A liver-targeted gene editing system based on endogenous promoter hijacking and application thereof

Abstract

The application discloses a liver-targeted gene editing system based on endogenous promoter hijacking and application, and belongs to the field of biological medicine. The system is composed of an LNP-wrapped modified Cas nuclease mRNA (first component) and a promoter-free viral vector carrying a therapeutic transgene donor (second component). The system uses LNP to realize the transient burst expression of Cas nuclease in the liver, mediates the generation of double-strand breaks at the site of endogenous high-expression genes, induces the site-specific integration of therapeutic transgenes without exogenous promoters, and hijacks the expression driven by endogenous promoters by using the splice acceptor (SA) mechanism. The application solves the risk of carcinogenesis caused by random integration of exogenous strong promoters and the immunotoxicity of long-term expression of nucleases through a "double safety lock" design. Experimental results prove that the system has high editing efficiency, long-term stability and no off-target, and can be used for various liver-derived metabolic diseases such as hemophilia, hypercholesterolemia and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and gene therapy technology, and particularly relates to a liver-targeted gene editing system and its application based on endogenous promoter hijacking. Background Technology

[0002] In recent years, thanks to the maturity of lipid nanoparticle (LNP) delivery systems, mRNA technology has rapidly expanded from COVID-19 vaccines to fields such as protein replacement therapy for genetic diseases, personalized cancer vaccines, and in vivo CRISPR / Cas9 gene editing. However, the transition of mRNA from "transient immune activation" to "continuous treatment of chronic diseases" is still constrained by two major bottlenecks: short intracellular half-life and challenges in quality control during industrial-scale production.

[0003] The 3′ poly(A) tail of mRNA is a key element determining its stability and translation efficiency. Poly(A) tails longer than 100 nt can recruit polyadenylate-binding protein (PABP), forming a "closed-loop model" with the 5′ cap, promoting ribosome recycling and inhibiting deadenylase degradation. Therefore, constructing long poly(A) tails has become an industry consensus. However, in in vitro transcription (IVT) production, long poly(A) sequences on plasmid templates are highly susceptible to replication slip during bacterial amplification, leading to shortened poly(A) tail length or severe batch-to-batch heterogeneity, directly violating GMP requirements and reducing final therapeutic efficacy.

[0004] To mitigate the aforementioned plasmid instability, existing technologies have proposed a "segmented Poly(A) tail" strategy, which involves inserting short non-A linkers / spacers into consecutive A sequences. The current industry gold standard (such as the design used by BioNTech in its BNT162b2 vaccine) uses an "A30-L10-A70" structure, inserting a 10-nucleotide random linker between 30 and 70 A sequences. While this design improves plasmid retention to some extent compared to a pure Poly(A) tail, significant unresolved challenges remain under the high standards required for chronic disease treatment: One risk is the hidden risk of plasmid topoisomerization and dimerization. Even with a segmented design, homologous recombination can still easily occur during high-density fermentation, forming dimers / multimers that are difficult to detect with Sanger sequencing. This leads to failure of enzyme digestion linearization, heterogeneous transcription templates, and a significant increase in purification costs.

[0005] Secondly, there is the irrationality of the connector design and spatial steric hindrance. Existing long connectors (10 nt) do not accurately match the PABP binding footprint, easily forming "naked" areas, which accelerates nuclease attack and cannot actively block the CCR4-NOT complex.

[0006] Thirdly, the long-term in vivo effect is insufficient to meet the needs of alternative therapies. Existing designs have been optimized primarily for vaccine development, with the core objective being the transient expression of antigens to induce an immune response. However, for protein replacement therapies such as those for hemophilia that require long-term (days to weeks) maintenance of effective blood drug concentrations, the in vivo half-life of existing designs remains relatively short, leading to frequent dosing by patients and significantly limiting clinical adherence.

[0007] In summary, there is an urgent need in the field for a next-generation mRNA 3' end architecture based on rational design. This architecture should be able to fundamentally eliminate plasmid recombination and dimerization in bacteria, achieving "absolutely homogeneous" industrial-grade manufacturing, while significantly extending the functional half-life of mRNA in mammals through synergistic mechanisms, so as to truly meet the application needs of liver-targeted gene editing and long-term treatment of chronic genetic diseases. Summary of the Invention

[0008] This invention aims to resolve a fundamental safety contradiction prevalent in the existing field of liver gene therapy: how to simultaneously eliminate the oncogenic risk caused by random integration of exogenous promoters into viral vectors and the immune and off-target toxicity caused by long-term expression of gene editing tools within a single system, and on this basis, to construct a universal, modular, and safe expression strategy that can be applied to the treatment of various liver metabolic diseases.

[0009] Specifically, this invention provides a universal liver-targeted gene editing system based on endogenous promoter hijacking. This system overcomes the insertion mutation risks common to traditional all-AAV systems when treating diseases such as hemophilia A / B and familial hypercholesterolemia through a "dual security lock" design.

[0010] In a first aspect, the present invention provides a liver-targeted gene editing system based on endogenous promoter hijacking, the system comprising: a first component: nuclease mRNA encapsulated by lipid nanoparticles (LNP), wherein the mRNA encodes a CRISPR / Cas nuclease; The second component is a promoterless viral vector carrying a therapeutic transgene donor, wherein the viral vector does not contain an exogenous promoter or enhancer sequence that drives the expression of the therapeutic transgene; The liver-targeted gene editing system exhibits high editing efficiency and long-term stability in vivo.

[0011] Preferably, the liver-targeted gene editing system operates by: using the first component to generate double-strand breaks in the intron or exon regions of endogenous high-expression gene sites in the hepatocyte genome, and then site-specifically integrating the therapeutic transgenic donor from the second component into the break site; After integration, the expression of the therapeutic transgene is driven by the endogenous promoter of the endogenous highly expressed gene site via the splice acceptor (SA) mechanism.

[0012] Preferably, the endogenously highly expressed gene locus is the albumin (Alb) gene locus.

