Construction of engineered exosome targeted delivery system EMT-Cas12a and application of engineered exosome targeted delivery system EMT-Cas12a in treatment of AIDS

Abstract

The invention discloses an engineered exosome targeting delivery system (EMT-Cas12a) and application thereof in AIDS (acquired immune deficiency syndrome) treatment. The targeting delivery system is loaded with a functional component for cutting HIV (human immunodeficiency virus) previrus DNA (deoxyribonucleic acid) on the basis of CRISPR-Cas12a (clustered regularly interspaced short palindromic repeats). According to the targeting delivery system, a nano antibody Nb1 of targeting CD4 is expressed on the surface of EMT-Cas12a, so that the efficiency of targeting delivery of CRISPR-Cas12a to CD4 + cells is improved. According to the targeted delivery system, a multi-crRNA series strategy is designed, a conserved region shared by a targeted HIV-1 B subtype and Chinese epidemic strains (07BC, 08BC and B ') is conserved, the crRNA and Cas12a mRNA form a functional component for cutting HIV previrus DNA, the functional component and Cas12a mRNA are jointly packaged into EMT-Cas12a, and the risk of virus escape is reduced. According to the targeting delivery system, in cells cultured in vitro (cell lines infected with HIV-1 and PBMC derived from HIV-1 positive donors), and in vivo tests of intravenous injection of EMT-Cas12a in humanized mice infected with HIV-1, good biological safety characteristics and excellent antiviral efficacy are proved.

Description

Technical Field

[0001] This invention relates to the field of targeted delivery technology, and in particular to the construction of an engineered exosome targeted delivery system EMT-Cas12a and its application in the treatment of AIDS. Background Technology

[0002] Human immunodeficiency virus (HIV), the virus that causes AIDS, is a retrovirus that infects human CD4+ cells, gradually weakening the human immune system and increasing the risk of opportunistic infections and cancer development. HIV is transmitted through specific bodily fluids, primarily blood, semen, vaginal secretions, rectal secretions, and breast milk. The most common routes of transmission are unprotected sex, sharing HIV-contaminated needles or syringes (e.g., during drug use), mother-to-child transmission (during pregnancy, childbirth, or breastfeeding), and transfusion of contaminated blood or blood products (which is now very rare under strict screening). HIV can integrate its proviral DNA into the host cell genome, using the host cell to replicate and continuously destroying CD4+ cells, leading to a gradual weakening of the patient's immune system. Without effective antiretroviral therapy (ART), after a latency period that can last for years or even longer (during which the infected person may not show obvious symptoms but is still infectious), the patient's immune system will gradually collapse, progressing to the latest stage, AIDS. Patients at this stage are highly susceptible to various opportunistic infections (such as tuberculosis, pneumonia, and fungal infections) and certain types of cancer (such as Kaposi's sarcoma and lymphoma), which can ultimately be life-threatening. Based on the genetic structure, immune response, and geographical distribution of HIV, it can be classified into human immunodeficiency virus type I (HIV-1) and human immunodeficiency virus type II (HIV-2). HIV-1 and HIV-2 both belong to the retrovirus genus, the lentivirus group of primates, with HIV-1 being the dominant group. The currently available standard treatment for HIV-1 is combination antiretroviral therapy (cART).

[0003] CAR-T therapy works by combining at least three antiretroviral drugs with different mechanisms of action (such as nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), and integrase strand transfer inhibitors (INSTIs)) to synergistically inhibit viral replication across multiple targets. While currently available treatments (mostly prescribed in combination) can effectively suppress HIV replication, often reducing viral load to undetectable levels, they do not eradicate the virus. Lifelong treatment is required to maintain viral suppression, and viral rebound is rapid upon discontinuation, posing a life-threatening risk. CAR-T therapy is generally well-tolerated, but complications such as long-term toxicity, drug resistance, and adherence issues still exist. Furthermore, CAR-T therapy has a dual effect on the immune system: on the one hand, it reduces the autoimmune responses associated with chronic HIV infection, manifesting as immune activation, chronic inflammation, and subsequent tissue damage; on the other hand, in the context of immune reconstitution, CAR-T therapy may induce or exacerbate existing autoimmune diseases, potentially leading to autoimmune thyroiditis, autoimmune hepatitis, systemic sclerosis, or lupus-like syndrome.

[0004] Therefore, there is a need to develop novel therapies with curative potential or those capable of achieving sustained viral suppression and immune reconstitution. Permanently eliminating the latent HIV reservoir (which mediates viral rebound and transmission risks) would fundamentally address the limitations of existing treatments that only aim at viral control. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an engineered exosome targeted delivery system, a method for constructing the system, and its application in the treatment of AIDS, so as to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a crRNA that specifically targets conserved gene regions of HIV, wherein the sequence of the crRNA is selected from at least one of SEQ ID No. 1-7.

[0007] In a second aspect, the present invention provides an engineered exosome-based targeted delivery system, wherein the EMT-Cas12a nanobody Nb1 targeting CD4 is expressed on its surface; the targeted delivery system is loaded with a functional component based on CRISPR-Cas12a cleavage of HIV proviral DNA, the functional component including the aforementioned crRNA sequence. The targeted delivery system is obtained by transfecting HEK293F cells with three plasmids, including pCD63-L7Ae plasmid, pLamp2b-Nb1 plasmid, and pC / D-Cas12a-crRNA plasmid, wherein the pC / D-Cas12a-crRNA plasmid includes the aforementioned crRNA sequence.

[0008] It should be noted that CRISPR-Cas12a (also known as Cpf1, derived from the genera Prevotella and Francisella) belongs to the Class 2 V CRISPR / Cas system, which is more efficient and accurate than the original CRISPR-Cas9 system.

[0009] Common delivery methods for CRISPR systems in human T cells include electroporation, lentiviral vectors, adeno-associated viruses (AAVs), and lipid nanoparticles (LNPs). However, all of these delivery methods have key limitations, necessitating the development of novel delivery vectors with superior targeting specificity, higher loading capacity, transduction efficiency, and safety profiles. Exosomes are a subgroup of extracellular vesicles (EVs) secreted by most cells in the body. Based on their biogenesis, EVs can be divided into three subgroups: exosomes (30-150 nm in diameter), microvesicles (150-1000 nm), and apoptotic bodies (50-2000 nm). Due to their low immunogenicity, diverse drug loading capacity, excellent biocompatibility, strong in vivo stability, and ability to penetrate biological barriers, exosomes have become very important nanocarriers.

