In vivo therapeutic methods based on engineered exosome delivery of LwaCas13a-crRNA complexes to target degradation of HIV RNA

Abstract

The application discloses an in-vivo treatment method for targeting and degrading HIV RNA based on an engineered exosome delivering an LwaCas13a-crRNA complex. The method assembles an LwaCas13a protein and a crRNA targeting a conserved region of HIV into a complex, and uses an engineered exosome displaying a CD4 molecule to load the complex. The drug-loaded exosome targets and delivers the LwaCas13a-crRNA complex into an HIV-infected cell through the specific binding of CD4 to gp120 on the surface of the infected cell, and realizes the efficient cutting and degradation of the HIV genomic RNA. The application combines the low immunogenicity, targeted delivery capability of the exosome and the RNA cutting activity of CRISPR / Cas13a, and provides a safe and effective new strategy for the functional cure of AIDS.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an in vivo treatment method that uses a crRNA complex to target and deliver HIV-infected cellular exosomes as a delivery vector to deliver LwaCas13a protein HIV RNA and treat AIDS. Background Technology

[0002] Acquired immunodeficiency syndrome (AIDS) is mainly caused by human immunodeficiency virus (HIV) infection, and the challenge in its treatment lies in the presence of a viral reservoir. While existing antiretroviral therapy (ART) can effectively suppress viral replication, it cannot eliminate proviral DNA integrated into the host genome or residual viral RNA.

[0003] The CRISPR / Cas13a system is an RNA-guided RNA-targeting cleavage tool. Unlike Cas9, which targets DNA, LwaCas13a (derived from *Leptotrichia wadei*) specifically cleaves single-stranded RNA and exhibits a "collateral cleavage" effect, making it a promising candidate for eliminating RNA viruses. However, efficiently, safely, and specifically delivering the LwaCas13a effector protein and its corresponding crRNA to latently HIV-infected cells remains a technological challenge.

[0004] Exosomes, as natural nanoscale vesicles, possess low immunogenicity, good biocompatibility, and tissue penetration, making them ideal drug delivery carriers. However, there are currently no reports of combining engineered exosomes with the LwaCas13a system for in vivo therapy targeting the elimination of HIV RNA. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an in vivo treatment method based on engineered exosome delivery of the LwaCas13a-crRNA complex to target and degrade HIV RNA.

[0006] To achieve the above objectives, the present invention provides the following technical solution: An in vivo therapeutic method based on engineered exosome delivery of the LwaCas13a-crRNA complex to target and degrade HIV RNA includes the following steps: 1. Design and synthesis of HIV-targeting crRNAs: Specific crRNAs were designed targeting multiple highly conserved regions of the HIV genome. The crRNAs contain a backbone sequence capable of binding to the LwaCas13a protein and a guide sequence capable of specifically recognizing HIV RNA.

[0007] 2. Construction of engineered exosomes: Donor cells were genetically modified to express the fusion protein Lamp2b-CD4. The Lamp2b-CD4 fusion protein is formed by fusing the exosome membrane protein Lamp2b with the extracellular domain of the HIV receptor protein CD4, resulting in exosomes displaying CD4 molecules on their surface that can recognize the HIV envelope protein gp120.

[0008] 3. Loading the LwaCas13a-crRNA complex: The LwaCas13a protein and the crRNA described in step 1 were assembled in vitro to form the LwaCas13a-crRNA ribonucleoprotein complex. The complex was loaded into the engineered exosomes obtained in step 2 by electroporation or co-incubation to obtain drug-loaded exosomes.

[0009] 4. In vivo administration: The drug-loaded exosomes obtained in step 3 are administered to the subjects via intravenous or local injection. The drug-loaded exosomes target and bind to gp120 on the surface of HIV-infected cells via CD4 molecules displayed on their surface. After endocytosis into the cells, they release the LwaCas13a-crRNA complex, which specifically binds to and cleaves HIV RNA, thereby inhibiting HIV replication.

[0010] In a preferred embodiment, in step 1, the conserved regions of HIV are selected from the non-coding regions or long terminal repeat (LTR) sequences of the ggag, pol, and env genes of HIV.