[0013] Preferably, the therapeutic transgenic donor is a nucleotide sequence encoding coagulation factor VIII (F8); The therapeutic transgenic donor is a cDNA sequence containing only the coding region.

[0014] Preferably, the therapeutic transgenic donor is operatively linked upstream to a splice acceptor (SA) sequence and a polypeptide self-cleavage sequence (2A) or a ribosome entry site (IRES) sequence; The splice acceptor is configured to capture the transcription product of the upstream exon of the endogenously highly expressed gene, forming a fusion mRNA.

[0015] Preferably, the viral vector is a recombinant adeno-associated virus (rAAV) vector; the serotype of the rAAV vector is selected from one of AAV8, AAV9, AAVrh10, AAV3B, AAV-LK03 or their engineered variants.

[0016] Preferably, the nuclease mRNA is a chemically modified Cas nuclease mRNA; the uridine in the Cas nuclease mRNA sequence is completely replaced by N1-methylpseudouridine, and it has a 5' Cap-1 cap and a 3' Poly(A) tail structure; the Cas nuclease is selected from SpCas9 or SaCas9; when the Cas nuclease is SpCas9, the plasmid template sequence of the Cas nuclease mRNA is shown in SEQ ID No. 14; when the Cas nuclease is SaCas9, the plasmid template sequence of the Cas nuclease mRNA is shown in SEQ ID No. 15.

[0017] Preferably, the lipid components of the lipid nanoparticles (LNPs) include: 30%~60% molar ratio of ALC-0315, 30%~60% molar ratio of cholesterol, 5%~20% molar ratio of DSPC, 5%~20% molar ratio of DOPE, 0.5%~4% molar ratio of DMG-PEG2000, and 2%~20% molar ratio of mannose lipids or glycerocholic acid lipids.

[0018] Preferably, the lipid components of the lipid nanoparticles (LNPs) include: 40% molar of ALC-0315, 40% molar of cholesterol, 5% molar of DSPC, 5% molar of DOPE, 0.5% to 4% molar of DMG-PEG2000, and 2% to 20% molar of mannose lipids or glycerocholic acid lipids.

[0019] Secondly, the present invention provides the use of the above-described system in the preparation of a medicament for treating liver metabolic diseases.

[0020] Preferably, the liver metabolic disease is hemophilia A.

[0021] The beneficial effects of this invention are as follows: 1. This invention possesses extremely high security: Unlike traditional all-AAV delivery systems, this invention utilizes LNP to deliver Cas9 mRNA. The Cas9 protein is metabolically degraded approximately 48 hours after expression in vivo (e.g., Figure 15 As shown in the figure, this avoids the cytotoxic T cell (CTL) immune response induced by the long-term presence of nucleases, successfully achieving immune escape and protecting the edited hepatocytes from being cleared by the immune system.

[0022] Secondly, the donor vector of this invention does not contain a strong exogenous promoter; it can only be expressed by "hijacking" the endogenous promoter after precise integration into the target endogenous site (such as the Alb site). This fundamentally reduces the risk of insertional mutations or oncogenesis caused by AAV random integration near oncogenes.

[0023] 2. This invention possesses excellent editing efficiency and long-lasting effectiveness. Experimental data proves (e.g.) Figure 14 The transient high-level expression of Cas9 achieved by LNP delivery of mRNA has a significantly higher Indel efficiency (average 48.5%) than the slow and sustained expression of the full AAV system (approximately 39.5%), effectively improving efficiency by about 1.5 times.

[0024] like Figure 11 and 15 As shown, the F8 activity in hemophilia mice remained consistently above 100% after treatment, and no decline in activity was observed in the long-term observation period, similar to that in the AAV group. This demonstrates the therapeutic potential of this system to achieve long-term benefits with a single dose.

[0025] 3. This invention possesses precise targeting and an extremely low off-target rate. Verification using NP-seq technology (such as...) Figure 16The therapeutic donor fragment exhibits an extremely high proportion of forward splicing within the Alb intron region. This direction-specific homologous recombination ensures that the therapeutic gene can accurately capture endogenous transcriptional flow.

[0026] Deep sequencing (OliTag-seq) results show (e.g.) Figure 7 , 13 This system achieves efficient editing without detecting any significant off-target events, ensuring the accuracy of gene editing.

[0027] 4. This invention possesses excellent industrialization potential and batch uniformity. The Cas9 mRNA design used in this invention avoids the instability problem of traditional long Poly(A) tails during plasmid amplification. Through optimized LNP formulation and microfluidic technology, high encapsulation efficiency (e.g., ...) is achieved. Figure 3 ) and uniform particle size distribution (e.g. Figure 2 It meets the requirements of industrial-grade production and GMP quality control.

[0028] 5. This invention possesses strong versatility and broad application potential. This invention has not only been successfully validated in a hemophilia A model, but also demonstrated its broad applicability in treating various hepatogenic metabolic diseases such as familial hypercholesterolemia and hemophilia B through the design of sgRNAs targeting multiple key gene loci, including TTR, ApoE, and LDLR (see Tables 3 and 4). Figure 20 ). Attached Figure Description