[0010] In this invention, the pCD63-L7Ae plasmid expresses a fusion protein of the exosome tetraspanic membrane marker protein CD63 and the archaea ribosomal protein L7Ae, with a dissociation constant KD=0.8nM, forming an exosome membrane-anchored RNA packaging device. pLamp2b-Nb1 plasmid: a fusion protein encoding the CD4-targeting nanobody Nb1 and the lysosome-associated membrane protein Lamp2b, whose transmembrane region contains pH-sensitive helices (AA 1-40) to facilitate endosome escape; pC / D-Cas12a-crRNA plasmid: carries a functional element containing a Box C / D kink-turn motif, enabling the self-assembly of the Cas12a mRNA and crRNA complex.

[0011] A third aspect of the present invention provides a method for preparing the engineered exosome targeted delivery system described above, comprising: (1) pCD63-L7Ae plasmid, pLamp2b-Nb1 plasmid and pC / D-Cas12a-crRNA plasmid were co-transfected into cells, cultured, and cell supernatant was collected. The pC / D-Cas12a-crRNA plasmid contained the sequence of the crRNA described above. (2) The cell supernatant was initially screened using a hollow fiber membrane tangential flow system to remove free proteins and cell debris; then it was finely sorted by size exclusion chromatography to collect the exosome eluent; the exosome eluent was then centrifuged at high speed to obtain engineered exosomes.

[0012] Furthermore, in step (1), the cells are HEK293F cells, and the culture time is 72h.

[0013] Furthermore, in step (2), the molecular weight cutoff of the hollow fiber membrane tangential flow system is 280~320 kDa, and the initial screening injection pressure is 0.15 MPa.

[0014] Furthermore, in step (2), when performing fine sorting by size exclusion chromatography, a chromatography medium with an average molecular weight exclusion value of 700 kDa is selected, the injection flow rate is 90~150 cm / h, and the column pressure difference is less than 2.5 bar.

[0015] Furthermore, in step (2), the ultracentrifugation is performed at 2~8℃ with an ultracentrifugation rate of 90000~110000×g for 3~4 h.

[0016] The present invention also provides the application of the engineered exosome targeted delivery system described above in the preparation of drugs for treating HIV infection.

[0017] The present invention also provides a drug comprising the engineered exosome targeted delivery system described above, and a pharmaceutically acceptable carrier.

[0018] The beneficial effects of this invention include at least the following: The engineered exosome-targeted delivery system constructed in this invention uses CD4-targeting nanobodies combined with exosome delivery characteristics to deliver a multi-crRNA array and Cas12a mRNA to CD4+ cells. This cleaves the HIV proviral DNA in CD4+ cells, thereby destroying the HIV proviral DNA and achieving the goal of curing HIV infection. It has the following advantages: 1. Using exosomes as a delivery tool for CRISPR Cas12a ensures good safety; 2. Using CD4-targeting nanobodies achieves specific targeted delivery of CRISPR Cas12a; 3. The use of a multi-crRNA array targeting the conserved region of HIV effectively prevents HIV escape and exhibits excellent antiviral efficacy. Attached Figure Description

[0019] Figure 1 This describes the localization of the crRNA target sequence on the HIV-1 genome.

[0020] Figure 2 Establishment of a high-throughput visual screening method for crRNA in vitro; (A) Schematic diagram of screening crRNA using fluorescence-based reporter gene experiments; (B) Validation of the specificity of the crRNA visual screening system.

[0021] Figure 3 To enable high-throughput visualization screening of anti-HIV-1 crRNA in vitro.

[0022] Figure 4 A schematic diagram of anti-HIV EMT-Cas12a targeting CD4+ cells.

[0023] Figure 5 This is a schematic diagram of the exosome purification process.

[0024] Figure 6 The results of the analysis of physicochemical properties and surface markers of EMT-Cas12a are as follows: (A) Morphological characteristics of EMT-Cas12a exosomes under transmission electron microscopy; (B) Histogram of particle size distribution frequency of EMT-Cas12a determined by NTA; (C) Characteristic protein markers of EMT-Cas12a exosomes analyzed by Western blot: CD63-L7Ae fusion protein, TSG101 and CD81; (D) Flow cytometry analysis of EMT-Cas12a by magnetic bead trapping: CD63, CD9, CD81.

[0025] Figure 7 The results show the purity of the purified EMT-Cas12a engineered exosomes; (A) Nanoflow cytometry analysis of membrane-stained exosomes; (B) Western blot detection of exosome marker proteins and organelle proteins.

[0026] Figure 8 To analyze the surface markers (CD63, CD9, CD81), Nb1, and nucleic acid content of EMT-Cas12a and blank control exosomes using nanoflow cytometry.

[0027] Figure 9 L7Ae and C / Dbox significantly enhanced the loading and delivery of Cas12a mRNA in EMT-Cas12a; (A) Real-time quantitative analysis of exosomal mRNA content. Compared with the control group, L7Ae-C / D box significantly increased the amount of Cas12a mRNA entering exosomal cells. Error bars represent three independent SEMs; (B, C) Immunoblot analysis of Cas12a expression after incubation with Jurakat cells using Ctrl EXO, Cas12a EXO without L7Ae, Cas12a EXO without C / Dbox, and EMT-Cas12a. Data are expressed as mean SD (n = 3). One-tailed t-tests were used, and P < 0.05 was considered statistically significant.

[0028] Figure 10 Enhanced CD4 targeting mediated by Nb1 + T cell uptake efficiency of EMT-Cas12a; (A) PBMCs were incubated with EMT-Cas12a (ZsGreen-labeled), target-free Cas12a-crRNA exosomes (ZsGreen-labeled), and Blank EXO (non-fluorescently labeled), and the nuclei were reverse-stained with DAPI and analyzed by confocal microscopy; (B, C) Flow cytometry analysis was performed to analyze Nb1-mediated EMT-Cas12a specific targeting of CD4+ cells in PBMCs. Data are expressed as mean SD (n = 3). Statistical significance was determined using a one-tailed t-test, **p = 0.0030.

[0029] Figure 11To test the antiviral activity and Cas12a delivery capacity of single or multi-stranded crRNA arrays in Jurkat cells; (A) Schematic diagram of in vitro antiviral activity study of EMT-Cas12a in Jurkat cells; (B) Analysis of antiviral activity of sicr-EMT-Cas12a, with the mean NanoLuc level of uninfected Jurkat cells (blank) as the baseline; (C) Fluorescence reporter analysis of crRNA arrays; (D) Evaluation of delivery efficiency of EMT-Cas12a, with protein extracted from Jurkat cells co-incubated with EMT-Cas12a for 48 hours for experimental analysis, using GAPDH as an internal control; (E) In vitro processing of pre-crRNA by Cas12a, lanes 1-2: RNAladders, lane 3: Pre-crRNA incubated with Cas12a, showing 41 nt and 43 nt products (mature crRNA) and the remaining uncleaved pre-crRNA. (245nt), Lane 4: Untreated pre-crRNA control, confirming no spontaneous degradation; Lane 5: Synthetic mature crRNA control; (F) Antiviral activity analysis of multi-strand crRNA array EMT-Cas12a, with the mean NanoLuc level of uninfected Jurkat cells (blank) as the baseline; Real-time qPCR detection of HIV-1 Gag RNA (G) and DNA (H) levels in EMT-Cas12a-treated cells, with signals normalized to GAPDH mRNA and β-globulin DNA levels, respectively. Data are expressed as SD mean (n = 3) (ND = below the qPCR detection limit). Statistical significance was determined by one-way ANOVA, ****p<0.0001.

