Cell specific gene delivery system based on coronavirus Spike and hACE2 interaction mediation

Abstract

The invention relates to a cell specific gene delivery system based on the interaction mediation of coronavirus Spike and hACE2. Comprising the following steps: replacing a modified lentivirus of a VSVG envelope protein with a new coronavirus Spike protein as a gene delivery vector, wherein a cell is a cell expressing hACE2. The delivery system successfully delivers the fluorescent protein (FP) to the cells expressed by the hACE2. Besides, according to the delivery system disclosed by the invention, after tail intravenous injection of rats and mice, specific delivery of liver tissues expressing hACE2 is successfully realized, and animals subjected to injection have no immune and behavioral abnormality reaction within half a year.

Description

Technical Field

[0001] The present invention generally relates to a gene delivery system, and more specifically to a method for specifically delivering genes to cells expressing hACE2 using a modified lentiviral vector. Background Art

[0002] Lentiviral vectors are widely used in gene therapy due to their stable gene transduction efficiency and low immunogenicity. Lentiviruses enter host cells through membrane fusion mediated by fusion proteins. The traditional lentiviral vector surface fusion protein is VSVG, which recognizes low-density lipoprotein receptor (LDLR) to enter host cells. Due to the widespread expression of LDLR, VSVG envelope lentiviral vectors have a wide range of tropism and can bind to a variety of cell types, which has limitations in specific delivery. In order to improve the targeting of lentiviral vectors, researchers have been exploring new strategies to improve the specificity and efficiency of lentiviral vectors. The use of myomaker and myomerger to engineer lentiviral envelope proteins can achieve targeting to skeletal muscle. By delivering mDystrophin to skeletal muscle, the pathological manifestations and skeletal muscle function of the Duchenne muscular dystrophy (DMD) mouse model were effectively alleviated (Hindi, Petrany et al. 2023). In addition, the ability of lentiviral vectors to infect specific cell types can be enhanced by engineering design through ligand-receptor interactions (Yu, Shi et al. 2022). Despite significant progress, there is still an urgent need to optimize viral vectors to expand tissue and organ tropism, improve transduction efficiency, and minimize potential immunotoxicity and genotoxicity. Summary of the invention

[0003] The object of the present invention is to provide a gene delivery system, which is at least capable of using a modified lentiviral vector to perform specific gene delivery to cells expressing hACE2.

[0004] According to one aspect of the present invention, a cellular gene delivery system is provided, comprising a modified lentivirus in which the VSVG envelope protein of the SARS-CoV-2 is replaced by the Spike protein of the SARS-CoV-2 as a gene delivery vector, wherein the cells are cells expressing hACE2.

[0005] The present invention modifies the lentiviral vector (S-LV) by replacing the original VSVG protein of the lentivirus with the Spike protein (S protein) of the new coronavirus (SARS-CoV-2), and specifically recognizes and binds to the surface of cells expressing human angiotensin-converting enzyme 2 (hACE2) through this modified lentiviral vector, thereby achieving specific delivery to these cells. According to the delivery system of the present invention, the Spike protein used is preferably mutated, and the mutation site is D614G of the Spike protein. The mutation site can enhance the binding affinity of the Spike protein to the hACE2 receptor, thereby improving the infection efficiency of the virus.

[0006] According to the delivery system of the present invention, the Spike protein used is preferably truncated at the C-terminus by 19 amino acids (D614G+C19del), which can affect the stability and functionality of the Spike protein and further regulate the interaction between the virus and the host cell.

[0007] According to the delivery system of the present invention, it is further preferred to use the Spike protein in combination with the VSVG mutant (K47Q+R354A). Since VSVG loses its ability to bind to LDLR after mutation but retains its ability to promote fusion of lentivirus, the infection efficiency can be further improved while retaining the characteristics of targeting.

[0008] According to another aspect of the present invention, a gene delivery method is also provided, comprising using the modified lentiviral vector to deliver genes to cells expressing hACE2.

[0009] According to the present invention, the cells expressing hACE2 may be lung cells or liver cells of rodents.

[0010] The delivery system of the present invention successfully delivered fluorescent protein (FP) to hACE2-expressing cells and hACE2 transgenic mouse lung tissue. In addition, the delivery system of the present invention, especially after tail vein injection in rats, successfully achieved specific delivery to liver tissue expressing hACE2, and the injected animals had no immune and behavioral abnormalities within half a year. This further illustrates the safety and effectiveness of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Figure 2 is a diagram showing the working principle of the gene delivery system according to the present invention.

[0012] Figure 2A This is a diagram showing the infection of red fluorescent protein in cells after the lentiviral vector according to Example 1 of the present invention is co-cultured with different cell lines; Figure 2BThis is a diagram showing the specific infection of red fluorescent protein in the lungs of mice after nasal drops of the lentiviral vector according to Example 1 of the present invention were administered to hACE2 mice.