[0029] Figure 1 A schematic diagram of the preparation process for the LNP-mRNA universal delivery system; Figure 2 This is a graph showing the particle size distribution of the LNP particles prepared in Example 1. Figure 3 The diagram shows the encapsulation efficiency of CRISPR sgRNA at different target gene sites. Figure 4 Figure showing the results of GFP mRNA transfection validation in HEK293T cells; Figure 5 The image shows the validation results of in vivo imaging of Luc mRNA delivered by LNP. Figure 6 A statistical graph showing the gene editing efficiency of the LNP delivery system at different sites; Figure 7 This is a graph showing the off-target events of LNP delivery system editing at specific target sites, obtained from high-throughput sequencing detection. Figure 8A timeline of gene editing in mice using the AAV8-BDDF8 and LNP-SpCas9 systems; Figure 9 A schematic diagram showing the targeted integration of the AAV8-BDDF8-N6 vector into the Alb gene locus in mice; Figure 10 This diagram illustrates the expression of the LNP-SpCas9 system at the DNA, mRNA, and protein levels after gene editing. Figure 11 Figure 1 shows the F8 activity of hemophilia A mice treated with LNP-delivered SpCas9 mRNA and sgRNA at different time points. Figure 12 The graph shows the results of liver function biochemical indicators (including ALB, AST, ALT, TBIL and UREA) in mice in the treatment group and control group. Figure 13 This is a graph showing the gene editing results of the ALB-Intron13 target obtained from high-throughput sequencing. Figure 14 HE-stained sections of major organ tissues (heart, liver, spleen, lung, and kidney) from mice in the treatment and control groups. Figure 15 This figure shows the comparison of gene editing efficiency in vivo between the AAV-LNP delivery system and the full AAV sustained expression system of this invention; Figure 16 This is a comparison of the AAV-LNP delivery system of this invention and the sustained expression of F8 in mice after full AAV editing. Figure 17 A diagram showing the positive validation results for the direction of targeted integration of therapeutic donor fragments; Figure 18 This is a schematic diagram illustrating the construction of a promoterless vector for hemophilia B. Figure 19 A schematic diagram illustrating the construction of a vector targeting familial hypercholesterolemia; Figure 20 Diagram of the general platform scalability mechanism of the LNP delivery system of this invention. Detailed Implementation

[0030] This invention discloses a composition for gene editing and its uses. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. Furthermore, those skilled in the art can clearly modify or appropriately alter and combine the content described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0031] The sequences involved in this invention are described in Table 1: Table 1 Sequence Description

[0032] Example 1 Preparation of promoter-free gene editing compositions (construction of a dual-security lock system) This embodiment provides a method for preparing a liver-targeted gene editing drug composition that is "promoter-free throughout the system", including a first safety lock (transiently expressed Cas mRNA+sgRNA wrapped in LNP) and a second safety lock (promoter-free AAV therapeutic transgenic donor vector).

[0033] 1. Preparation of transiently expressed Cas nuclease mRNA mRNA was synthesized in vitro using the plasmid encoding SpCas9 (SEQ ID No. 14) or SaCas9 (SEQ ID No. 15) as a template and the HiScribe™ T7 High Yield RNA Synthesis Kit.

[0034] During transcription, all uridines are replaced with N1-methylpseudouridine-5'-triphosphate, and Cap-1 capping and Poly(A) tail modification are performed simultaneously. This allows the modified mRNA to be translated immediately after entering the cell and to be rapidly degraded by the cell after completing the editing task, avoiding the risk of long-term continuous expression of Cas9 protein in vivo by traditional AAV-Cas9 DNA vectors.

[0035] 2. Design and synthesis of sgRNAs targeting endogenously highly expressed gene loci Intron regions of highly expressed liver genes were selected as integration "landing sites," and sgRNAs (with 2'-O-methyl + thiophosphoryl backbone modification to improve stability) were designed and chemically synthesized. Specific target sequences are as follows: Table 2 sgRNA target sequence listing

[0036] 3. Preparation and encapsulation of liver-targeting lipid nanoparticles (LNPs) The SpCas9 mRNA and sgRNA prepared above were pre-dissolved in a 50 mM sodium citrate solution with a pH of 4. The lipid components included ALC-0315 (molar ratio 30%~60%, 40% in this example), cholesterol (molar ratio 30%~60%, 40% in this example), DSPC (molar ratio 5%~20%, 10% in this example), DOPE (molar ratio 5%~20%, 5% in this example), DMG-PEG2000 (molar ratio 0.5%~4%, 2% in this example), and mannose lipids / glycerocholic acid lipids (molar ratio 2%~20%, mannose lipids in this example, molar ratio 3%).

[0037] All of these lipid components are dissolved in anhydrous ethanol. Based on the N / P ratio (charge ratio of nucleic acid to cationic lipid) of 6 and the required nucleic acid mass, the various lipid components are mixed to form the oil phase, while the sodium citrate solution containing nucleic acid forms the aqueous phase.

[0038] By using microfluidic pumps and microfluidic chip technology, precise mixing and self-assembly of the oil and water phases were achieved, with the flow rate ratio controlled between 1:2 and 1:4 (1:3 in this example), thereby efficiently preparing liposome nanoparticles encapsulating SpCas9 mRNA and sgRNA.

[0039] Subsequently, dialysis was performed using a SpectraPor® Biotech Grade Cellulose Ester (CE) Dialysis Membrane (Spectrum lab) with a molecular cutoff of 300 kDa, with 1xPBS as the dialysate, for 3 hours. Finally, the LNP was filtered through a 0.22-micron Millex®-GV Filter Unit (Millipore). A schematic diagram of the LNP preparation process is shown below. Figure 1 As shown.

[0040] 4. Effect detection The average particle size, polydispersity index (PDI), and zeta potential of LNPs were determined using dynamic light scattering (DLS) with a Zetasizer Nano (Malvern Instruments, Malvern, UK). The encapsulation efficiency of LNP mRNA was determined using the Quant-iT™ RiboGreen™ RNA assay kit (Thermo Fisher Scientific, Waltham, MA). The particle size distribution of LNP particles is shown in the figure below. Figure 2 As shown.

[0041] from Figure 2The results show that dynamic light scattering (DLS) analysis reveals that LNPs with different formulations have uniform particle sizes, distributed in the range of 100-150 nm, indicating that the preparation of LNPs has high repeatability and stability.

[0042] The encapsulation efficiency results for different target gene sites are as follows: Figure 3 As shown.

[0043] Figure 3 The results showed that by optimizing the LNP formulation, high efficiency in CRISPR sgRNA encapsulation was achieved, targeting Alb-Exon2, LDLR-Exon4, TTR-Exon4, PAH-Exon7, F9-Exon1, PCSK9-Exon1, and SERPINC1-Exon3.

