A pil vector-based targeted organ gene editing method
By constructing peptide-ionizable lipid (PIL) vectors and introducing specific modifications and functional modules, the problems of insufficient targeting, editing efficiency, and stability of LNP vectors were solved, achieving efficient and safe targeted organ gene editing.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- TIANLUN BIOTECHNOLOGY (SHENZHEN) CO LTD
- Filing Date
- 2026-04-09
- Publication Date
- 2026-07-21
AI Technical Summary
Existing lipid nanoparticle (LNP) vectors suffer from poor targeting, low editing efficiency, insufficient stability, and limited functionality during gene delivery, making it difficult to achieve efficient, safe, and precise targeted organ gene editing.
By constructing peptide-ionizable lipid (PIL) carriers and modifying specific amino acids or molecules, PIL-LNPs are prepared. Targeting ligands, membrane lysis molecules, and homeostasis regulators are introduced to match the pH environment of the target organ's endosomes, enabling precise targeted delivery and efficient editing.
This technology enables precise targeted delivery of PIL-LNP to target organs, improves the release and editing efficiency of gene editing tools within target cells, enhances stability in the blood, reduces the risk of distribution to non-target tissues, and improves therapeutic efficacy and safety.
Abstract
Description
Technical Field
[0001] This invention relates to the field of gene editing technology, specifically to a targeted organ gene editing method based on the PIL vector. Background Technology
[0002] Gene editing technology, as a cutting-edge technology in the biomedical field, has brought new hope for the treatment of various diseases. Among them, the emergence of gene editing tools such as the CRISPR-Cas9 system and lead editing technology has made precise modification of the genome possible. However, how to safely and efficiently deliver these gene editing tools to target cells and organs has always been a key issue restricting the clinical application of gene editing technology.
[0003] Currently, commonly used gene delivery vectors mainly include viral vectors and non-viral vectors. Viral vectors, such as adenoviruses and lentiviruses, have high transfection efficiency, but pose safety risks such as immunogenicity and insertion mutations, limiting their widespread clinical application. Among non-viral vectors, lipid nanoparticles (LNPs) have become a research hotspot in the field of gene delivery due to their good biocompatibility, low immunogenicity, and ability to be mass-produced.
[0004] However, the existing technology still has the following shortcomings: (1) Poor targeting: Existing LNP vectors lack the ability to effectively target specific organs when delivering gene editing tools. Most LNPs are widely distributed in the body and are difficult to precisely accumulate in the target organ, resulting in the distribution of gene editing tools in non-target tissues. This not only reduces the editing efficiency in the target organ, but may also cause gene mutations in non-target tissues, increasing safety risks. For example, when treating liver-related diseases, existing LNPs may also accumulate to a certain extent in other organs such as the lungs and spleen, affecting the treatment effect and bringing potential adverse reactions. (2) Limited editing efficiency: Even if some LNPs can reach the target organ, the release and editing efficiency of the gene editing tools they carry in the target cells needs to be improved. This may be because the structure and properties of LNPs do not match the endosome environment of the target cells, resulting in the gene editing tools being unable to be effectively released from LNPs into the cell nucleus for editing. For example, after some LNPs enter the cell, due to factors such as the acidic environment in the endosomes, they cannot break down in time to release gene editing components such as mRNA, resulting in low editing efficiency and difficulty in achieving the ideal therapeutic effect. (3) Insufficient stability: During blood circulation, LNPs are easily affected by various factors, leading to aggregation, degradation, and other problems, resulting in structural instability and affecting the delivery of gene editing tools. For example, proteins in the blood may adsorb onto the surface of LNPs, altering their surface properties and making them easier for the immune system to recognize and clear, thereby reducing the circulation time and delivery efficiency of LNPs in vivo. (4) Single function: Most existing LNP vectors only have delivery functions and lack other auxiliary functional modules, such as targeting ligands and membrane cleavage molecules. This makes it difficult for LNPs to achieve efficient targeted delivery and intracellular release in the face of complex in vivo environments. For example, LNPs without targeting ligands are difficult to specifically recognize receptors on the surface of target cells, increasing the risk of non-specific delivery; LNPs without membrane cleavage molecules are difficult to penetrate membrane structures in acidic endosome environments, affecting the release of gene editing tools. Summary of the Invention
[0005] The purpose of this invention is to provide a targeted organ gene editing method based on PIL vectors, so as to overcome the shortcomings of existing gene delivery vectors in terms of targeting, editing efficiency, stability and functional diversity, and achieve efficient, safe and precise targeted organ gene editing.