[0011] In a preferred embodiment, in step 2, the donor cells are human embryonic kidney epithelial cells (HEK293T) or mesenchymal stem cells.

[0012] In a preferred embodiment, in step 3, the parameters of the electroporation are voltage 250-400V and capacitance 100-300μF.

[0013] In a preferred embodiment, the method further includes a step of purifying exosomes, specifically by collecting vesicles with a particle size between 30 and 150 nm by ultracentrifugation or size exclusion chromatography.

[0014] After adopting the above technical solution, the beneficial effects of the present invention are: 1. This invention utilizes CD4 molecules displayed on the surface of engineered exosomes, which can specifically recognize and bind to the envelope glycoprotein gp120 on the surface of HIV-infected cells. This "key-and-lock" recognition mechanism ensures that the therapeutic complex is primarily enriched in viral reservoirs and actively infected cells, rather than normal tissue cells.

[0015] 2. Exosomes, as natural intercellular communication carriers, are characterized by their small particle size (30-150 nm) and high biocompatibility. They can cross the blood-tissue barrier and even the blood-brain barrier, entering the lymph nodes, central nervous system, and other major anatomical havens for HIV latency. This allows the LwaCas13a-crRNA complex to reach latently infected cells that are difficult for ART drugs to access, making it possible to completely eliminate the latent viral reservoir and achieve "functional cure" or even "sterile cure."

[0016] 3. This invention employs the LwaCas13a system to cleave HIV RNA rather than DNA. By designing crRNAs that target highly conserved regions of HIV (such as LTR and gag), it is possible to accurately distinguish HIV RNA from the host's own RNA, reducing off-target cleavage. Addressing the high mutation rate caused by the error-prone nature of HIV reverse transcriptase, this invention allows for the design of multiple crRNAs that can be mixed and loaded, simultaneously targeting multiple conserved regions. Even if a virus mutates at a certain site, other crRNAs can still exert their cleavage function, effectively preventing viral escape. The non-specific RNA cleavage activity activated after Cas13a binds to the target RNA can further disrupt the viral transcriptional microenvironment, enhancing the inhibitory effect.

[0017] In summary, this invention constructs an HIV treatment system that integrates "precise identification, safe delivery, and efficient clearance" by delivering the LwaCas13a-crRNA complex via CD4-targeted exosomes, thus solving the three major pain points of existing therapies: difficult delivery, high off-target risk, and inability to clear latent reservoirs. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of the in vivo treatment process of the present invention, which is based on the delivery of LwaCas13a-crRNA complex via engineered exosomes to target and degrade HIV RNA. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0021] Design and synthesis of HIV-targeting crRNA To cover mutations in multiple HIV-1 subtypes, three conserved sequences from the LTR and gag genes of HIV-1 NL4-3 strain were selected as targets. Corresponding crRNAs were designed, with sequences conforming to the LwaCas13a crRNA scaffold requirements, which are known conserved structures. In the actual design, a guide sequence targeting HIV was attached to the 3' end of the scaffold. This guide sequence was designed as a 28-nt RNA complementary to the conserved HIV region. The crRNA guide sequence specifically recognizes and binds to the target sequence on the HIV RNA. The crRNA was synthesized and purified through in vitro transcription. Example

[0022] Preparation of engineered exosomes 1. Construction of expression vector: Synthesize the fusion gene encoding human Lamp2b protein (without signal peptide) and the extracellular region (D1-D4 domain) of human CD4 molecule, clone it into pCDH-CMV-MCS-EF1-Puro lentiviral expression vector to obtain pCDH-Lamp2b-CD4 plasmid.

[0023] 2. Obtaining engineered cell lines: pCDH-Lamp2b-CD4 plasmid and packaging plasmids psPAX2 and pMD2.G were co-transfected into HEK293T cells to package lentivirus; viral supernatant was collected to infect new HEK293T cells, and cell lines stably expressing Lamp2b-CD4 (293T-CD4+) were obtained by selection with puromycin.