[0044] The transfection efficacy of the LNP formulation was verified by transfecting HEK293T cells with GFP mRNA. The results are as follows: Figure 4 As shown.

[0045] Figure 4 The results showed that HEK293T cells successfully expressed GFP after LNP delivery of GFP mRNA, and the transfected group showed significant fluorescence signal (FITC / APC double positive) compared with the wild-type (WT) control group.

[0046] The delivery efficacy of the LNP formulation was verified using in vivo fluorescence imaging of Luc mRNA delivered by LNP. Results are as follows: Figure 5 As shown.

[0047] Figure 5 The results showed that after injecting LNP-encapsulated GFP-Luc mRNA into mice, a significant fluorescence signal was detected by in vivo imaging technology, verifying the high efficiency of the LNP delivery system in vivo.

[0048] The results of gene editing using LNP-packaged SpCas9 mRNA and sgRNA targeting multiple gene sites in vivo are as follows: Figure 6 , 7 As shown.

[0049] Figure 6 and Figure 7 The results show that the LNP delivery system in this embodiment has high editing efficiency at different sites, all gene editing events occurred at the target site, and no off-target events were detected.

[0050] Comparative Example 1 AAV8-BDDF8-Donor packaging The AAV8-BDDF8-Donor vector was packaged using the method described in Zhang, JP et al. Curing hemophilia A by NHEJ-mediated ectopicF8 insertion in the mouse. Genome Biol 20, 276, doi:10.1186 / s13059-019-1907-9(2019).

[0051] The transfection conditions, plasmid composition, culture, and purification steps for the AAV8-BDDF8-Donor vector packaging process are the same as those described in the paper.

[0052] The AAV8-BDDF8-N6 Donor plasmid sequence is shown in SEQ ID No. 16.

[0053] Example 2 Animal experiment effect verification 1. Injected into the tail vein of hemophilia A mice.

[0054] Hemophilia A (HA) mice (129 × B6 mice carrying exon 16 knockout of the F8 gene) were purchased from the Jackson Laboratory (Bar Harbor, ME). These mice were originally conceived by Dr. H. Kazazian (University of Pennsylvania) and were housed at the National Key Laboratory of Blood and Health.

[0055] Animal experiments were conducted according to protocols approved by the SKLEH Animal Care and Use Committee. The basic method for tail vein injection involved dissolving AAV8-BDDF8-Donor prepared in Comparative Example 1 in a lactated Ringer's solution equivalent to 10% of the mouse's body weight, and then injecting it via the tail vein into 5-8 week old HA mice over 5-6 seconds. The injection dose was 2E+12vg / kg.

[0056] Two weeks later, the LNP-SpCas9 mRNA-sgRNA prepared in Example 1 was injected again at a total RNA dose of 1 mg / kg. To prevent bleeding, each mouse was simultaneously injected intravenously with 0.5 IU of F8 protein (Xyntha; Wyeth Pharmaceuticals) along with AAV / LNP.

[0057] 2. Immunosuppression.

[0058] For short-term immunosuppressive therapy, cyclophosphamide (50 mg / kg / dose) and / or methylprednisolone (50 mg / kg / dose) were administered intraperitoneally on the same day as AAV injection. The immunosuppressive regimen was once a week for 3 weeks (4 times in total).

[0059] 3. Blood and plasma were collected from mice and separated.

[0060] Mouse plasma was isolated using the tail vein method. Before collection, a 1.5 mL EP tube was prepared, and 10 μL of 3.2% sodium citrate solution (1:9) was added as an anticoagulant. The tail vein was gently incised with a blade, and the required blood volume (100 μL) was collected using a pipette. After blood collection, hemostasis was achieved at the mouse wound using MiracleCorp hemostatic powder. The blood sample was centrifuged at 2000 × g, 25 °C for 20 min, and the supernatant was collected as plasma. This plasma was transferred to a new test tube, immediately frozen on dry ice, and stored at -80 °C. Before determining F8 bioactivity, the plasma sample was rapidly thawed at 37 °C.

[0061] The experiment verified the time flow and mechanism of gene editing in mice using the AAV8-BDDF8 system prepared in Comparative Example 1 and the LNP-SpCas9 system of Example 1 of this invention.

[0062] The experimental timeline is as follows Figure 8 As shown.

[0063] A schematic diagram of the AAV8-BDDF8-N6 vector targeting and integrating into the mouse Alb gene locus is shown below. Figure 9 As shown.

[0064] A schematic diagram illustrating gene editing at the DNA, mRNA, and protein levels is shown below. Figure 10 As shown.

[0065] 4. F8 activity detection.

[0066] The coagulation activity of F8 was determined using a Sysmex CA1500 system (Sysmex, Kobe, Japan). Siemens reagents (Siemens; Marburg, Germany) included Dade Actin activated cephaloplastin reagent (Siemens; B4218-1) and coagulation factor F8 deficient plasma (Siemens; OTXW17).

[0067] Mouse plasma samples were diluted 4-fold with Dade Owren's Veronal Buffer (Siemens; B4234-25).

[0068] The F8 activity assay consisted of 5 μl of the test sample + 45 μl of OV Buffer + 50 μl of F8-deficient plasma + 50 μl of aPTT reagent, incubated at 37°C for 2 min; coagulation began after the addition of 50 μl of 25 mm calcium chloride; the clot formation time was measured using a Sysmex CA1500 system; a standard curve was prepared by diluting human standard plasma (Siemens), with normal mouse plasma used as a positive control. Results are as follows: Figure 11 As shown.

[0069] Figure 11 The results showed that F8 activity was significantly increased after treatment.

[0070] 5. Analysis of liver injury markers.