[0006] Specifically, the technical solution provided by this invention is: a method for targeted organ gene editing based on a PIL vector, comprising the following steps:
[0007] Step 1: Construct PIL vectors. Using solid-phase supported synthesis technology, natural amino acids or functional modules are combined with artificially alkylated ionizable Fmoc-protected amino acids through modular synthesis to prepare ionizable peptide lipids.
[0008] Step 2: Based on the target organ type, PIL is modified with specific amino acids or molecules. For lung targeting, lysine or arginine is introduced; for liver targeting, cysteine, histidine, tyrosine, or phenylalanine is introduced; for spleen targeting, glutamic acid, aspartic acid, proline, tryptophan, or Nα-acetylated lysine / arginine is introduced; for thymus targeting, lysine-tyrosine dipeptide is introduced; and for bone targeting, alendronate is introduced.
[0009] Step 3: Prepare PIL-LNP. Modified PIL is mixed with mRNA and helper lipids using microfluidic mixing technology to prepare lipid nanoparticles. The pKa value of the LNP is in the range of 5.5-7.0 to match the pH environment of the endosomes in the target organ.
[0010] Step 4: Delivery and editing. PIL-LNP is delivered to the target organ via intravenous injection or local administration.
[0011] Preferably, the alkyl chain length of PIL is 12 carbon atoms, the number of AIFAs is 4, and the lysine analogue is 2,4-diaminobutyric acid.
[0012] Preferably, the target organ is at least one of the lung, liver, spleen, thymus, or bone; the editing efficiency of the PIL-LNP in lung tissue is not less than 7%, and the editing efficiency in liver tissue is not less than 13%.
[0013] Preferably, the preparation of PIL-LNP further includes the following steps:
[0014] Step 3.1: Verify the chemical structure of PIL using 1H NMR and mass spectrometry;
[0015] Step 3.2: Use cryo-TEM to observe the morphology of LNPs and confirm that they contain "solid nuclei", "vesicles" or "empty double-layered vesicles" structures.
[0016] Step 3.3: Measure the pKa value of LNP using TNS to ensure it matches the pH of the endosome in the target organ;
[0017] Step 3.4: Analyze the fluorescence intensity of LNP in the target organ by flow cytometry to verify its tissue specificity.
[0018] Preferably, the gene editing is therapeutic gene editing, and the target genes include pathogenic genes related to lung genetic diseases, spleen immune diseases, and bone metabolic diseases.
[0019] Preferably, the carrier includes at least one of the following functional modules:
[0020] (1) Targeting ligands: used to recognize specific receptors on the surface of target organ cells;
[0021] (2) Membrane-splitting molecules: used to enhance the membrane permeability of LNPs in acidic endosome environments;
[0022] (3) Homeostasis regulators: used to maintain the stability of LNP in the blood circulation and prevent mRNA degradation.
[0023] Preferably, the targeting ligand is an antibody fragment, peptide, or aptamer; the membrane lysis molecule is a pH-sensitive ionizable lipid; and the homeostasis regulator is polyethylene glycol or a derivative thereof.
[0024] Preferably, the general chemical formula of PIL is AnBm, where "A" represents the type of alkyl tail chain, including saturated alkyl chain, hydroxyl-containing alkyl chain, amide bond or ester bond; "n" represents the length of alkyl chain, ranging from 8 to 18 carbon atoms; "B" represents lysine or its analogues, including Orn, Dab, and Dap; and "m" represents the number of AIFAs, ranging from 2 to 6.
[0025] Preferably, PIL-LNP achieves at least one of the following functions in the target organ:
[0026] (1) Deliver Cre recombinase mRNA to activate LoxP site-mediated gene recombination;
[0027] (2) Delivering a leader editor and engineered guide RNA to achieve single base substitution, insertion or deletion at the locus;
[0028] (3) Deliver the CRISPR-Cas9 system to achieve gene knockout or knock-in.
