Compositions for inducing immune tolerance and their use in gene therapy

Abstract

Compositions and methods are provided for reducing pre-existing antibodies against viral vectors or gene editing-related proteins. The compositions have shown a reduction in various antibody titers by administering liposome compositions complexed with viral vector proteins or fragments thereof. The liposomes contain phosphatidylcholine and phosphatidylserine (PS), with some or all of the PS present as lyso-PS. The compositions and methods can be used in conjunction with gene therapy and nucleic acid-based vector-based vaccination and therapy.

Description

Cross-reference of related applications

[0001] This application claims priority under U.S. Provisional Patent Application No. 63 / 139,602, filed on January 20, 2021, the disclosure of which is incorporated herein by reference.

[0002] Sequence List This application includes a sequence listing submitted electronically in ASCII format, the entirety of which is incorporated herein by reference. The ASCII copy, created on January 12, 2022, is named "011520_01662_SEQ_ID_ST25.txt" and has a size of 520 bytes. Background of the Disclosure

[0003] In the treatment of single-gene disorders through gene introduction and modification, one of the main challenges is successfully delivering the therapeutic gene to its target. New discoveries in this field have revealed that viruses, which have naturally evolved to deliver and replicate their nucleic acids in host cells, are useful tools for delivering therapeutic genes. Viral vectors are the most common vectors for delivering transgenes. One such viral vector is adeno-associated virus (AAV). Identification of 13 different serotypes has demonstrated that this viral vector is a promising tool in gene therapy due to its high transduction efficiency in target cells and robust, sustained transgene expression. Adenoviruses are small (approximately 25 nm) non-enveloped DNA viruses belonging to the parvovirus family. Each serotype has different cell transduction characteristics, and there is great potential for their use as delivery vehicles in gene therapy. AAV (including AAV5 and AAV8) has been of particular interest in targeted gene therapy. AAV has been used in preclinical and clinical trial stages in gene therapy for various diseases, including hemophilia B, Leber's congenital amaurosis (LCA), Pompe disease, and diabetes mellitus (DM).

[0004] Despite all the advantages of AAV or other viral vectors, numerous obstacles remain to the successful delivery of therapeutic transgenes. One of the most significant of these is pre-existing immunogenicity to the viral capsid. The target modified gene and the viral capsid itself are two main causes of the host immune response encountered in vector-mediated gene therapy. Serological epidemiological studies show that approximately 40% of the general population possesses neutralizing antibodies (NAbs) against AAV8, making it the least seroprevalent of all AAV serotypes (Vandamme et al., Hum Gene Ther. 2017;28(11):1061-74). The presence of anti-capsid antibodies, such as neutralizing antibodies, in circulation can prevent viral entry into cells, even at minimal titer levels. These pre-existing anti-AAV8 antibodies significantly impact the delivery efficiency of viral vectors and, consequently, inhibit transgene expression. They are also known to affect vector specificity, instead of migrating to the spleen. Apart from NAbs, patients are also known to possess capsid-specific binding antibodies. Unlike NAbs, BAbs can recognize the viral capsid but do not neutralize it. Binding or non-neutralizing antibodies are known to tag the capsid, accelerating its clearance from the system and leading to premature transgene expression. Due to high homology between different serotypes, these existing antibodies are also cross-reactive. Studies have shown that subjects with already established antibody titers against AAV1 or AAV2 may also exhibit cross-reactive antibody reactions against AAV8 (Calcedo Front Immunol. 2013;4:341).

[0005] Current strategies to evade the host's pre-existing immune response include screening for neutralization-resistant AAV variants using error-prone PCR, modifying the immunogenic domain to evade recognition by NAb, and capsid shuffling to create chimeric vectors through immunoselective selection. These approaches limit the number of viable vectors that can be produced because they can reduce packaging capacity and alter the infectious profile. Other strategies include limiting gene therapy to only naive patients, and reducing patients' NAb titer levels through plasmapheresis or administration of empty capsids and immunosuppression. These approaches limit the number of patients who can participate in gene therapy, are ineffective for patients with high NAb titers, and even create a global immunosuppression problem that exposes patients to a high risk of infection (Calcedo Front Immunol. 2013;4:341). All of the above complications in current approaches strongly indicate the need for a viable strategy that can reduce the antigenicity of AAV8 and suppress such pre-existing humoral responses in patients with high NAb titers without affecting the infectivity, transduction efficiency, specificity, or expression profile of the viral capsid. Similarly, existing antibodies against Cas9 in the general population may affect the safety and efficacy of CRISPR / Cas gene editing approaches by neutralizing Cas9 activity (Simhadri et al, Mol. Ther.Methods. Clin. Dev. 2018: 10, 105-112). CRISPR / Cas is expected to treat several clinical conditions as well as being a biological tool. Therefore, technologies that reverse existing antibody responses to gene therapy vectors and CRISPR platforms would have a positive impact by expanding the number of patients participating in these life-saving therapies. [Overview of the project]