[0071] Blood was collected from the orbital region of mice. After the blood samples coagulated naturally at room temperature for 1 hour, they were centrifuged at 2000×g and 25°C for 20 minutes. The supernatant serum was collected, transferred to a new test tube, and immediately stored at -80°C. The serum was rapidly thawed at 37°C immediately before testing. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and bilirubin levels were measured using a diagnostic kit (Beckman Coulter, Inc. Teco Diagnostics). Serum ALT, AST, albumin, and total bilirubin were measured using an Olympus AU5400 (IDEXX Memphis, TN).

[0072] The results are as follows Figure 12 As shown, the results indicate that there were no significant differences (ns) between the two groups in any of the indicators, suggesting that the combined LNP and AAV delivery system had no significant adverse effects on liver function in mice.

[0073] 6. Paraffin sections of liver tissue and HE staining.

[0074] Freshly dissected livers were fixed in 4% paraformaldehyde for 48 hours, followed by dehydration. Water was replaced sequentially with a gradient of ethanol solutions, and ethanol was removed with xylene. The livers were then immersed in molten paraffin to embed the tissue samples within the paraffin blocks. After the paraffin solidified, the blocks were sliced ​​into 4–5 micrometer thick sections using a microtome and mounted on glass slides. The sections were dewaxed after baking in a 60°C oven and rehydrated using a gradient of ethanol solutions. Next, the sections were stained with hematoxylin to visualize the cell nuclei, followed by contrast staining of the cytoplasm with eosin. After staining, the sections were dehydrated and cleared using a gradient of ethanol and xylene, and finally mounted with neutral resin for microscopic observation of the tissue structure.

[0075] The results are as follows Figure 14 As shown in the figure. The results indicate that both groups of tissues had normal structures and no obvious pathological changes, further demonstrating the safety of the delivery system.

[0076] 7. Sanger sequencing analysis of gene editing efficiency.

[0077] Genomic DNA was isolated from mouse liver and other organs using the DNeasy Blood & Tissue Kit (Qiagen). Gene editing efficiency was analyzed using DNA samples from treated and untreated hemophiliac mice. Primers were designed using Primer3Plus to amplify a 300-500 bp fragment surrounding the on-target sequence (gene loci are shown in Tables 1 and 2). The target sequence was amplified using KAPA HiFi DNA polymerase. PCR reaction conditions were: 98℃ for 2 min; 98℃ for 5 s, 64℃ for 10 s, 72℃ for 5 s, for 30 cycles. After obtaining the sequencing files, gene editing efficiency was analyzed using the Synthego website. Results are as follows: Figure 6 As shown, the experimental results indicate that the gene editing efficiency of different sgRNAs (sgAlb-Intron13, sgLDLR-Exon4, sgPAH-Exon4, sgF9-Exon7, sgPCSK9-Exon1, sgSERPINC1-Exon3) varies to some extent, with the percentage of indels ranging from approximately 30% to 60%. The data demonstrate that the LNP delivery system can achieve high editing efficiency at different gene loci.

[0078] Table 3. Gene loci mediated by LNP-SpCas9 / SaCas9 mRNA-sgRNA gene editing for AAV-BDDF8 integration.

[0079] Table 4. LNP-SpCas9 / SaCas9 mRNA-sgRNA targeting key gene loci in multiple diseases for gene editing.

[0080] 8. Off-target events were analyzed using paired-end sequencing combined with the OligoTag-Seq Pipeline.

[0081] This experiment employed OliTag-seq technology, a CRISPR / Cas9 gene editing off-target detection workflow based on published research, to analyze off-target events of LNP-SpCas9 mRNA-sgRNA (see Yang, ZX et al. OliTag-seq enhances in cellulo detection of CRISPR-Cas9 off-targets. CommunBiol 7, 696, doi:10.1038 / s42003-024-06360-w (2024).). In short, this experiment used 1 μg of gene-edited mouse gDNA, which was fragmented to 500–700 bp using a Covaris S220 instrument, and then purified using ZymoResearch's Select-a-Size DNA Clean & Concentrator MagBeads. The DNA fragments were then subjected to end repair, A-tail addition, and adapter ligation using the KAPAHyper Prep Kit. The purified DNA underwent two rounds of nested PCR amplification, and the amplified products were used for 150 bp paired-end sequencing. Sequencing data were analyzed for off-target events using a customized OliTag-seq workflow.

[0082] The results are as follows Figure 7 , 13 As shown in the figure. The experimental results indicate that all detected gene editing events occurred at the target site, and no off-target events were detected.

[0083] 9. Statistical analysis in this embodiment.

[0084] In this embodiment, Graphpad Prism 7.0 (Graphpad software, San Diego, CA) was used for plotting and statistical analysis. The mean ± SEM was measured for each treatment group in each independent experiment. Welch's unpaired t-test was used to determine the significance between the treatment and control groups. A p-value < 0.05 was considered significant: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0085] Example 3 Editing efficiency detection of the LNP-CRISPR method of this invention 1. Experimental steps: 1. Experimental Animals and Grouping: In this embodiment, F8 gene knockout (F8-KO) male mice were used as the experimental model. The specific strain information of this mouse is B6;129S-F8^tm1Kaz / J (JAX Strain #004424; RRID: IMSR_JAX:004424). Healthy male mice aged 5-8 weeks and weighing 20-25g were randomly divided into two groups of 3 mice each (n=3): AAV-CRISPR control group: Two doses were administered using a full AAV sustained expression system; LNP-CRISPR experimental group: AAV-donor injection was first used, and two weeks later, LNP-encapsulated SpCas9 mRNA and sgRNA prepared according to the present invention were used for targeted editing.

[0086] 2. Drug administration procedure: Animal experiments were conducted in accordance with the protocol approved by the SKLEH Animal Care and Use Committee.