[0029] The advantages of this invention compared with the prior art are as follows: (1) Precise targeting: This invention modifies PIL with specific amino acids or molecules and introduces targeting ligands, enabling PIL-LNP to specifically recognize receptors on the surface of target organ cells, thus achieving precise targeted delivery. Compared with the prior art, this greatly reduces the distribution of gene editing tools in non-target tissues, lowers safety risks, and improves editing efficiency in target organs. For example, in lung targeted therapy, PIL-LNP modified with lysine or arginine can more effectively accumulate in lung tissue, improving the gene editing effect on lung genetic diseases. (2) Highly efficient editing: The pKa value of PIL-LNP prepared by this invention ranges from 5.5 to 7.0, which matches the pH environment of the target organ endosome. At the same time, the introduction of membrane cleavage molecules enhances the membrane penetration ability of LNP in the acidic endosome environment, enabling gene editing tools to be released from LNP in time and enter the cell nucleus for editing. Compared with the prior art, this significantly improves the release and editing efficiency of gene editing tools in target cells, achieving higher editing efficiency. (3) Good stability: This invention introduces a homeostatic regulator, such as polyethylene glycol or its derivatives, into the PIL-LNP, which can form a protective film on the LNP surface, maintaining the stability of the LNP in blood circulation and preventing mRNA degradation. Compared with the prior art, it reduces the aggregation and degradation of LNP in the blood, improves the circulation time and delivery efficiency of LNP in vivo, and enables more gene editing tools to reach the target organ. (4) Diverse functions: The PIL-LNP vector of this invention contains multiple functional modules such as targeting ligands, membrane cleavage molecules, and homeostatic regulators, and has multiple functions such as targeted delivery, efficient intracellular release, and maintenance of stability. Compared with the single-function LNP vectors in the prior art, it can better cope with the complex in vivo environment and achieve efficient, safe, and precise gene editing of targeted organs. Detailed Implementation
[0030] Example 1
[0031] This embodiment provides a PIL-based targeted organ gene editing method, including the following steps:
[0032] I. Constructing the PIL Carrier
[0033] Using solid-phase supported synthesis, natural amino acids or functional modules are modularly synthesized with artificially alkylated ionizable Fmoc-protected amino acids to prepare ionizable peptide lipids (PILs). The PIL has an alkyl chain length of 12 carbon atoms, 4 AIFAs, and its lysine analogue is 2,4-diaminobutyric acid. The general chemical formula of the PIL is AnBm, where "A" represents the type of alkyl tail chain, including saturated alkyl chains, hydroxyl-containing alkyl chains, amide bonds, or ester bonds; "n" represents the alkyl chain length, ranging from 8 to 18 carbon atoms; "B" represents lysine or its analogues, including Orn, Dab, and Dap; and "m" represents the number of AIFAs, ranging from 2 to 6.
[0034] II. Modifying PIL
[0035] Depending on the target organ type, specific amino acid or molecular modifications are made to PIL. For lung targeting, lysine or arginine is introduced; for liver targeting, cysteine, histidine, tyrosine, or phenylalanine is introduced; for spleen targeting, glutamate, aspartic acid, proline, tryptophan, or Nα-acetylated lysine / arginine is introduced; for thymus targeting, lysine-tyrosine dipeptide is introduced; and for bone targeting, alendronate is introduced.
[0036] III. Preparation of PIL-LNP
[0037] The modified PIL was mixed with mRNA and helper lipids to prepare lipid nanoparticles using microfluidic mixing technology. The pKa value of the LNPs ranged from 5.5 to 7.0 to match the pH environment of the endosomes in the target organ. The preparation of PIL-LNPs also included the following steps:
[0038] (1) The chemical structure of PIL was verified by 1H NMR and mass spectrometry.
[0039] (2) The morphology of LNP was observed using cryo-TEM to confirm that it contains a “solid nucleus”, “vesicle” or “empty double-layered vesicle” structure.
[0040] (3) The pKa value of LNP was determined by TNS to ensure that it matches the pH of the endosome in the target organ.
[0041] (4) Flow cytometry was used to analyze the fluorescence intensity of LNP in the target organ to verify its tissue specificity.
[0042] IV. Target delivery and editing
[0043] PIL-LNP is delivered to the target organ via intravenous injection or local administration. The target organ is at least one of the following: lung, liver, spleen, thymus, or bone; the editing efficiency of PIL-LNP in lung tissue is not less than 7%, and the editing efficiency in liver tissue is not less than 13%.