[0006] This disclosure provides compositions and methods for suppressing the immune response to viral vector proteins. These compositions and methods are at least in part based on the demonstration of induction of low reactivity by using lyso-PS liposomes conjugated with viral vector proteins. The low reactivity of lyso-PS is antigen-specific. As an example, this disclosure provides a 16-mer peptide, AAV8 581-596 This provides data demonstrating the ability of lyso-PS liposomes to induce immune tolerance to (IVADNLQQNTAPQIG (SEQ ID NO: 1)). This 16-amino acid sequence, located in the viral protein III region of the AAV8 vector capsid, was found to be highly neutralized by the monoclonal antibody ADK8, which is used to determine the antigenic epitope of the capsid (Tseng et al., Front Immunol. 2014;5:9). The data provided herein demonstrate that lyso-PS can induce immune tolerance to immunogenic AAV8 581-596 This invention demonstrates the effective induction of oral immune tolerance to the peptide. Furthermore, this disclosure provides data showing that the composition and method can reverse pre-established capsid-specific antibody titers by oral administration. This may be due to lysoPS acting on plasma cells and memory B cells. The composition and method of the present invention can be used in gene therapy, vector vaccines, and CRISPR-based technologies.

[0007] In one embodiment, the Disclosure provides a composition comprising lipid particles (e.g., liposomes) containing phosphatidylserine (PS), wherein some or all of the PS may be present as lysophosphatidylserine (lyso-PS), and the liposomes are conjugated to a protein or peptide of a delivery vehicle (such as a viral vector used in gene therapy, gene editing, or vector vaccines). The Disclosure also provides a method for using such a composition to induce immune tolerance to a delivery vehicle protein or to reduce antibody titers against a delivery vehicle protein.

[0008] The compositions of this disclosure comprise a complex of a viral vector protein or its antigenic fragment with a liposome or lipid structure, wherein the liposome or lipid structure comprises phosphatidylserine (PS) and phosphatidylcholine (PC). At least a portion of the PS exists as lyso-PS. In some embodiments, all of the PS may exist as lyso-PS. In addition to PS and PC, the liposome may also contain phosphatidylethanolamine (PE). The protein may be any protein or peptide derived from the viral vector, or a modified version thereof.

[0009] The administration strategies disclosed herein can induce tolerance to immunogenic proteins or peptides used for the delivery of gene therapeutics. For example, oral pre-exposure (e.g., immunization) to specific liposome compositions (referred to here as tolerogenic compositions or priming compositions) conjugated with target proteins (such as proteins or peptides derived from bacterial or viral vectors) can result in reduced responsiveness to the proteins during subsequent gene delivery using those delivery vehicles. Induction of immune tolerance can lead to decreased expression of co-stimulatory signals on dendritic cells, CD4 + This can manifest as one or more of the following: decreased T cell proliferation and / or induction of immunomodulatory cytokine secretion such as TGF-β and IL-10, and / or decreased antibody titers.

[0010] This composition and method are useful for inducing immune tolerance to administered viral vector proteins (particularly via the oral route). This composition and method are also useful for reducing existing antibody titers.

[0011] The process of reversing titers against AAV and Cas9 may be mechanistically different from reversing the immune response to autologous proteins and allergens necessary for treating autoimmune diseases. For example, reversing the immune response to autologous proteins in type 1 diabetes requires defense using cell-mediated tolerance.

[0012] In one aspect, the present disclosure provides a method for reducing existing antibodies against viral vector proteins. In a plurality of embodiments, the existing antibodies may be against adenoviral vector proteins (such as one or more of various capsid proteins). In a plurality of embodiments, the existing antibodies may be against the AAV8 capsid protein.