[0030] Figure 12For the analysis of the targeting and off-target activities and cytotoxicity evaluation of EMT-Cas12a; (A) HIV-1 DNA indels induced by Cas12a-P7 / T1 crRNA in HIV-1 infected Jurkat cells, the frequency of various insertions and deletions and the distribution of different mutation types (base substitution, deletion and delins) were studied. Next-generation sequencing was performed on genomic DNA extracted from Jurkat cells treated with Array3 or Array5 EMT-Cas12a once. The sequence marked "reference" corresponds to the homologous region of NL4-3 strain, and the green bars indicate the binding position of crRNA on the DNA fragment; (B) Computer prediction of anti-HIV crRNA off-target sites and CFD scores was performed using the GRCh38 reference genome; (C) The off-target activity of anti-HIV crRNA was analyzed by T7 endonuclease I method. DNA of HIV-infected cells treated with EMT-Cas12a was analyzed. Five days later, primers flanking the off-target site were used to amplify the PCR product of the predicted off-target site (top figure), and off-target activity was detected by gel electrophoresis (bottom figure). Lane M is the DNA Ladder; (D) Alamar blue assay was used to evaluate the cytotoxicity of EMT-Cas12a to the Jurkat cell line. Jurkat cells were treated with EMT-Cas12a every two days and cultured for 14 days.

[0031] Figure 13 Evaluation of intracellular targeting activity of Array3 or Array5-EMT-Cas12a; next-generation sequencing of genomic DNA extracted from Jurkat cells after a single treatment with Array3-EMT-Cas12a or Array5-EMT-Cas12a. Sequences marked with "reference" correspond to homologous regions of NL4-3 strain. Green bars correspond to the binding sites of crRNA on DNA fragments. (A) Sequencing analysis of HIV-1 DNA insertion / deletion mutations induced by Cas12a in the L4 targeting region; (B) Sequencing analysis of HIV-1 DNA insertion / deletion mutations induced by Cas12a in the G6 targeting region; (C) Sequencing analysis of HIV-1 DNA insertion / deletion mutations induced by Cas12a in the P88 targeting region; (D) Sequencing analysis of HIV-1 DNA insertion / deletion mutations induced by Cas12a in the G16 targeting region; (E) Sequencing analysis of HIV-1 DNA insertion / deletion mutations induced by Cas12a in the E1 targeting region.

[0032] Figure 14Evaluation of intracellular targeting activity of G16-EMT-Cas12a; Genomic DNA was extracted from HIV-1-infected Jurkat cells treated with G16-EMT-Cas12a and analyzed by next-generation sequencing. The green graphic represents the binding site of crRNA on the DNA fragment. (A) Insertion-deletion mutation in the targeting region G16; (B) Insertion-deletion mutation in the non-targeting region P7; (C) Insertion-deletion mutation in the non-targeting region T1.

[0033] Figure 15 EMT-Cas12a inhibition of HIV improved the survival rate of PBMC humanized mice; (A) Schematic diagram of the experimental procedure for testing EMT-Cas12a control of HIV-1 infection in PBMC humanized mice; (B, C) Real-time monitoring of NanoLuc expression (threshold: >1×10⁻⁶) 5 (Photons / second / square centimeter) The study investigated the dynamic changes of HIV-1 infection in PBMC humanized mice treated with different EMT-Cas12a regimens (crRNA-free, T1, L4, Array1, Array4, Array5). NanoLuc expression was measured using the IVIS system (B) and quantitatively analyzed using the Aura program (C). Bioluminescent signals were displayed in pseudocolor, and each data point represents the mean ± standard deviation (n=3). (D) Kaplan-Meier survival curves show the median survival under different EMT-Cas12a treatment regimens.

[0034] Figure 16Multi-level antiviral analysis of EMT-Cas12a in vivo: immunological assessment, molecular virological validation, and in vivo uptake analysis; (A, B) Flow cytometry analysis of CD4+ and CD8+ T cells in humanized mouse models of PBMCs treated with different methods. PBMCs isolated from mouse blood were stained with anti-CD4 APC and anti-CD8 BV421 antibodies. Data are expressed as mean ± standard deviation (n = 3); (C) Plasma p24 concentration in humanized mouse models of PBMCs treated with different methods was detected by ELISA. Data are expressed as mean ± standard deviation (n = 3). Statistical significance was determined using a t-test. Compared with the group without crRNA: T1 group *p = 0.0003, L4 group *p = 0.0169, Array1 / Array4 / Array5 groups ****p<0.0001; Compared with T1 group: Array1 group #p = 0.0165, Array4 group ##p = 0.0080, Array5 group #p = 0.0337; (D) Determination of viral infectivity in mouse plasma: Mouse plasma was diluted 1:10 (0.1-fold dilution) and then used to infect C8166 cells. NanoLuc luciferase activity was measured 72 hours after infection. Data are expressed as mean ± standard deviation (n = 3). One-way ANOVA was used to determine statistical significance. Compared with the group without crRNA: T1 group *p = 0.0173, L4 group *p = 0.0169, Array1 group *p = 0.0168, Array4 group *p = 0.0168, Array5 group *p = 0.0169; Compared with T1 group: Array1 group ##p = 0.0036, Array4 group ##p = 0.0032; The levels of HIV-1 Gag RNA in mouse spleen (E), HIV-1 Gag RNA in blood (F), and Viagra in spleen were detected by real-time quantitative PCR (qPCR). RNA levels (G) and signal values ​​were standardized to human GAPDH mRNA levels. Data are expressed as mean ± standard deviation (n = 3). One-way ANOVA was used to determine statistical significance. Compared with the group without crRNA: blood Gag RNA—T1 and Array1-5 groups ****p<0.0001; spleen Gag RNA—T1 group**p=0.0057, L4 group**p=0.0043, Array1 group**p=0.0040, Array4 group**p=0.0039, Array5 group**p=0.0039; spleen Vif RNA—T1 and Array1-5 groups ****p<0.0001; (H,I) Analysis of EMT-Cas12a uptake in vivo. Flow cytometry results showed that tail vein injection of zsGreen-labeled EMT-Cas12a (H,I) Figure 16 H (left image, zsGreen group) and unlabeled exosomes ( Figure 16 (Figure H, right, Ctrl group) The proportion of zsGreen positive CD4+ T cells in peripheral blood 12 hours later. Data are expressed as mean ± standard deviation (n = 3).