[0013] Figure 3A A schematic diagram of the mutation and truncation sites of the Spike protein of the novel coronavirus according to Example 2 of the present invention; Figure 3B The flow cytometry results of different modified lentiviral vectors in Example 2 of the present invention infected 293FT cells.

[0014] Figure 4A The expression of red fluorescent protein in different tissues after S-LV 3.0 in Example 2 of the present invention was injected into mice expressing hACE2 in the liver via the tail vein; Figure 4B The expression of red fluorescent protein in different tissues after S-LV 3.0 in Example 3 of the present invention was injected into rats expressing hACE2 in the liver via the tail vein; Figure 4C The expression levels of cytokines TNF-α, IL-1β and INF-β in serum of mice expressing hACE2 in the liver were measured six months after S-LV 3.0 in Example 3 of the present invention was injected into the liver of mice via the tail vein.

[0015] Figure 5A The expression of hPCSK9 in different tissues after S-LV 3.0-hPCSK9 in Example 4 of the present invention was injected into rats expressing hACE2 in the liver via the tail vein; Figure 5B The total cholesterol level in serum of rats expressing hACE2 in the liver after S-LV 3.0-hPCSK9 was injected into the rat tail vein according to Example 4 of the present invention; Figure 5C The triglyceride level in serum of rats expressing hACE2 in the liver after S-LV 3.0-hPCSK9 was injected into the rat tail vein according to Example 4 of the present invention; Figure 5D The level of low-density lipoprotein in serum after S-LV 3.0-hPCSK9 in Example 4 of the present invention was injected into rats expressing hACE2 in the liver via the tail vein; Figure 5E This is the level of high density lipoprotein in serum after S-LV 3.0-hPCSK9 in Example 4 of the present invention was injected into rats expressing hACE2 in the liver via the tail vein. DETAILED DESCRIPTION

[0016] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0017] Figure 1 A diagram showing the working principle of the gene delivery system according to the present invention is shown, in which a lentiviral vector containing the novel coronavirus Spike protein replacing VSVG is specifically delivered to hACE2-expressing cells.

[0018] Example 1: Construction of a lentiviral vector (S-LV) in which the SARS-CoV-2 Spike protein replaces VSVG and verification of its delivery specificity in hACE2-expressing cells and mice.

[0019] 1. Vector construction: First, insert the Spike protein gene into the lentiviral envelope plasmid to replace the original VSVG gene. Cloning is performed through restriction enzyme sites to ensure the correct insertion and direction of the Spike protein gene. DNA sequencing technology is used to verify the sequence of the inserted Spike protein gene to ensure that there are no mutations and insertion errors.

[0020] 2. Virus packaging and titer determination: The three-plasmid packaging system was used to package the lentivirus. The packaging plasmid encoding the lentiviral core protein, the transfer plasmid expressing the red fluorescent protein mRFP, and the envelope plasmid containing the VSV-G or Spike protein sequence were co-transfected into 293FT cells. The cell supernatant was collected 48h and 72h after transfection, and the lentivirus was obtained by ultracentrifugation at 100,000×g. By optimizing the transfection conditions, including the selection of transfection reagents, the ratio of DNA to transfection reagents, and the culture conditions after transfection, a high-titer lentiviral vector was obtained, and the titer of the lentiviral vector was determined by the qRT-PCR method.

[0021] 3. Verification at the cell level: The constructed VSVG-LV and S-LV were co-cultured with different cell lines. After 72 hours of infection, the infection efficiency and specificity of the lentiviral vector were detected by immunofluorescence staining. Figure 2A It was shown that VSVG-LV can widely infect a variety of cell lines, while S-LV specifically delivers mRFP to African green monkey kidney cells Vero E6 and human liver cancer cell lines HepG2 that express hACE2, but does not infect rat brain glioma cell lines C6. After further transfection of a plasmid expressing hACE2 in C6 cells, the delivery of red fluorescent protein was observed.

[0022] 4. Verification of in vivo delivery in mice: Using a mouse model expressing hACE2, the S-LV vector was used for intranasal infection. The delivery efficiency and specificity of the viral vector in the mouse lung tissue were detected by in vivo imaging. Figure 2B It was shown that S-LV successfully delivered mRFP to the lung tissue of mice expressing hACE2, while the lung tissue of mice not expressing hACE2 was not infected.