[0013] The method of the present invention can be used in individuals known to have immune intolerance (immunological intolerance) to viral vector target proteins, or in individuals whose immune status to the target protein is unknown. For example, the composition can be used in individuals who are about to receive gene therapy or vaccine therapy using a delivery vehicle containing the target protein or peptide, but who have never previously shown immune intolerance to that protein, or naive individuals (i.e., individuals who have never previously been administered the peptide or protein). If there is no detectable titer of antibodies against the protein, the individual is considered not to have shown immune intolerance to that protein.

[0014] In a plurality of embodiments, the present disclosure includes inducing tolerance in an individual to one or more antigens (in the individual, antibodies against this antigen can inhibit the function of nucleic acid editing). Nucleic acid editing includes, but is not necessarily limited to, therapeutic and prophylactic approaches that introduce a nucleic acid editing system into an individual to facilitate editing. Such nucleic acid systems include, but are not necessarily limited to, viral systems and CRISPR systems. Thus, the present disclosure includes inducing tolerance in an individual who is a candidate for nucleic acid editing (e.g., gene therapy) to one or more antigens in one of the related editing systems.

[0015] In multiple embodiments, the present disclosure provides a method of inducing tolerance to viral vector peptides used in vector vaccines. Vector vaccines are generally constructed from carrier viruses such as adenoviruses or poxviruses and engineered to carry related genes derived from a virus (e.g., SARS-CoV-2). In this embodiment, while an immune response to the target protein derived from a pathogenic organism inserted within the genetic material of the delivery viral vehicle is desired, attenuation of the immune response to the viral vector protein itself is desired. Thus, the compositions and methods of the present invention can be used for the selective suppression of existing or novel immune responses to the delivery vehicle component of a vaccine composition.

[0016] In multiple embodiments, tolerance is induced to any antigen included in any of the following virus-based delivery nucleic acid editing systems: adenovirus, such as recombinant adeno-associated virus (rAAV), retrovirus, lentivirus, herpes simplex virus (HSV), and baculovirus

[0017] In a plurality of non-limiting embodiments, tolerance is induced in individuals having existing anti-adenovirus (Ad) neutralizing antibodies (AdNAb). In multiple embodiments, such antibodies bind to antigens associated with any type of adenovirus, including but not limited to adenovirus serotype 2, adenovirus serotype 5, and adenovirus serotype 8. In multiple embodiments, such antibodies bind to the adenovirus capsid protein. In multiple embodiments, the antibodies bind to the adenovirus fiber and / or penton base protein.

[0018] In several embodiments, tolerance is induced to any CRISPR-related agent intended to function in human or non-human organisms capable of producing antibodies. In several embodiments, tolerance is induced to any CRISPR-related antigen contained in any CRISPR-related enzyme (e.g., Cas enzyme) (including any member of any type or class of Cas enzyme). In several embodiments, Cas is a type I, type II, or type III Cas enzyme. In several non-limiting embodiments, Cas is Cas9 or Cas12a (also known as Cpf1). [Brief explanation of the drawing]

[0019] [Figure 1] Design of an in vivo reversal study Antigen (e.g., AAV8 capsid, AAV8) 581-596 To induce antibody titers specific to peptides (AAV5, Cas9), mice were given subcutaneous injections for 4-6 weeks (1-6 times per week). After antibody titer induction, these mice were divided into treatment groups and administered orally weekly starting from weeks 5-7 (until week 17). This was followed by a 1-2 week washout period. To monitor antibody titer levels, blood was collected weekly by saphenous vein puncture from week 1 throughout the study period.

[0020] [Figure 2] Induction of anti-AAV8 antibody titer by subcutaneous administration Free AAV8 581-596 Panel a shows the anti-AAV8 antibody titers of 20 animals in Group 1 that were administered 1 μg of peptide, and panel b shows the antibody titers of 30 animals that were administered 1 μg of free AAV8 capsid protein.