[0087] First administration (Day 0): The AAV-CRISPR control group and the LNP-CRISPR experimental group were simultaneously administered via tail vein injection. AAV8-BDDF8-Donor prepared in Comparative Example 1 was dissolved in lactated Ringer's solution at 10% of the mouse's body weight and injected into the mice at a uniform rate over 5–6 seconds. The AAV injection dose was strictly controlled at 2E+12 vg / kg. To prevent acute immune responses induced by the viral vector, cyclophosphamide (50 mg / kg / dose) and methylprednisolone (50 mg / kg / dose) were simultaneously administered intraperitoneally on the day of AAV injection. This transient immunosuppressive regimen was administered once a week for 3 weeks (4 times in total).

[0088] Second administration (Day 14): Two weeks after the first administration, the LNP-CRISPR experimental group received a second tail vein injection of LNP-SpCas9 mRNA-sgRNA prepared in Example 1 at a total nucleic acid dose of 1 mg / kg; the AAV-CRISPR control group received an equal dose of AAV-SpCas9-sgRNA. To prevent bleeding at the tail vein injection puncture site, each mouse was simultaneously injected intravenously with 0.5 IU of recombinant F8 protein (Xyntha; Wyeth Pharmaceuticals) during the second administration.

[0089] 3. Tissue Sampling: Eight weeks after the last administration (Day 70), mice were euthanized via cervical dislocation. Intact liver tissue was rapidly dissected, and the surface was thoroughly washed with 1x PBS pre-cooled to 4°C to remove blood. Using sterile forceps and surgical scissors, approximately 20 mg of tissue was collected from each of the left, middle, right, and caudate lobes of the mouse liver. The tissue samples were mixed and placed in centrifuge tubes containing 300 μL of tissue lysis buffer. The mixture was thoroughly homogenized on ice, and 0.5 μL of protein kinase K (PKase) was added. The mixture was incubated overnight at 56°C for digestion.

[0090] Detection method: Genomic DNA (gDNA) was extracted from mixed liver tissue of mice using the DNeasy Blood & Tissue Kit (Qiagen) following the standard procedure outlined in the instruction manual.

[0091] Specific primers were designed using the Primer3Plus online tool to amplify a region of approximately 400 bp surrounding the On-target (Alb-Intron13) sequence.

[0092] High-fidelity amplification was performed using KAPA HiFi DNA polymerase. The PCR reaction volume was 25 μL, and the reaction program was set as follows: 98℃ pre-denaturation for 2 min; followed by 30 cycles (98℃ denaturation for 5 s, 64℃ annealing for 10 s, 72℃ extension for 5 s); and finally, 72℃ complete extension for 5 min.

[0093] After the PCR products were verified as single target bands by agarose gel electrophoresis, they were sent to a commercial company for Sanger first-generation sequencing. The obtained .ab1 sequencing files were imported into the Synthego ICE Analysis online bioinformatics platform, and the insertion / deletion (Indel) editing efficiency percentage of the target site was quantified by comparing them with wild-type sequences.

[0094] Analysis of experimental results and technical advantages: The results of the editing efficiency test are as follows: Figure 15 As shown. From Figure 15 As can be seen, the proposed formulation (using LNP-CRISPR as an example) mediated an average Indel efficiency of up to 48.5% at the liver Alb site; while the control group using the full AAV sustained expression system had an editing efficiency of only about 39.5%. These results demonstrate that the transient high expression of Cas9 achieved by LNP-delivered mRNA in this invention is significantly superior to the slow sustained expression of the full AAV system, improving effectiveness by approximately 1.5 times.

[0095] Example 4 Long-term stability of the LNP-CRISPR method of this invention Experimental steps: The mouse breed, age, grouping, and initial drug treatment were the same as in Example 3. This example focuses on long-term dynamic observation after drug administration.

[0096] Detection method: 1. Dynamic blood collection monitoring: At fixed time points of weeks 1, 2, 4, 6, 8, 10 and 12 after the last administration, blood was collected from the tail vein of each group of mice.

[0097] Collect 90 μL of whole blood sample and immediately drop it into a centrifuge tube pre-filled with 10 μL of 3.8% sodium citrate anticoagulant (the blood to anticoagulant volume ratio is strictly controlled at 9:1). Gently invert and mix to prevent blood clotting.

[0098] Place the mixed blood sample in a refrigerated centrifuge at 4°C and centrifuge at 2000 xg (approximately 2500 rpm) for 20 minutes.

[0099] Carefully aspirate the upper plasma layer (taking care not to disturb the middle leukocyte layer), quickly freeze it in dry ice, and then transfer it to an ultra-low temperature freezer at -80℃ for long-term storage until concentrated F8 activity analysis. 2. Quantitative detection of F8 procoagulant activity (one-stage method): The one-stage clotting assay was performed using a Sysmex CA1500 fully automated coagulation analyzer (Sysmex, Kobe, Japan). Before the experiment, the plasma samples to be tested were rapidly thawed in a 37℃ water bath and placed on ice for later use.

[0100] The test reagents used were original Siemens reagents, including Dade Actin activated cephaloplastin reagent (Siemens; catalog number B4218-1) and coagulation factor F8 deficient plasma (Siemens; catalog number OTXW17). Mouse plasma samples were precisely diluted 1:4 using Dade Owren's Veronal Buffer (Siemens; catalog number B4234-25).

[0101] Reaction system: The instrument automatically aspirates 5 μL of diluted test sample, 45 μL of OV Buffer, 50 μL of F8-deficient plasma, and 50 μL of aPTT reagent, mixes them, and incubates at 37°C for 2 minutes. Subsequently, the instrument automatically injects 50 μL of 25 mM calcium chloride solution to trigger the coagulation cascade reaction and optically records the clot formation time.

[0102] Data Analysis: A standard curve was prepared using serially diluted mixed plasma from normal mice. The instrument's software automatically calculated and output the relative percentage of F8 activity in the samples based on the standard curve. The final data were imported into GraphPadPrism 7.0 software for plotting, and Welch's unpaired t-test was used to analyze the statistical significance of differences between the two groups (p < 0.05 was considered significant).

[0103] Experimental results: The results of long-term stability testing are as follows: Figure 16 As shown.