[0035] Figure 17To verify the antiviral activity of EMT-Cas12a in in vitro cultured HIV-positive donor peripheral blood cells; (A, G) Frequency of CD4+ and CD8+ T cells in PBMCs of different treatment groups (HIV-positive donor 1, HIV-positive donor 2), PBMCs were stained with anti-CD4 APC and anti-CD8 BV421 antibodies; (B, H) Quantitative analysis of the CD4 / CD8 ratio of PBMCs derived from HIV-positive donor 1 and HIV-positive donor 2 after treatment. Data are expressed as mean ± standard deviation (n = 3). Statistical significance was determined using a t-test. HIV-positive donor 1: Compared with the group without crRNA, Array 1-5 groups showed p < 0.0001; compared with the T1 group... Groups 1-5: p < 0.0001; Subject 2: Compared with the group without crRNA, T1 and Array 1-5: p < 0.0001; (C,I) Real-time quantitative PCR (qPCR) detection of HIV-1 RNA levels in PBMCs from HIV-positive donors 1 and 2 after processing. Signal values ​​were standardized to GAPDH mRNA levels. Data are expressed as mean ± standard deviation (n = 3). One-way ANOVA was used to determine statistical significance. HIV-positive donor 1: Compared with the group without crRNA, Array 1: p = 0.0185, Array 4: p = 0.0026, Array 5: p = 0.0005; HIV-positive donor 2: Compared with the group without crRNA, T1 and Array 1-5: p < 0.0001; Compared with T1: Array 1: p = 0.0377, Array 2: p < 0.0001. Group 4 ##p=0.0051, Group 5 ###p=0.0009; (D,J) Real-time quantitative PCR (qPCR) detection of HIV-1 DNA levels in PBMCs from HIV-positive donors 1 and 2 after processing. Signal values ​​were standardized to β-Globin DNA levels. Data are expressed as mean ± standard deviation (n = 3).One-way ANOVA was used to determine statistical significance. For HIV-positive donor 1: compared with the group without crRNA, Array 1-5 groups showed p < 0.0001; compared with the T1 group, Array 1-5 groups showed p < 0.0001. For HIV-positive donor 2: compared with the group without crRNA, Array 1 group showed p = 0.0001; T1 and Array 4-5 groups showed p < 0.0001; compared with the T1 group, Array 1 group showed p < 0.0001; Array 4 group showed p = 0.0032. (E) Analysis of the HIV RNA to DNA ratio of PBMCs from HIV-positive donor 1 after treatment. Data are expressed as mean ± standard deviation (n = 3). One-way ANOVA was used to determine statistical significance. For subject 1: compared with the positive control group, p < 0.0001; compared with the T1 group, Array 1-5 groups showed p < 0.0001; compared with the T1 group, Array 1-5 groups showed p < 0.0001. Group 1 #p = 0.0324, Group 4 #p = 0.0324, Group 5 #p = 0.0323; (K) Analysis of the HIV RNA to DNA ratio of PBMCs from Subject 2 after treatment. Data are expressed as mean ± standard deviation (n = 3). Statistical significance was determined using a t-test. HIV-positive donor 2: Group 1 *p = 0.0222, Group 4 *p = 0.0110. (F,L) Determination of replicable virus using the TZM-bl method. Determination of infectious virus in the supernatant of cultured PBMCs in vitro: The supernatant of treated PBMCs was used to infect TZM-bl cells using a sequential 10-fold serial dilution method (3 replicates per dilution). Data are expressed as mean ± standard deviation (n = 3). Detailed Implementation

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0038] The following specific embodiments illustrate the solution proposed in this invention: Example 1: Design and preliminary screening of anti-HIV crRNA 1. Design of anti-HIV crRNA To better address the diversity and high variability of HIV strains, designing crRNAs targeting highly conserved regions of the HIV genome is crucial. This example integrates genome databases of mainstream HIV strains in China, including 39 recombinant CRF07_BC strains, 36 recombinant CRF08_BC strains, 59 B' subtype strains, and the international standard strain NL4-3, targeting HIV-1 subtype B and prevalent strains in China. Whole-genome conservation analysis was performed using the Clustal Omega multiple sequence alignment algorithm. Despite the high variability (mutation rate >15%) in the coding regions of envelope proteins such as env, core functional regions in the viral life cycle—such as the integrase domain of the gag-pol gene (chrX:7,154,632–7,155,019,hg38), the 5'-LTR promoter core region (chr19:45,678,221–45,678,590), and the RNA packaging signal Ψ (chr2:32,901,447–32,901,812)—remain highly conserved under evolutionary pressure (homology >98%). These regions were established as priority targets for crRNA design. Using the Benchling cloud-based design tool in conjunction with the CRISPOR off-target prediction system, crRNAs were screened, ultimately yielding 42 crRNAs. From these, 7 broad-spectrum, highly efficient, and highly specific crRNAs were selected. Figure 1 The crRNA target regions are: L4 (targeting the LTR region), G6 (targeting the Gag and Pol regions), G16 (targeting the Gag region), P7 and P88 (targeting the Pol region), T1 (targeting the Tat region), and E1 (targeting the Env region). The crRNA target locations, sequences, and scores are shown in Table 1.

[0039] Table 1. Anti-HIV-1 crRNA sequence, location, and scoring.

[0040] 2. Establishment of a high-throughput visualization screening method for crRNA in vitro The effectiveness of crRNA was validated using the FAM-BHQ1 dual-labeled molecular beacon reporter system, which works based on the trans-cleavage activity of Cas12a: when crRNA guides Cas12a to recognize and bind to target DNA, the non-specific nuclease activity of Cas12a is activated, cleaving the free fluorescent probe in the system. The probe is labeled with a FAM fluorescent group (maximum excitation wavelength 485 nm) at its 5' end and a BHQ-1 quencher group (maximum emission quenching wavelength 535 nm) at its 3' end. In the inactive state, the probe's hairpin structure makes FAM and BHQ-1 spatially adjacent, and fluorescence resonance energy transfer (FRET) quenches the fluorescence signal; however, after Cas12a activation, it cleaves the probe, releasing the spatial constraint and releasing the fluorescence signal. Figure 2 ).