[0021] [Figure 3] Anti-AAV8 capsid / viral particle antibody (potency level reversed after oral administration)

[0022] [Figure 4] Anti-AAV8 peptide-specific antibody titer after oral administration

[0023] [Figure 5] Far-ultraviolet CD spectra of Cas9 and LysoPS-Cas9

[0024] [Figure 6] Unfolding profiles of Cas9 and LysoPS-Cas9 obtained by far-ultraviolet CD spectroscopy

[0025] [Figure 7] Comparison of anti-cas9 antibody titers between week 7 and week 17 in the treatment group. Each sample is represented by a black circle, and a positive control sample (mouse plasma sample induced with Cas9 for 3 weeks but without any treatment) is added as an internal control and represented by a black triangle. Samples with a dashed line indicate an increase in anti-Cas9 antibody, and the number on the graph represents the number of animals with a decrease in antibody titer. The concentration of Cas9 antibody is expressed in ng / ml using a standard antibody, and is shown as a 50-fold dilution.

[0026] [Figure 8] Elevated levels of LAG3+CD49b+CD8+ cells were observed in the spleen of LysoPS-AAV8 supply animals after oral administration. Detailed explanation of disclosure

[0027] As used herein, the term “target protein” means any protein for which immunological low responsiveness is desired. For example, the target protein may be a protein or peptide derived from a microorganism or a variant thereof used as a delivery vehicle (e.g., for polynucleotide delivery).

[0028] A "specific" immunological response means that the immune response to unrelated proteins (proteins that did not complex with the liposomes of the priming composition) is unaffected.

[0029] "To be administered" or "administered" means that a protein or peptide is delivered to an individual or introduced into the body of an individual by any means or route of delivery.

[0030] The "inhibitory" titer or antibody against a protein or antigen refers to a specific antibody produced against that protein or antigen.

[0031] "Lipid structures" refer to liposomes and other structures (micelle structures, liposomes, cochleates, molecular assemblies, etc.).

[0032] As used herein in relation to phospholipids, the term "lyso" means that the glycerol portion of the molecule has only one acyl chain (rather than two). For example, lyso-PS has only one acyl chain, whereas PS has two.

[0033] A liposome-complexed protein or a liposome-complexed protein means that the protein can associate with a particle in one or more of the following configurations: the protein is located in the lumen of the particle, partially or completely intercalates into the bilayer, or binds to or adsorbs to the surface of the particle. As an example, data on liposome-protein complexes for adeno-associated virus (AAV) capsid proteins are provided herein.

[0034] Liposomes may be referred to herein as lipid nanoparticles or nanoparticles. Phospholipids for preparing liposomes can be obtained, for example, from any available source (e.g., from plants or animals). Phospholipids are commercially available or can be synthesized by known methods. For example, phosphatidylserine (PS) can be obtained from porcine brain PS or plant-derived soy (e.g., soybean) PS. Lyso-PS is also commercially available. In this specification, examples of protein complexes and induction of immune tolerance may refer to specific proteins, but these are equally applicable to other proteins.

[0035] Abbreviation: Adeno-associated virus (AAV); viral protein (VP); inverted terminal repeats (ITR); recombinant adeno-associated virus (rAAV); neutralizing antibody (NAb); wild-type AAV (wtAAV); conjugating antibody (BAb); immunoglobulin G (IgG); dendritic cells (DC); interleukin-10 (IL-10); transforming growth factor β (TGF-β); regulatory T cells (Treg); T cell-independent antigen (TI); T cell-dependent antigen (TD); microfold cells (M-cells); gut-associated lymphoid tissue (GALT).

[0036] This disclosure provides compositions and methods for suppressing the immune response to viral vector proteins in therapeutic or prophylactic treatment of pathological conditions using viral vector delivery vehicles. The methods of this disclosure involve administering to an individual in whom suppression of the immune response is desired a composition comprising liposomes or other lipid structures comprising phosphatidylcholine (PC) and PS, wherein some or all of the PS exists as lyso-PS, and the liposomes or lipid structures form complexes with viral vector proteins or peptides, or fragments or modified versions (modifiers) of such proteins or peptides. For example, any protein or fragment thereof encoded by an adenovirus vector (or any other viral vector) or a peptide can be used (e.g., AAV serotypes from AAV1 to AAV11, all subtypes from AdA to AdG). The sequences of these and other proteins are well known. The UniProt acceptance numbers are A0A0S0C249, A0A0S0DU13, A0A0S0DSV8 (VP1 fragment), Q6JC62 (capsid protein VP1), Q8JQF8 (capsid protein), B4Y886 (capsid protein VP1 fragment), Q9YIJ1 (AAV5), Q8JQF8 (AAV8), Q99ZW2 (spCas9), and P04133 (ad5 hexon). Further acceptance numbers are saCas9: J7RUA5, stCas9: G3ECR1, cas12a: U2UMQ6, cas13a: P0DOC6, P0DPB7, C7NBY4, and U2PSH1.