[0023] Example 2: Verification of infection efficiency in cells after optimizing lentiviral vector by mutation of Spike protein 1. Spike protein mutation: (1) Mutation design and plasmid construction: At the 614th amino acid position of the Spike protein, glycine (G) was replaced by aspartic acid (D) (D614G) to enhance the binding affinity of the Spike protein to the hACE2 receptor; the Spike gene of the lentiviral envelope plasmid was subjected to the D614G point mutation by site-directed mutagenesis. First, a pair of primers containing the D614G mutation site was designed, and the envelope plasmid containing the Spike sequence was amplified using the primers to generate DNA containing the mutation site. The DNA was then digested using the DpnⅠ restriction endonuclease, which can recognize and cut methylated DNA, thereby digesting the template plasmid. The digested product was transformed into Escherichia coli for sequence verification to obtain the D614G mutant plasmid.

[0024] In addition, the 19 amino acids at the C-terminus of the Spike protein were deleted (C19del) to affect the stability and functionality of the Spike protein; the 19 amino acids at the C-terminus were truncated. The Spike envelope plasmid containing the D614G mutation was used as a template, and primers containing C19del were designed for PCR amplification. After digestion with DpnⅠ restriction endonuclease, the plasmid was transformed into Escherichia coli for sequence verification, thereby obtaining the D614G+C19del mutant plasmid. Figure 3A The Spike protein domains as well as mutation and truncation sites are indicated.

[0025] VSVG protein specifically binds to LDLR through its CR2 and CR3 domains. K47 and R354 are the key residues for VSVG protein to bind to the LDL-R CR domain. K47Q and R354 mutations will destroy this binding ability, but do not affect the fusion activity of VSVG, which can still mediate the fusion of virus and cell membrane. Using the envelope plasmid containing VSVG as a template, primers for K47Q and R354 were designed, PCR amplification was performed, and the residues were digested with DpnⅠ restriction endonuclease and transformed into Escherichia coli for sequence verification to obtain mutant plasmids.

[0026] (2) Virus packaging: The modified lentiviral vector was packaged using a three-plasmid system. The packaging plasmid encoding the lentiviral core protein, the transfer plasmid expressing the red fluorescent protein mRFP, and the envelope plasmid containing the D614G+C19del mutant Spike protein sequence were co-transfected into 293FT cells. The cell supernatant was collected 48h and 72h after transfection, and the S-LV 2.2 modified lentivirus was obtained by ultracentrifugation at 100,000×g.

[0027] The packaging plasmid encoding the lentiviral core protein, the transfer plasmid expressing the red fluorescent protein mRFP, the envelope plasmid containing the D614G+C19del mutation of the Spike protein sequence, and the VSVG envelope plasmid containing the K47Q+R354 mutation were co-transfected into 293FT cells. The cell supernatant was collected at 48h and 72h, and the S-LV 3.0 modified lentivirus was obtained by ultracentrifugation at 100000×g. The correct construction and high-titer production of the mutant vector were ensured by sequence verification and virus titer determination.

[0028] 2. Verification of infection efficiency: The modified lentiviral vectors were co-cultured with 293FT cells expressing or not expressing hACE2, and the infection efficiency was detected by flow cytometry. The infection efficiency of S-LV 2.2, S-LV 3.0 and the original S-LV vector was compared to evaluate the effect of mutations on infection efficiency and specificity. Figure 3B The flow cytometry results of different modified lentiviruses infecting 293FT cells are shown. The results show that the modified lentiviral vector specifically infects hACE2-expressing cells, and obvious mRFP-positive cells can be detected, while 293FT cells that do not express hACE2 are not infected; in contrast, VSVG-LV has no difference in infection of the two cells, showing non-specific infection characteristics. The infection efficiency of S-LV 3.0 in 293FT cells expressing hACE2 is significantly higher than that of S-LV and S-LV2.2. By comparing these three modified lentiviral vectors, the effects of mutations on infection efficiency and specificity can be evaluated, which is of great significance for viral vector design, etc.

[0029] Example 3: Evaluation of the delivery specificity of S-LV 3.0 in mice and rats expressing hACE2 in the liver 1. Animal model construction: Mouse and rat models with liver-specific expression of hACE2 were constructed by genetic engineering technology. The delivery efficiency and specificity of the viral vector in the liver of mice and rats were evaluated by tail vein injection of S-LV 3.0 carrying red fluorescence.

[0030] 2. Specificity evaluation: Using a mouse and rat model with liver-specific expression of hACE2, S-LV 3.0 was injected through the tail vein. Three weeks after infection, the expression of red fluorescent protein in mouse and rat tissues was detected by fluorescence imaging technology to evaluate the virus delivery efficiency and specificity. Figure 4A The expression of red fluorescent protein in various tissues of mice was shown. The results showed that red fluorescent signals were observed in the liver of mice expressing hACE2, while there were no red fluorescent signals in the lungs, kidneys, spleen and heart. There were no positive signals in various tissues of mice that did not express hACE2, indicating the specificity of S-LV 3.0 delivery to mouse liver cells.