[0044] The carrier includes at least one of the following functional modules:
[0045] (1) Targeting ligand: used to recognize specific receptors on the surface of target organ cells, and can be antibody fragments, peptides or aptamers. By introducing a targeting ligand, PIL-LNP can specifically bind to receptors on the surface of target cells, achieving precise targeted delivery and solving the problem of poor targeting in the prior art.
[0046] (2) Membrane cleavage molecules: These enhance the membrane permeability of LNPs in acidic endosome environments and are pH-sensitive ionizable lipids. After entering the cell, when LNPs are in an acidic endosome environment, membrane cleavage molecules can play a role in helping gene editing tools to be released from LNPs and enter the cell nucleus for editing, thus improving editing efficiency.
[0047] (3) Homeostasis regulators: These are used to maintain the stability of LNPs in the bloodstream and prevent mRNA degradation. They can be polyethylene glycol or its derivatives. Homeostasis regulators can form a protective film on the surface of LNPs, reduce the adsorption of proteins in the blood, reduce the risk of LNPs being recognized and cleared by the immune system, and improve the circulation time and delivery efficiency of LNPs in the body.
[0048] The gene editing is therapeutic gene editing, and the target genes include pathogenic genes associated with lung genetic diseases, spleen immune diseases, and bone metabolic diseases. The PIL-LNP achieves at least one of the following functions in the target organ:
[0049] (1) Deliver Cre recombinase mRNA to activate gene recombination mediated by LoxP site.
[0050] (2) Delivering a leader editor and engineered guide RNA to enable single base substitution, insertion or deletion at loci.
[0051] (3) Deliver the CRISPR-Cas9 system to achieve gene knockout or knock-in.
[0052] Example 2
[0053] This embodiment provides a specific implementation method for lung-targeted gene editing. Using solid-phase supported synthesis technology, natural amino acids and artificially alkylated ionizable Fmoc-protected amino acids are modularly synthesized to prepare a polypeptide ionizable lipid with an alkyl chain length of 12 carbon atoms, 4 AIFAs, and 2,4-diaminobutyric acid (2,4-diaminobutyric acid) as the lysine analog. Lysine is introduced to modify the PIL for lung targeting. The modified PIL, along with mRNA encoding Cre recombinase and helper lipids, is prepared into lipid nanoparticles using microfluidic mixing technology. The chemical structure of PIL is verified by 1H NMR and mass spectrometry; the morphology of LNP is observed using cryo-TEM to confirm its structure; and the pKa value of LNP is determined by TNS to ensure pH matching with lung tissue endosomal pH. PIL-LNP is delivered to mouse lung tissue via intravenous injection. After a period of time, detection shows that Cre recombinase in the lung tissue successfully activates LoxP-mediated gene recombination, achieving an editing efficiency of 8%.
[0054] Example 3
[0055] This embodiment provides a specific implementation method for liver-targeted gene editing, constructing the PIL vector according to the method in Example 1. Cysteine residues were introduced to modify the PIL for liver targeting. The modified PIL, along with mRNA encoding the CRISPR-Cas9 system and helper lipids, was prepared into lipid nanoparticles, and the relevant structures were verified and pKa values were determined. PIL-LNP was delivered to mouse liver tissue via intravenous injection. The results showed that gene knockout was successfully achieved in liver tissue, with an editing efficiency of 15%.
[0056] Example 4
[0057] This embodiment provides a specific implementation method for spleen-targeted gene editing. Using solid-phase supported synthesis technology, natural amino acids and artificially alkylated ionizable Fmoc-protected amino acids are modularly synthesized to prepare a polypeptide ionizable lipid with an alkyl chain length of 12 carbon atoms, 4 AIFAs, and a lysine analogue of 2,4-diaminobutyric acid. Glutamic acid is introduced to modify the PIL for spleen-targeted editing. The modified PIL, along with mRNA encoding a lead editor and engineered guide RNA, and helper lipids, is prepared into lipid nanoparticles, and relevant detection is performed. PIL-LNP is delivered to mouse spleen tissue via intravenous injection. Detection shows that single-base substitution at the locus is successfully achieved in the spleen tissue, with an editing efficiency of 10%.
[0058] In summary, the PIL-based targeted organ gene editing method of the present invention can achieve precise targeted delivery and efficient gene editing, and has significant effects in gene editing of organs such as the lungs, liver, and spleen, providing a new and effective means for the treatment of hereditary diseases.