[0104] from Figure 16 As can be seen, in the long-term observation after injection in mice, the F8 activity of the control group (AAV-CRISPR) showed a significant progressive decline after reaching its initial peak; while the activity of the group in this application (LNP-CRISPR) remained stable above 100%. This is because the LNP-CRISPR system used in this invention benefits from the metabolic degradation of Cas9 within 48 hours, preventing the presentation of strong exogenous antigens on the surface of hepatocytes, thus successfully achieving immune escape and ensuring the long-lasting stability of the therapeutic effect.

[0105] Example 5 Experimental steps: The mouse breeds, groups, and treatment methods were the same as in Example 3.

[0106] Detection method: To verify the quality and directionality of the therapeutic donor fragment's site-specific integration into the endogenous Alb site in hepatocytes, this embodiment employs long-range PCR combined with targeted nanopore sequencing (NP-seq) technology for in-depth analysis.

[0107] 1. Sample Extraction and Library Construction: Mouse liver tissue from the observation endpoint (week 12) was selected, and high-purity large-fragment genomic DNA (gDNA) was extracted using the DNeasyBlood & Tissue Kit (Qiagen). 1 μg of gDNA was randomly fragmented into 500–700 bp fragments using a Covaris S220 ultrasonic disruptor. Subsequently, the target length fragments were screened and purified using magnetic beads with Select-a-Size DNA Clean & Concentrator MagBeads (Zymo Research). The purified DNA fragments were then subjected to end smoothing repair, 3' A-tailing, and ligation with dedicated sequencing adapters using the KAPA Hyper Prep Kit.

[0108] 2. Targeted Enrichment and Deep Sequencing: To specifically enrich sequences that have undergone gene editing and integration, the ligation products were amplified using two rounds of nested PCR. The primer design strategy was as follows: one primer was anchored inside the AAV donor fragment (e.g., the BDDF8 sequence), and the other primer was anchored to the endogenous Alb genomic sequence outside the homologous arm. This design ensured that only DNA fragments that had successfully undergone targeted integration were amplified exponentially. The purified nested PCR enrichment products were sent to the Illumina platform for 150 bp paired-end sequencing.

[0109] 3. Bioinformatics Analysis and Forward Assembly Confirmation: After acquiring the raw FastQ sequencing data, low-quality reads were filtered using a customized OliTag-seq bioinformatics analysis workflow, and then mapped to a mouse reference genome containing the expected recombination structure of the AAV donor sequence using the BWA algorithm. The directional characteristics of reads crossing the integration linker (forward vs. reverse integration) were analyzed to calculate the precise proportion of donor fragment forward assembly.

[0110] Experimental results: The verification results of forward stitching are as follows Figure 17 As shown, in all detected integration events, the vast majority of donor fragments exhibited forward splicing. The high proportion of forward splicing strongly demonstrates that the CRISPR / Cas9-guided homologous recombination designed in this invention has extremely high direction specificity, ensuring that therapeutic genes (such as F8) can be precisely integrated into the Alb intron region via forward insertion, thereby correctly capturing the transcriptional flow driven by the endogenous Alb promoter and achieving efficient and stable protein expression.

[0111] Example 6 Design scheme for platform extension applications of this invention 1. Platform Universality and Feasibility Analysis Those skilled in the art should understand that the "dual security lock" platform provided by this invention is not limited to the treatment of hemophilia A (F8). Its core design of "removing exogenous promoters" and "transient editing" endows the system with extremely high universality. By removing exogenous strong promoters (such as TBG, LP1, etc., typically occupying 300-600bp) and enhancer sequences that usually occupy valuable space in AAV vectors, the promoterless AAV vector of this invention releases a huge payload capacity. This makes the platform expected to easily accommodate the coding regions (CDS) of various large therapeutic genes that are originally difficult to load or approach their packaging limits after loading, while maintaining ultimate security.

[0112] Based on human reference genome data analysis, this platform is expected to have significant adaptation advantages for the following key therapeutic genes, which forms the basis for the implementation of this invention's broad scope of protection: Coagulation factor IX (F9): Targets hemophilia B. Its CDS length is only about 1383 bp (461 aa). Due to its short sequence, combined with the promoter-free strategy of this invention, it is expected to be easily adapted to single-stranded (ssAAV) or self-complementary (scAAV) vectors. In particular, although scAAV has half the capacity, it can still be easily loaded in this system while saving space through promoter removal, thus potentially achieving a faster onset of action.

[0113] Low-density lipoprotein receptor (LDLR): Targets familial hypercholesterolemia. Its CDS length is approximately 2580 bp (860 aa). Despite its large genomic span (approximately 45 kb), this system only requires the delivery of the CDS. Compared to traditional methods that require space to accommodate strong promoters, this method is expected to more easily package the full-length LDLR CDS, avoiding potential functional loss from truncated proteins.

[0114] Phenylalanine hydroxylase (PAH): Targets phenylketonuria (PKU). Its CDS length is approximately 1356 bp (452 ​​aa). Although the gene has a complex genomic structure, its CDS is compact. Utilizing the promoter-free strategy of this invention, it is expected to completely eliminate the potential risk of liver cancer caused by random AAV integration in traditional PKU gene therapy.

[0115] 2. Design of a promoterless strategy for treating hemophilia B (targeting the F9 gene) Design objective: To clarify the application and construction scheme of this platform in the hemophilia B model.

[0116] Method design: Editing elements: LNPs will be used to encapsulate SpCas9 mRNA and sgRNA targeting mouse Alb gene intron 13 (same as in Example 1).

[0117] Donor vector: Construct a promoterless AAV8-F9-Donor vector. The expected vector structure includes: ITR-homologous arm-SA-P2A-hF9 CDS-PolyA-ITR. The hF9 CDS is approximately 1.4 kb in length.