[0024] 3. Exosome collection: Expand 293T-CD4+ cells and culture them in serum-free medium for 48 hours; collect the supernatant, remove cell debris by differential centrifugation (300g for 10 min, 2000g for 20 min, 10000g for 30 min), and then precipitate the exosomes by ultracentrifugation (100000g for 70 min); after resuspending in PBS, further purify by particle size exclusion chromatography; identify the morphology by transmission electron microscopy, analyze the particle size by nanoparticle tracking, and detect the expression of exosome markers (CD9, CD63) and the fusion protein Lamp2b-CD4 by Western blotting. Example

[0025] Loading LwaCas13a-crRNA complex 1. Expression of LwaCas13a protein: The gene sequence encoding LwaCas13a was cloned into the pET-28b vector and transformed into Escherichia coli BL21(DE3); after expression induced by IPTG, the LwaCas13a protein was purified by Ni-NTA affinity chromatography.

[0026] 2. In vitro assembly: The purified LwaCas13a protein was mixed with the crRNA synthesized in Example 1 at a molar ratio of 1:2 and incubated in binding buffer at 37°C for 30 minutes to form the LwaCas13a-crRNA ribonucleoprotein complex (RNP).

[0027] 3. Loading: Take 100 μg of engineered exosomes prepared in Example 2 and mix them with 50 μg of RNP complex in electroporation buffer; use an electroporator with a voltage of 300 V, a capacitance of 200 μF, and a time constant of 5-10 ms for electroporation; after electroporation, incubate at 37°C for 30 min to restore the exosome membrane structure; remove unbound free RNPs by ultracentrifugation, and the precipitate is the drug-loaded exosome. Example

[0028] In vitro targeting and functional validation 1. Cell model: The CEMT cell line (CEM / HIV cells) was infected with HIV-1NL4-3 strain to establish a chronic infection cell model.

[0029] 2. Targeting detection: Drug-loaded exosomes were labeled with PKH67 fluorescent dye and co-incubated with CEM / HIV cells and uninfected CEM cells for 4 hours. Confocal microscopy showed that PKH67-labeled exosomes were mainly enriched around CEM / HIV cells, while the signal was weaker in uninfected CEM cells, proving that exosomes displaying CD4 on their surface have specific targeting ability for HIV-infected cells.

[0030] 3. Functional assay: Drug-loaded exosomes were added to CEM / HIV cell cultures; after 72 hours, the cell culture supernatant was collected, and the viral protein concentration was detected by a p24 ELISA kit; the results showed that compared with the blank exosome group without RNP loading, the p24 antigen level in the experimental group supernatant was reduced by about 85%; the intracellular HIV RNA copy number was detected by RT-qPCR, and the results showed that the HIV RNA level was significantly reduced, confirming that the LwaCas13a-crRNA complex was successfully delivered and performed RNA degradation function. Example

[0031] Evaluation of in vivo treatment efficacy 1. Animal model: 6-8 week old NSG mice were selected and injected with 2x10^5 Hela-CD4 / HIV-infected cells via the tail vein to establish an acute HIV infection mouse model.

[0032] 2. Group treatment: Mice were randomly divided into three groups: PBS control group, blank exosome group, and drug-loaded exosome group (prepared in Example 3); 8 mice in each group; the drug-loaded exosome group was treated by injecting 200ug of drug-loaded exosomes into the tail vein twice a week for four consecutive weeks.

[0033] 3. Detection indicators: Blood was collected weekly to separate plasma and detect HIV p24 antigen levels; after treatment, mice were sacrificed, and spleen, lymph nodes, and liver tissue were taken to extract total RNA. HIV RNA levels were detected by RT-qPCR and pathological section analysis was performed.

[0034] 4. Results: After treatment, the plasma p24 antigen level in mice in the drug-loaded exosome group was significantly lower than that in the PBS control group (P<0.01), and the HIV RNA load in the spleen and lymph nodes decreased by about 2-3 log values. No obvious pathological damage was observed in the major organs by HE staining, and the serum liver and kidney function indicators were normal, indicating that the treatment method has good safety and significant in vivo anti-HIV effect.