[0041] 3. Verify the broad-spectrum targeting ability of candidate crRNAs against major prevalent HIV-1 strains in China. A multi-strain parallel validation strategy was employed, using infectious clone plasmids of the major circulating HIV strains B' subtype, CRF07_BC, and CRF08_BC in China, as well as the classic strain NL4-3 (pNL4-3, NIH#114) as template DNA to construct a high-throughput visualization validation platform. Validation showed that all seven crRNAs could guide the Cas12a protein to effectively cleave the four HIV template DNAs. Figure 3 ). Example 2 Construction of an anti-HIV EMT-Cas12a system To achieve efficient and specific delivery of LbCas12a and anti-HIV-1 crRNA, thereby improving gene editing efficiency, this invention constructs an exosome-mediated targeted CRISPR-Cas12a delivery system (EMT-Cas12a) based on exosomes targeting CD4+ cells. Figure 4Modular assembly was achieved by co-transfecting HEK293F cells with three plasmids: pCD63-L7Ae plasmid expressed a fusion protein (KD=0.8nM) of the exosome tetraspanic membrane marker protein CD63 and the archaeosome ribosomal protein L7Ae, forming an exosome membrane-anchored RNA packaging device; pLamp2b-Nb1 plasmid encoded a fusion of the CD4-targeting nanobody Nb1 and the lysosome-associated membrane protein Lamp2b, with its transmembrane region containing a pH-sensitive helix (AA 1-40) to promote endosome escape; pC / D-Cas12a-crRNA plasmid carried a functional element containing a Box C / D kink-turn motif, enabling the self-assembly of the Cas12a mRNA (with the 5'UTR inserted into the IRES element to avoid cytotoxicity) and crRNA complex; The sequence of pCD63-L7Ae is shown in SEQ ID No. 8, the sequence of pLamp2b-Nb1 is shown in SEQ ID No. 9, and the sequence of pC / D-Cas12a-crRNA is shown in SEQ ID No. 10.

[0042] Example 3: Production and Purification of EMT-Cas12a Exosomes To effectively separate and enrich high-purity exosomes and avoid interference from impurity proteins, lipoproteins, and cell debris in the sample, this invention carefully designed and employed a combined purification system based on TFF (tangential flow filtration), SEC (size exclusion chromatography), and ultracentrifugation. Specifically, after transfecting HEK293F cells with the three plasmids required for preparing EMT-Cas12a, the cell supernatant was collected after 72 hours of transfection. The cell supernatant underwent three-stage treatment: first, initial screening was performed using a hollow fiber membrane tangential flow system (300 kDa molecular weight cutoff) at an injection pressure of 0.15 MPa, effectively removing 98.3% of free proteins and cell debris; subsequently, fine sorting was performed using size exclusion chromatography with an average molecular weight exclusion value of 700 kDa, an injection flow rate of 90–150 cm / h, and a column pressure difference of less than 2.5 bar. Elution peak analysis showed that the target exosomes were distributed in the flow-through region between the loading and washing phases. Figure 5 (Flowchart); Finally, ultracentrifugation at 100,000×g (4℃, 3.5 h) was used to increase the concentration. The obtained exosome precipitate was reconstituted with PBS, and the protein concentration was quantified by BCA method to reach 2.3±0.4 mg / mL.

[0043] Example 4: EMT-Cas12a Exosome Quality Control The purified EMT-Cas12a was characterized and analyzed using transmission electron microscopy, and the results are as follows: Figure 6As shown in Figure A, the engineered exosomes exhibit a typical biconcave cup morphology with an intact surface membrane structure. The concentration of the purified EMT-Cas12a protein was determined to be 2.3 ± 0.4 mg / mL using the BCA method, and NTA characterization showed a particle concentration of 2.4 × 10⁻⁶. 12 The particle size distribution was uniform, with an average particle size of 112.3 ± 8.7 nm (PDI = 0.19) as determined by dynamic light scattering. Particles with a size distribution in the 80-150 nm range accounted for 93.4% of the total. Figure 6 B), which meets the quality standards of the International Society for Extracellular Vesicles (ISEV). Figure 6 Western blot analysis of C showed that EMT-Cas12a exhibited a specific CD63-L7Ae fusion protein band at 65 kDa, while the blank control group showed no such signal. Simultaneously, the expression of TSG101 (44 kDa) and CD81 (26 kDa) validated the integrity of the exosome membrane structure. Flow cytometry analysis based on CD63 antibody-conjugated magnetic bead capture also confirmed the expression of surface exosome markers of EMT-Cas12a. Figure 6 (D) The results showed that the positive rates of CD63, CD9 and CD81 in the magnetic bead-captured population were all above 90% (the binding rate of the isotype control group was <0.8%), revealing that this engineering strategy is an effective biosynthetic pathway for targeting exosomes.

[0044] To evaluate the purity of the EMT-Cas12a engineered exosomes obtained after multi-step purification, this embodiment used nanoflow cytometry and Western blot methods for systematic validation. Nanoflow cytometry analysis showed that ( Figure 7 (A) 96% of the particles were membrane dye positive. The principle behind this result is that the lipophilic fluorescent dye used can specifically embed and label vesicle structures with complete lipid bilayers, but cannot label impurities without membrane structures (such as protein aggregates, nucleic acids, etc.). Therefore, the high proportion of membrane dye positive particles fully demonstrates that complete vesicle structures were successfully enriched in the extract, and non-target impurities were effectively removed.

[0045] Western blot results further confirmed the high purity of exosomes at the molecular marker level. Figure 7 B): Engineered exosomes exhibited strong positive signals for typical exosome marker proteins (CD9, TSG101), while the Golgi apparatus marker protein GM130 was almost undetectable. These results indicate that the final EMT-Cas12a exosome sample had high purity and no significant residual organelle contamination.

[0046] In summary, the results based on membrane integrity testing (nanoflow cytometry) and specific marker detection (Western blot) fully demonstrate that the purification process established in this study can successfully obtain high-purity EMT-Cas12a engineered exosomes, meeting the requirements of subsequent functional experiments and application research. This confirms the feasibility of high-purity preparation and standardized production of EMT-Cas12a engineered exosomes.

