.
TABLE 2
Claims (10)
1. A polynucleotide expression cassette, comprising:
1) An expression regulatory element;
2) A coding sequence encoding an anti-VEGF protein; the anti-VEGF protein comprises ectodomain 2 of VEGFR1, ectodomain 3 of VEGFR 2; preferably, the anti-VEGF protein comprises ectodomain 2 of VEGFR1, ectodomain 3 of VEGFR2 and an immunoglobulin Fc fragment; more preferably, the anti-VEGF protein has an amino acid sequence set forth in SEQ ID NO. 1.
2. The polynucleotide expression cassette of claim 1, wherein the coding sequence encoding an anti-VEGF protein comprises the sequence of SEQ ID NO:2, and a nucleotide sequence as described in 2.
3. The polynucleotide expression cassette of claim 1, wherein the polynucleotide expression cassette comprises flanking Inverted Terminal Repeats (ITRs); preferably, the ITR is an adeno-associated virus ITR of serotype selected from the group consisting of AAV1ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, AAV9 ITR, AAV10 ITR, AAV11 ITR and AAV12 ITR.
4. The polynucleotide expression cassette of claim 1, wherein the polynucleotide expression cassette comprises, in order from 5 'to 3':
1)AAV 5'ITR；
2) A CMV promoter sequence;
3) A coding sequence encoding an anti-VEGF protein operably linked to a promoter sequence;
4) HGH polyadenylation signal sequence;
5) AAV 3' itrs; or alternatively
The polynucleotide expression cassette comprises, in order from 5 'to 3':
1)AAV 5'ITR；
2) A CMV promoter sequence;
3) A coding sequence encoding an anti-VEGF protein operably linked to a promoter sequence;
4) An SV40 polyadenylation signal sequence;
AAV 3' itrs; or alternatively
The polynucleotide expression cassette comprises, in order from 5 'to 3':
1)AAV 5'ITR；
2) A CMV promoter sequence;
3) A coding sequence encoding an anti-VEGF protein operably linked to a promoter sequence;
4) A rabbit beta globin polyadenylation signal sequence;
AAV 3'ITR。
5. the polynucleotide expression cassette of claim 1, wherein the polynucleotide expression cassette comprises the sequence of SEQ ID NO: 3. SEQ ID NO:4 or SEQ ID NO:5, and a nucleotide sequence described in seq id no.
6. A plasmid comprising the polynucleotide expression cassette of any one of claims 1-5.
7. A host cell comprising the polynucleotide expression cassette of any one of claims 1-5 or the plasmid of claim 6; preferably, the host cell is a mammalian cell, a yeast cell, a bacterial cell or an insect cell.
8. A recombinant virus comprising a capsid protein and the polynucleotide expression cassette of any one of the preceding claims 1-5; preferably, the recombinant virus is a recombinant adeno-associated virus; preferably, the capsid protein is a serotype capsid protein selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or a variant of any of the foregoing.
9. A pharmaceutical composition comprising the polynucleotide expression cassette of any one of claims 1-5, or the plasmid of claim 6, or the recombinant virus of claim 8 and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is an intravitreal injection, subretinal injection, intracoronary injection, intravenous injection, intratumoral injection or intramuscular injection.
10. Use of the polynucleotide expression cassette of any one of claims 1-5, or the plasmid of claim 6, or the recombinant virus of claim 8, in the manufacture of a medicament for the treatment of a VEGF-related disease, preferably an ocular neovascular disease; more preferably, the ocular neovascular disease is selected from the group consisting of age-related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion, macular edema due to branch retinal vein occlusion, macular edema secondary to retinal vein occlusion, polypoidal choroidal vasculopathy, wet age-related macular degeneration of very low vision, choroidal neovascularization secondary to pathological myopia.
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