[0059] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.
Claims
1. A method for targeted organ gene editing based on a PIL vector, characterized in that... Includes the following steps: Step 1: Construct PIL vectors. Using solid-phase supported synthesis technology, natural amino acids or functional modules are combined with artificially alkylated ionizable Fmoc-protected amino acids through modular synthesis to prepare ionizable peptide lipids. Step 2: Based on the target organ type, PIL is modified with specific amino acids or molecules. For lung targeting, lysine or arginine is introduced; for liver targeting, cysteine, histidine, tyrosine, or phenylalanine is introduced; for spleen targeting, glutamic acid, aspartic acid, proline, tryptophan, or Nα-acetylated lysine / arginine is introduced; for thymus targeting, lysine-tyrosine dipeptide is introduced; and for bone targeting, alendronate is introduced. Step 3: Prepare PIL-LNP. Modified PIL is mixed with mRNA and helper lipids using microfluidic mixing technology to prepare lipid nanoparticles. The pKa value of the LNP is in the range of 5.5-7.0 to match the pH environment of the endosomes in the target organ. Step 4: Delivery and editing. PIL-LNP is delivered to the target organ via intravenous injection or local administration.
2. The method for targeted organ gene editing based on a PIL vector according to claim 1, characterized in that: The PIL has an alkyl chain length of 12 carbon atoms, an AIFA number of 4, and a lysine analogue of 2,4-diaminobutyric acid.
3. The method for targeted organ gene editing based on a PIL vector according to claim 1, characterized in that: The target organ is at least one of the following: lung, liver, spleen, thymus, or bone; the editing efficiency of PIL-LNP in lung tissue is not less than 7%, and the editing efficiency in liver tissue is not less than 13%.
4. The method for targeted organ gene editing based on a PIL vector according to claim 1, characterized in that: The preparation of the PIL-LNP also includes the following steps: Step 3.1: Verify the chemical structure of PIL using 1H NMR and mass spectrometry; Step 3.2: Use cryo-TEM to observe the morphology of LNPs and confirm that they contain "solid nuclei", "vesicles" or "empty double-layered vesicles" structures; Step 3.3: Measure the pKa value of LNP using TNS to ensure it matches the pH of the endosome in the target organ; Step 3.4: Analyze the fluorescence intensity of LNP in the target organ by flow cytometry to verify its tissue specificity.
5. The method for targeted organ gene editing based on a PIL vector according to claim 1, characterized in that: The gene editing is therapeutic gene editing, and the target genes include pathogenic genes related to lung genetic diseases, spleen immune diseases, and bone metabolic diseases.
6. The method for targeted organ gene editing based on a PIL vector according to claim 1, characterized in that... The carrier includes at least one of the following functional modules: (1) Targeting ligands: used to recognize specific receptors on the surface of target organ cells; (2) Membrane-splitting molecules: used to enhance the membrane permeability of LNPs in acidic endosome environments; (3) Homeostasis regulators: used to maintain the stability of LNP in the blood circulation and prevent mRNA degradation.
7. The method for targeted organ gene editing based on a PIL vector according to claim 6, characterized in that: The targeting ligand is an antibody fragment, peptide, or aptamer; the membrane cleavage molecule is a pH-sensitive ionizable lipid; and the homeostasis regulator is polyethylene glycol or a derivative thereof.
8. The method for targeted organ gene editing based on a PIL vector according to claim 1, characterized in that: The general chemical formula of the PIL is AnBm, where "A" represents the type of alkyl tail chain, including saturated alkyl chain, hydroxyl-containing alkyl chain, amide bond or ester bond; "n" represents the length of alkyl chain, ranging from 8 to 18 carbon atoms; "B" represents lysine or its analogues, including Orn, Dab, and Dap; and "m" represents the number of AIFAs, ranging from 2 to 6.
9. A method for targeted organ gene editing based on a PIL vector according to claim 1, characterized in that... The PIL-LNP performs at least one of the following functions in the target organ: (1) Deliver Cre recombinase mRNA to activate LoxP site-mediated gene recombination; (2) Delivering a leader editor and engineered guide RNA to achieve single base substitution, insertion or deletion at the locus; (3) Deliver the CRISPR-Cas9 system to achieve gene knockout or knock-in.