[0047] Example 5: Evaluation of the effectiveness of the anti-HIV EMT-Cas12a system To overcome the technical bottlenecks of the CRISPR-Cas12a system in cell-targeted delivery, this invention innovatively integrates the natural delivery characteristics of exosomes with synthetic biology modification strategies to construct an EMT-Cas12a engineered exosome delivery system capable of targeting CD4+ cells. This system integrates three core components into exosomes using genetic engineering technology: the exosome membrane-anchored RNA packaging device CD63-L7Ae, the targeting nanobody Nb1, and Cas12a mRNA and crRNA. Specifically, the CD63-L7Ae fusion protein specifically recognizes the CD / box RNA motif (C / Dbox kink-turn domain) on Cas12a mRNA and crRNA through its KOW domain, forming a stable molecular anchoring mechanism. Meanwhile, the Nb1 nanobody, derived from an alpaca single-domain antibody, achieves high-density loading through surface display technology; its unique antigen-binding site specifically recognizes CD4 molecules. This design overcomes the non-specific immune response caused by the Fc fragment of traditional antibodies.

[0048] Based on the design goal of targeting the delivery of Cas12a mRNA and crRNA to CD4+ cells using the EMT-Cas12a system, this invention systematically validated the expression and functional characteristics of each component in engineered exosomes using cross-platform technology. To clarify the impact of engineered modifications on the function of natural exosomes, nanoflow cytometry was used to quantitatively analyze the biomarker levels, surface nanobody density, and nucleic acid loading of EMT-Cas12a and blank exosomes (Blank EXO). Figure 8Data showed that the CD63 positivity rate of the EMT-Cas12a group transfected with CD63-L7Ae plasmid was as high as 82.4%, significantly higher than the 16.83% of the Blank EXO group, while the expression levels of CD9 and CD81 did not show significant differences. Immunolabeling experiments using anti-VHH antibody confirmed that the positivity rate of Nb1 nanobodies on the surface of EMT-Cas12a particles reached 15.3%, while the non-specific binding rate of Blank EXO was less than 1%. Nucleic acid quantification analysis showed that the total nucleic acid signal intensity of EMT-Cas12a was 25.4% higher than that of Blank EXO. RT-qPCR further confirmed that its Cas12a mRNA content was significantly higher than that of the L7Ae-deficient type (L7Ae-EXO) and the CD / box sequence deletion type (CD / box-EXO), suggesting that the synergistic effect of the CD63-L7Ae scaffold and CD / box RNA motif significantly enhanced the targeting loading efficiency of the target RNA in exosomes. Figure 9 A).

[0049] To verify the delivery capability of the system, different engineered exosomes were co-incubated with Jurkat cells for 48 hours before Western blot analysis. The results showed that ( Figure 9 Compared to the L7Ae-deficient control group (B, 9C), the intracellular Cas12a protein expression level in the EMT-Cas12a treatment group was increased by more than 10-fold, indicating that the engineering strategy effectively promoted the intracellular release of functional proteins.

[0050] To verify the targeted delivery capability of EMT-Cas12a, zsGreen-labeled Nb1 and untargeted engineered exosomes were co-incubated with PBMCs. Confocal microscopy was used to observe whether the PBMCs successfully internalized the exosomes. The results showed that the fluorescence intensity of PBMCs after co-incubation with CD4-targeted (Nb1) exosomes was significantly higher than that of the untargeted group. Figure 10 A), suggesting that it accumulates more efficiently within cells. Flow cytometry data ( Figure 10 (B) shows that, compared with unmodified zsGreen-labeled exosomes, Nb1-modified exosomes achieved approximately 28-fold enrichment of fluorescence signal in the CD4+ cell population of PBMCs. Figure 10 (C) Consistent with confocal results. In summary, this study confirms that Nb1 endows the EMT-Cas12a system with targeting specificity for CD4+ cells, significantly improving its cell type-specific delivery efficiency. Multimodal experiments validated that the EMT-Cas12a system represents a highly efficient CRISPR-Cas12a delivery strategy for precisely targeting CD4+ cells.

[0051] Example 6: In vitro antiviral activity study of the EMT-Cas12a system The antiviral activity of different crRNAs EMT-Cas12a was evaluated in Jurkat cells infected with NL4-nanoluc. Figure 11A shows a schematic diagram of the experimental procedure. In this invention, single crRNA Sicr-EMT-Cas12a (e.g., T1-EMT-Cas12a targeting the T1 site) and multi-cRNA array system Arcr-EMT-Cas12a were constructed (Array1 containing four targets L4, G16, T1, E1; Array2 containing four targets L4, G16, P88, T1; Array3 containing five targets L4, G16, P88, T1, E1; Array4 containing five targets L4, G6, P7, T1, E1; and Array5 containing four targets L4, G6, P7, T1).

[0052] Engineered exosomes loaded with a single crRNA, Sicr-EMT-Cas12a, acted on cells. Viral replication dynamics were monitored by quantitative intracellular NanoLuc luciferase activity (Figure 11B). The principle is that the HIV-1 LTR promoter drives nanoluc gene expression, and its signal intensity is positively correlated with viral transcriptional activity. The experiment included two control groups: (i) an empty crRNA control group (EMT-Cas12a without crRNA, Ctr), and (ii) a positive control group (PBS-treated infected cells, Pos). NanoLuc levels were higher in the empty crRNA control group and the positive control group, while NanoLuc levels were significantly lower in the Sicr-EMT-Cas12a group.

[0053] The high mutation rate of HIV makes single-crRNA strategies susceptible to escape due to HIV genome evolution. This invention innovatively employs a multi-crRNA array targeting conserved regions strategy—simultaneously attacking multiple highly conserved functional regions in the viral genome (gag-pol integrase region, vif helper protein coding region, etc.) to construct multiple genetic barriers to prevent escape. This design fully utilizes the endogenous RNase activity of Cas12a, which can precisely cleave mature crRNA from the pre-crRNA array by recognizing the conserved direct repeat (DR) hairpin structure upstream (5') of the crRNA target sequence. This invention designs a CRISPR array integrating four to five crRNA units separated by DR sequences, and verifies its capability through in vitro fluorescent cleavage experiments. As shown in Figure 11E, the crRNA array (per-crRNA) can be processed into mature crRNA in vitro by Cas12a, and then guide Cas12a to recognize the target DNA sequence. This activates the DNase activity of Cas12a, thereby cleaving the target sequence and probe. Figure 11 C). To confirm that all EMT-Cas12a engineered exosomes mediated effective cellular delivery of Cas12a, Jurkat cells were co-incubated with different engineered exosomes for 48 hours, and protein lysates were extracted and analyzed by Western blot (Fig. 11D). Consistent with predictions, the combined crRNA approach significantly improved HIV inhibition compared to single crRNA (T1). All five multi-crRNA arrays reached baseline-level NanoLuc signals at the 288-hour endpoint (Fig. 11F). After 288 hours of EMT-Cas12a treatment, cells were cultured for another 72 hours in exosome-free medium, and then intracellular HIV RNA and DNA were quantitatively detected by qPCR (Fig. 11G and 11D). Figure 11 (H) The five crRNA arrays were compared with a positive control. The HIV gag DNA in Array1, Array4, and Array5 was below the detection threshold. These results clearly demonstrate the in vitro antiviral activity of EMT-Cas12a and the superiority of the combined array cure method.