[0035] This invention successfully achieved in vivo targeted delivery of the LwaCas13a-crRNA ribonucleoprotein complex by constructing engineered exosomes displaying CD4 molecules on their surface. In vitro experiments confirmed that the drug-loaded exosomes could specifically recognize HIV-infected cells and effectively cleave HIV RNA within the cells, significantly inhibiting viral replication (reducing p24 antigen levels by approximately 85%). In vivo pharmacodynamic studies showed that in an HIV-infected mouse model, treatment with this method significantly reduced viral load in peripheral blood and lymphoid tissues by 2-3 logarithmic orders of magnitude, and no significant abnormalities were observed in major organ pathological examinations and serum biochemical indicators, confirming the method's good safety and tolerability. This invention ingeniously combines the natural delivery advantages of exosomes with the precise RNA cleavage activity of CRISPR / Cas13a, not only overcoming the technical bottlenecks of low in vivo delivery efficiency and poor targeting in existing gene editing tools, but also providing a novel, safe, and effective treatment strategy for clearing the latent HIV viral reservoir and achieving functional cure of AIDS.

[0036] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An in vivo therapeutic method based on engineered exosome delivery of the LwaCas13a-crRNA complex for targeted degradation of HIV RNA, characterized in that, Includes the following steps: a. Design and synthesize crRNA targeting the conserved region of the HIV gene; b. Construct engineered exosomes, the surface of which displays targeting molecules capable of specifically recognizing the HIV envelope protein gp120; c. Assemble LwaCas13a protein with the crRNA described in step a to form an LwaCas13a-crRNA ribonucleoprotein complex, and load the complex into the engineered exosomes described in step b to obtain drug-loaded exosomes; d. The drug-loaded exosomes described in step c are administered to the subject, and the target molecules displayed on their surface are used to guide them to HIV-infected cells, delivering the LwaCas13a-crRNA complex into the cytoplasm, cleaving HIV RNA, and achieving treatment.

2. The in vivo treatment method based on engineered exosome delivery of the LwaCas13a-crRNA complex for targeted degradation of HIV RNA according to claim 1, characterized in that, In step b, the target molecule is CD4 protein or a functional fragment thereof with gp120 binding activity, which is expressed on the exosome membrane protein Lamp2b by fusion and displayed on the surface of the exosome.

3. The in vivo treatment method based on engineered exosome delivery of the LwaCas13a-crRNA complex for targeted degradation of HIV RNA according to claim 1, characterized in that, In step a, the guide sequence of the crRNA targets conserved regions selected from the LTR, ggag, and pol genes of HIV.

4. The in vivo treatment method based on engineered exosome delivery of the LwaCas13a-crRNA complex for targeted degradation of HIV RNA according to claim 1, characterized in that, In step c, the loading method is electroporation, liposome-mediated transfection, or chemical transfection.

5. The in vivo treatment method based on engineered exosome delivery of the LwaCas13a-crRNA complex for targeted degradation of HIV RNA according to claim 1, characterized in that, The exosomes are derived from genetically engineered mammalian cells, including but not limited to HEK293T cells, dendritic cells, or mesenchymal stem cells.

6. The in vivo treatment method based on engineered exosome delivery of the LwaCas13a-crRNA complex for targeted degradation of HIV RNA according to claim 1, characterized in that, In step d, the method of administration includes intravenous injection, intramuscular injection, or local injection.

7. A pharmaceutical composition for carrying out the method according to any one of claims 1-6, characterized in that, The drug-loaded exosomes contain an effective dose of the drug-loaded exosomes and a pharmaceutically acceptable carrier, wherein the surface of the drug-loaded exosomes displays CD4 protein or a functional fragment thereof and the internal contents are loaded with an LwaCas13a-crRNA ribonucleoprotein complex.

8. The pharmaceutical composition according to claim 7, characterized in that, The crRNA sequence consists of a backbone sequence that can bind to LwaCas13a and a guide sequence that can specifically bind to HIV-1 RNA.

9. An engineered exosome, characterized in that, The exosome membrane surface expresses the Lamp2b-CD4 fusion protein that targets HIV-infected cells, and internally loads the LwaCas13a-crRNA ribonucleoprotein complex.

10. The engineered exosomes according to claim 9, characterized in that, The exosomes have a particle size of 30-150 nm.

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