[0031] Figure 4B The red fluorescence signals in various tissues of rats were shown. The results showed that red fluorescence signals were observed in the liver of rats expressing hACE2, while there were no positive signals in various tissues that did not express hACE2, indicating the specificity of S-LV 3.0 delivery to rat liver cells.

[0032] 3. Safety assessment: Six months after the tail vein injection of mice, the animals showed no abnormalities in behavior and lifespan. The safety of S-LV 3.0 was further evaluated by the levels of cytokines in serum. The levels of cytokines in mouse serum were quantitatively analyzed by enzyme-linked immunosorbent assay (ELISA), including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interferon-β (INF-β). Figure 4C The ELISA test results are shown, in which the expression levels of TNF-α, IL-1β and INF-β were all within the normal range without significant abnormalities, which further confirmed the safety of the S-LV 3.0 modified lentiviral vector.

[0033] Example 4: Application of S-LV 3.0 to Specific Delivery of hPCSK9 in Rats Expressing hACE2 in the Liver 1. Rat tail vein injection: (1) Plasmid construction and viral packaging: The target gene fragment was amplified by PCR technology, and the amplified hPCSK9 gene fragment and the lentiviral transfer plasmid were double-digested with restriction endonucleases. The gene fragments after digestion were purified and connected to construct recombinant plasmids. The connected recombinant plasmids were transformed into competent Escherichia coli, and positive clones were screened and sequenced to obtain lentiviral transfer plasmids with the hPCSK9 gene correctly inserted. The packaging plasmid encoding the lentiviral core protein, the transfer plasmid expressing hPCSK9, and the envelope plasmid containing the Spike protein sequence with D614G+C19del mutations and the VSVG envelope plasmid containing K47Q+R354 mutations were co-transfected into 293FT cells to obtain the S-LV 3.0 modified lentivirus expressing hPCSK9.

[0034] (2) Tail vein injection: The hPCSK9-expressing S-LV 3.0 vector was injected into rats expressing hACE2 in the liver via the tail vein. The delivery efficiency and specificity of the viral vector in the rat liver were evaluated 3 weeks after infection.

[0035] 2. Detection of hPCSK9 expression level: The expression level of hPCSK9 in rat liver was detected by immunofluorescence staining. The expression level of hPCSK9 in the liver of rats injected with S-LV 3.0 vector expressing hPCSK9 was compared with that in the control group to evaluate the delivery efficiency and specificity of the viral vector. Figure 5AThe results showed that after S-LV 3.0 injection, significant hPCSK9 expression was observed in the liver of rats expressing hACE2, indicating that the S-LV 3.0 vector can effectively deliver the hPCSK9 gene into rat liver cells and achieve its expression. The hPCSK9 expression in the liver of rats after S-LV injection was significantly lower than that in S-LV 3.0, and no hPCSK9 expression was observed in the liver of rats in the control group, further confirming the specificity of the S-LV 3.0 vector.

[0036] 3. Delivery effect evaluation: The delivery effect of S-LV 3.0 vector expressing hPCSK9 was evaluated by detecting the changes in the levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) in rat serum. The results showed that compared with the control group, the rats injected with S-LV 3.0 vector had a lower level of TC ( Figure 5B )、TG( Figure 5C )、LDL( Figure 5D ) and HDL ( Figure 5E ) levels were significantly increased, and this trend of change indicated that the high expression of hPCSK9 after the delivery of S-LV 3.0 vector directly affected blood lipid metabolism.

[0037] In summary, by modifying the envelope protein on the surface of the lentiviral vector and replacing it with the new coronavirus Spike protein, specific delivery to hACE2-expressing cells can be achieved. Furthermore, by truncating the D614G point mutation of the Spike protein and the 19 amino acids at the C-terminus, combined with the VSVG mutant (K47Q+R354A), not only can the editing efficiency of the viral vector be improved, but also its targeting specificity can be retained. In addition, the application of the modified viral vector in animal models, especially after tail vein injection in rats, can achieve specific delivery to liver tissue expressing hACE2, and the injected animals have no abnormal immune and behavioral reactions within half a year.

Claims

1. A cellular gene delivery system, comprising a modified lentivirus in which the VSVG envelope protein of the novel coronavirus Spike protein is replaced as a gene delivery vector, wherein the cell is a cell expressing hACE2.

2. The delivery system according to claim 1, wherein the Spike protein used is mutated, and the mutation site is D614G of the Spike protein.

3. The delivery system according to claim 2, wherein the Spike protein used is further truncated at the C-terminus by 19 amino acids. The delivery system according to claim 3 , wherein the Spike protein is also used in combination with a VSVG mutant.

5. A gene delivery method, comprising delivering genes to cells expressing hACE2 using the modified lentiviral vector according to any one of claims 1 to 4.

6. The method according to claim 5, wherein the cells expressing hACE2 are lung cells or liver cells of rodents.

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