[0054] Example 7 Phenotypic and Genotypic Analysis of EMT-Cas12a-treated Cell Cultures This invention performs HIV genotyping on cell samples to map the HIV genome. Jurkat cells infected with HIV-1 NL4-NanoLuc were pretreated once with Array3-EMT-Cas12a and Array5-EMT-Cas12a (10⁻⁶ cells per cell). 6 (One exosome particle), then cultured for 5 days. Cell DNA was extracted and PCR amplified to target HIV genomic regions, followed by sequencing. Figure 12A and Figure 13 The sequences of the L4, G6, G16, P7, P88, T1, and E1 target regions obtained by NGS sequencing of PCR products are shown. Cells treated with EMT-Cas12a exhibited typical deletion regions within or around the Cas12a cleavage sites in the crRNA target regions. Control cells were treated with EMT-Cas12a loaded with G16-EMT-Cas12a. NGS analysis of the G16-treated group showed that indexing (…) was present only at the G16 target site. Figure 14 No significant sequence changes were observed at non-target sites.

[0055] Example 8: Off-target effects and drug toxicity detection of EMT-Cas12a This invention assessed potential off-target effects in edited cells and predicted possible off-target sites for crRNAs based on the human reference genome sequence (GRCh38). Of the seven crRNAs analyzed, only G16 and P88 showed off-target sites based on computational predictions. According to the list generated by CRISPOR, all predicted off-target sites had a cleavage frequency determination (CFD) score of 0.0 (…). Figure 12 (B) indicates an extremely low off-target risk. Genomic DNA was extracted from Jurkat cells treated with EMT-Cas12a loaded with G16 or P88, and the products near the crRNA target sites were amplified by PCR, followed by T7 endonuclease I (NEB) detection of indels. Gel analysis showed that no additional cleavage bands appeared at all predicted off-target sites compared to the untreated control (Figure 12C), experimentally validating the specificity of crRNA.

[0056] To evaluate the cytotoxic effects of EMT-Cas12a on Jurkat cells and primary human peripheral blood mononuclear cells (PBMCs) under chronic exposure, Alamar Blue cell viability assays were performed after co-incubation of engineered exosomes with the cells. Based on the results of cell characterization analysis, EMT-Cas12a treatment did not affect the cell morphology or metabolic activity of Jurkat cells and PBMCs, and no significant cytotoxicity was observed (Figure 12D).

[0057] Example 9: In vivo antiviral activity study of the anti-HIV EMT-Cas12a system This invention investigated the anti-HIV effect of EMT-Cas12a on humanized PBMC mice infected with HIV-1 (NL4-NanoLuc). The experimental procedure is as follows: Figure 15 A. The antiviral effects of Ctrl EXO (EMT-Cas12a without crRNA) and different EMT-Cas12as administered to mice via tail vein injection were tracked using an in vivo animal fluorescence imaging system. Based on the results of cell experiments, Sicr-EMT-Cas12a (T1 and L4) and three multi-strand crRNA arrays (Array1, Array4, and Array5) were selected for in vivo experiments. The results showed that each EMT-Cas12a significantly inhibited viral replication (Figures 15B and 15C) and reduced mortality (…). Figure 15 D), showing a superior therapeutic advantage over Ctrl EXO in a mouse model (Figure 15B). All mice in the Ctrl EXO group died before day 28 post-infection, while only one mouse in the T1-EMT-Cas12a treatment group died on day 36. Two mice in the Array4 group (numbers A101 and A129) and one mouse in the Array1 group (number A126) showed fluorescence signals reduced to background levels (signal <10). 6 ).

[0058] At the experimental endpoint, mice were euthanized, and their organs and blood were collected for subsequent analysis. Although CD4+ T cell depletion is one of the hallmark features of HIV infection, in recent years, the CD4 / CD8 ratio has been considered a more accurate reflection of clinical progression than CD4 count alone. Therefore, flow cytometry analysis was performed in this invention to assess the CD4 / CD8 ratio of humanized T cells in mouse spleens (Figures 16A and 16B). The results showed that all EMT-Cas12a treatment groups exhibited partial preservation of the CD4+ cell population, with the Array4 group showing the most significant effect, followed by the Array1 group. This contrasted sharply with the Ctrl EXO group, which almost completely depleted CD4+ T cells (CD4 / CD8 ratio <0.002). In the Array4 group, the CD4 / CD8 ratios of the two mice showing baseline fluorescence signals were 1.56 and 0.115, respectively, while the ratio in the Array1 group was 0.238.

[0059] Viral infection status was confirmed by measuring the level of viral capsid protein p24 in mouse plasma. Figure 16 C). Compared with the positive control group, plasma p24 levels were significantly reduced in all EMT-Cas12a groups loaded with crRNA. Furthermore, the reduction in p24 levels was more significant in the Array 1, Array 4, and Array 5 groups compared with the single crRNA T1 group.

[0060] To analyze the actively replicating viral reservoir in mouse plasma, C8166 cells were infected with mouse plasma (per 10 cells). 4 EMT-Cas12a-loaded mice were cultured with 10 μL of plasma per cell for 72 hours to amplify infectious virus. The NanoLuc signal level in the plasma re-infected cultures from EMT-Cas12a-treated mice was significantly lower than that in the control group without crRNA, indicating a significant reduction in infectious virus levels in mouse plasma (Fig. 16D). Plasma infectivity in Array 1 and Array 4 groups was significantly lower than that in the single crRNA T1 group. HIV-1 Gag and Vif transcripts were significantly reduced in mice treated with EMT-Cas12a loaded with crRNA (Fig. 16E-G). In particular, mice from the Array 4 subgroup, designated A101, had viral RNA levels below the detection limit. These results demonstrate the antiviral capacity of EMT-Cas12a in vivo, and that the multi-crRNA array (Arcr-EMT-Cas12a) exhibits higher antiviral efficiency in vivo compared to a single crRNA system.

[0061] Next, to verify whether EMT-Cas12a could be taken up by transplanted human CD4+ T cells, zsGreen-labeled or unlabeled EMT-Cas12a was injected intravenously into PBMC-humanized mice via the tail vein. Twelve hours after injection, peripheral blood mononuclear cells (PBMCs) were isolated and analyzed by flow cytometry to quantify zsGreen+ CD4+ T cells (…). Figure 16 H and Figure 16 (I), indicating that it can be efficiently and targetedly delivered to CD4+ cells.