[0118] Expected Results: Due to the smaller size of the F9 gene, AAV packaging efficiency is expected to be higher than that of F8. After treatment, the F9 transgene is expected to integrate into the Alb site, using the Alb promoter to drive expression, thereby restoring coagulation function in hemophilia B mice and exhibiting extremely low liver toxicity.

[0119] 3. Design of a promoter-free strategy for treating familial hypercholesterolemia (targeting the LDLR gene) Design objective: To elucidate the construction scheme for the applicability of this platform to membrane receptor proteins (non-secretory type).

[0120] Method design: Target selection: The Alb gene locus is proposed as the integration target.

[0121] Donor vector: Construct a promoterless AAV8-LDLR-Donor. The expected vector contains SA-P2A-hLDLR CDS (approximately 2.6 kb).

[0122] Expected Results: LDLR protein is expected to be efficiently localized and expressed on the cell membrane of hepatocytes, significantly reducing LDL-C levels in the serum of model mice. Due to the removal of the exogenous promoter, the risk of cellular metabolic stress or unintended activation that may result from long-term strong LDLR expression is expected to be avoided.

[0123] 4. Design to extend integration sites to TTR or ApoE sites Design objective: To clarify that the integration site of this invention is not limited to the Alb site, and other liver-highly expressed genes can be flexibly selected as the "promoter borrowing" object in the construction scheme.

[0124] Design Background: Besides Alb, other genes with strong promoter activity exist in the liver and are often used to develop liver-specific promoters (such as TBG, TTR, hAAT / ApoE). In this design embodiment, the endogenous sites of these genes are intended to be used directly as integration landing sites.

[0125] Method design: Option A (Targeting TTR): Design sgRNAs targeting introns of the TTR (thyroxine transporter) gene. TTR is expressed at high and stable levels in the liver, and its own promoter is often used to construct gene therapy vectors, demonstrating its strong driving ability.

[0126] Option B (Targeting ApoE / hAAT): Design sgRNAs targeting the ApoE or hAAT gene loci. The combination of the ApoE enhancer and the hAAT promoter is a commonly used and highly effective element in liver gene therapy, and direct targeting of this endogenous site is expected to achieve a similar strong driving effect.

[0127] Donor design: Construct a promoterless AAV-PAH-Donor (for phenylketonuria, CDS approximately 1.4 kb), with homologous arm sequences designed to perfectly match the genomic sequences flanking the TTR or ApoE target sites.

[0128] Expected Results: Under LNP-mRNA-mediated targeted cleavage, the promoterless PAH gene is expected to achieve site-specific integration into the TTR or ApoE site, utilizing the endogenous TTR or ApoE promoter to drive PAH expression and effectively reduce the concentration of phenylalanine in the blood. This design further demonstrates the flexibility and versatility of the "promoterless hijacking strategy" of this invention in multi-site selection.

Claims

1. A liver-targeted gene editing system based on endogenous promoter hijacking, characterized in that, The system comprises: a first component: a nuclease mRNA encapsulated by lipid nanoparticles (LNPs), the mRNA encoding a CRISPR / Cas nuclease; a second component: a promoterless viral vector carrying a therapeutic transgene donor, the viral vector containing no exogenous promoter or enhancer sequence driving the expression of the therapeutic transgene; the liver-targeted gene editing system exhibits high editing efficiency and long-term stability in vivo.

2. The liver-targeted gene editing system according to claim 1, characterized in that, The liver-targeted gene editing system operates as follows: the first component generates double-strand breaks in the intron or exon regions of endogenous highly expressed gene sites in the hepatocyte genome, and the therapeutic transgene donor in the second component is integrated into the break site; after integration, the expression of the therapeutic transgene is driven by the endogenous promoter of the endogenous highly expressed gene site through the splice acceptor (SA) mechanism.

3. The liver-targeted gene editing system according to claim 2, characterized in that, The endogenously highly expressed gene locus is the albumin (Alb) gene locus.

4. The liver-targeted gene editing system according to claim 3, characterized in that, The therapeutic transgene donor is a nucleotide sequence encoding coagulation factor VIII (F8); the therapeutic transgene donor is a cDNA sequence containing only the coding region.

5. The liver-targeted gene editing system according to claim 4, characterized in that, The therapeutic transgenic donor is operatively linked upstream to a splice acceptor (SA) sequence and a polypeptide self-cleavage sequence (2A) or ribosome entry site (IRES) sequence; the splice acceptor is configured to capture the transcription product of the upstream exon of the endogenously highly expressed gene to form a fusion mRNA.

6. The liver-targeted gene editing system according to claim 5, characterized in that, The viral vector is a recombinant adeno-associated virus (rAAV) vector; the serotype of the rAAV vector is selected from one of AAV8, AAV9, AAVrh10, AAV3B, AAV-LK03 or their engineered variants.

7. The liver-targeted gene editing system according to claim 6, characterized in that, The nuclease mRNA is a chemically modified Cas nuclease mRNA; the uridine in the Cas nuclease mRNA sequence is completely replaced by N1-methylpseudouridine, and it has a 5' Cap-1 cap and a 3' Poly(A) tail structure; the Cas nuclease is selected from SpCas9 or SaCas9; when the Cas nuclease is SpCas9, the plasmid template sequence of the Cas nuclease mRNA is shown in SEQ ID No. 14; when the Cas nuclease is SaCas9, the plasmid template sequence of the Cas nuclease mRNA is shown in SEQ ID No.

15.

8. The gene editing system according to claim 7, characterized in that, The lipid components of the lipid nanoparticles (LNPs) include: 30%~60% molar of ALC-0315, 30%~60% molar of cholesterol, 5%~20% molar of DSPC, 5%~20% molar of DOPE, 0.5%~4% molar of DMG-PEG2000, and 2%~20% molar of mannose lipids or glycerocholic acid lipids.

9. Use of the system according to any one of claims 1-8 in the preparation of a medicament for treating hepatic metabolic diseases.

10. The use according to claim 9, characterized in that, The liver metabolic disease mentioned is hemophilia A.

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