[0062] Example 10: Validation of the antiviral effect of EMT-Cas12a in in vitro culture of peripheral blood cells from HIV-1 subjects. To assess whether the results of the in vitro cell line experiments and in vivo mouse experiments conducted in this invention could be reproduced in peripheral blood mononuclear cells (PBMCs) from HIV-positive donors, PBMCs from two HIV-positive donors were selected. These cells were stimulated and expanded in vitro with CD3 / CD28-coupled magnetic beads, and then treated with different EMT-Cas12a (without crRNA, T1, Array1, Array4, and Array5). Flow cytometry, qPCR, and replication capacity analysis were performed. PBMCs from healthy donors were used as negative controls. In the PBMCs of HIV-positive donor 1 (CRF07_BC subtype), the CD4+ T cell level was significantly increased in the EMT-Cas12a treatment group compared to the positive control group (without crRNA). Figure 17 AF presents the in vitro analysis results of HIV-positive donor 1. Compared with the positive control group (CD4 / CD8 ratio <0.04), the proportion of CD4+ T cells and the CD4 / CD8 ratio were significantly increased in the Arcr-EMT-Cas12a treatment groups (Array1, Array4, Array5), while the treatment effect in the T1 subgroup was relatively poor (p<0.05) (Figures 17A and 17B). Intracellular viral RNA and DNA replication expression was detected by qPCR (Figures 17C and 17D). The results showed that EMT-Cas12a in Array4 and Array5 significantly reduced viral RNA transcription compared with the positive control, while the total cellular DNA replication showed the opposite trend of increasing in all treatment groups. The viral RNA / DNA ratio was significantly reduced in all treatment groups, indicating that EMT-Cas12a works by inhibiting proviral transcription and reducing infected cell death. Figure 17 E).

[0063] The production of infectious virus was quantified by adding 100 μL of cell culture supernatant diluted to 0.5, 0.05, and 0.005 micrograms to TZM-bl cells (which had been pre-cultured in 96-well plates for one day) (Fig. 17F). The data showed that Arcr-EMT-Cas12a effectively inhibited the production of infectious progeny viruses. The luciferase activity in the T1 group was higher than that in the positive control group, which may be due to (1) the sustained depletion of CD4+ T cells (the percentage of CD4+ T cells <3%) in the positive control group, which weakened virus production, and (2) the immune evasion characteristics of the T1 group.

[0064] Flow cytometry analysis of HIV-positive donor 2 showed that the proportion of CD4+ T cells and the CD4 / CD8 ratio trended in the same manner as in donor 1 across all treatment groups (Figs. 17G and 17H). qPCR results showed that all EMT-Cas12a treatment groups exhibited downregulation of HIV RNA and DNA levels compared to the positive control group (without crRNA) (Figs. 17I and 17J). Compared to the positive control group, cells in the Arcr-EMT-Cas12a treatment group showed a significant decrease in viral RNA and total cellular DNA, while no statistically significant difference was observed in the T1 group. Array5 showed an unexpectedly high viral RNA / proviral DNA ratio, which may be due to the total cellular DNA measurement being close to the detection limit (<10 copies / 10^6). 6 The technical error was caused by the presence of individual cells (Figure 17K). To verify this hypothesis, further analysis was conducted on infectious viral particles in the culture supernatant. Figure 17 These results indicate that all EMT-Cas12a treatments inhibited the production of infectious viruses, with EMT-Cas12a loaded with a multi-crRNA array showing the best inhibitory effect (Figure 17J).

[0065] In summary, this invention provides an engineered exosome-based targeted delivery system (EMT-Cas12a) that loads a functional component based on CRISPR-Cas12a for cleaving HIV proviral DNA. This targeted delivery system expresses the CD4-targeting nanobody Nb1 on the surface of EMT-Cas12a, improving the efficiency of targeted delivery of CRISPR-Cas12a to CD4+ cells. This invention employs a multi-crRNA tandem strategy targeting conserved regions shared by HIV-1 subtype B and prevalent Chinese strains (07_BC, 08_BC, B'). These crRNAs, along with Cas12a mRNA, serve as functional components for cleaving HIV proviral DNA and are co-packaged into EMT-Cas12a, reducing the risk of viral escape. This targeted delivery system has demonstrated good biocompatibility and excellent antiviral efficacy in in vitro cell cultures (HIV-1-infected cell lines and HIV-positive donor PBMCs) and in in vivo experiments involving intravenous injection of EMT-Cas12a into humanized mice infected with HIV-1 PBMCs.

[0066] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0067] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0068] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A crRNA that specifically targets conserved gene regions of HIV, characterized in that, The sequence of the crRNA is selected from at least one of SEQ ID No. 1-7.

2. An engineered exosome targeted delivery system, characterized in that, The targeted delivery system expresses the CD4-targeting nanobody Nb1 on the surface of EMT-Cas12a; the targeted delivery system is loaded with a functional component based on CRISPR-Cas12a cleavage of HIV proviral DNA, the functional component including the sequence of crRNA as described in claim 1.

3. The method for preparing the engineered exosome targeted delivery system according to claim 2, characterized in that, include: (1) pCD63-L7Ae plasmid, pLamp2b-Nb1 plasmid and pC / D-Cas12a-crRNA plasmid were co-transfected into cells, cultured, and cell supernatant was collected, wherein the pC / D-Cas12a-crRNA plasmid contained the sequence of crRNA as described in claim 1; (2) The cell supernatant was initially screened using a hollow fiber membrane tangential flow system to remove free proteins and cell debris; Subsequently, size exclusion chromatography was used for fine sorting, and the exosome eluent was collected; the exosome eluent was then subjected to ultracentrifugation to obtain engineered exosomes.

4. The method according to claim 3, characterized in that, In step (1), the cells are HEK293F cells, and the culture time is 72h.

5. The method according to claim 3, characterized in that, In step (2), the molecular weight cutoff of the hollow fiber membrane tangential flow system is 280~320 kDa, and the initial screening injection pressure is 0.15 MPa.

6. The method according to claim 3, characterized in that, In step (2), when performing fine sorting in size exclusion chromatography, a chromatography medium with an average molecular weight exclusion value of 700 kDa is selected, the injection flow rate is 90~150 cm / h, and the column pressure difference is less than 2.5 bar.

7. The method according to claim 3, characterized in that, In step (2), ultracentrifugation is performed at 2-8℃ with an ultracentrifugation rate of 90,000-110,000×g for 3-4 hours.

8. The use of the engineered exosome targeted delivery system prepared by any of the preparation methods of claims 2 or 1-7 in the preparation of drugs for treating HIV infection.

9. A drug comprising the engineered exosome targeted delivery system prepared by any of the methods of claims 2 or 1-7.

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