[0037] In several embodiments, the viral vector protein conjugated with lyso-PS liposomes may be any capsid protein or a fragment thereof. Generally, the fragment may consist of 6 to 32 amino acids (including all integer values ​​and ranges between them). For example, the capsid protein or fragment thereof may be derived from AAV2, AAV5, or AAV8. In several embodiments, the viral vector protein may be AAV8 581-596The resulting liposomes, which are complexed with viral vector proteins, are sometimes called liposome complexes.

[0038] This disclosure can be used to reduce existing antibodies against viral vectors used in DNA vaccine formulations. For example, AD5, AD26 and their variants (e.g., variants of AD26 derived from chimpanzee AD) can be used.

[0039] In several embodiments, the viral vector may be any viral vector used for the delivery of nucleic acids to a mammalian host. Examples include, but are not limited to, adenoviruses, such as recombinant adeno-associated virus (rAAV) (which may be referred to interchangeably as adenovirus vectors herein), retroviruses, lentiviruses, herpes simplex virus (HSV), and baculoviruses. Examples of proteins derived from viral vectors include capsid proteins and their antigenic fragments. The method may involve administering a composition comprising a viral vector protein or peptide or its antigenic fragment, conjugated in liposomes containing PC and lyso-PS, to an individual in need of treatment.

[0040] These liposomes can be prepared by trigger loading techniques as described herein and illustrated in the examples. The first step involves increasing the temperature and / or decreasing the pH to generate a structurally altered state of the protein or peptide. For example, AAV8 581-596The peptides can be loaded into liposomes by exposure to high temperatures (e.g., up to 70°C) that result in peptide unfolding. In several embodiments, heating can be performed from room temperature to 70°C, for example, from 30°C to 65°C. Alternatively, or in addition, a change in conformation can be induced by lowering the pH (e.g., from 3 to 8). Incubation with lyso-PS liposomes in this state allows the peptides to intercalate into the liposomal bilayer. The liposomes can then be cooled to room temperature (typically between 18 and 25°C, for example, 18, 19, 20, 21, 22, 23, 24, or 25°C). The pH range can be 3 to 8 (e.g., 3, 4, 5, 6, 7, or 8). The liposomal compositions of the present invention can be stored for several months at room temperature in a lyophilized state, up to 48 hours (hr) in a liquid or reconstituted state, several months in a freezer, and several months in a solid state and several weeks in a liquid state under refrigeration.

[0041] In some embodiments, the meeting may be at least 40%, 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, the meeting may be 80-99%, 75-99%, 75-95%, or 80-95%. In some embodiments, the meeting may be 80%, 81%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0042] Liposomes and other lipid structures contain PC and PS, some or all of which may be in the form of lyso-PS. Liposomes or other lipid structures may contain only PS (or lyso-PS) and PC as phospholipids. PS or lyso-PS may be present in a range of 10% to 50% of the total phospholipids in the bilayer, where the percentage is in moles. 1% to 100% of PS (and all values ​​and ranges in between) may be in the form of lyso-PS. For example, liposomes may have PC:lyso-PS in molar ratios of 90:10, 80:20, 70:30, 60:40, or 50:50. In one example, lyso-PS may be 15 to 50 mole%, with the remaining phospholipids being PC. For example, lyso-PS may be 15 to 30%. Whenever a range is mentioned in this disclosure, all values ​​within that range are also included. In one example, only (partially or entirely) of the PS is in the lyso-PS form, while all of the PC have two acyl chains. All phospholipid ratios in this disclosure are molar ratios unless otherwise specified.

[0043] For example, lipid structures such as liposomes (where 30-100% of PS exists as lyso-PS) further contain retinoid acids and / or rapamycin. Thus, lipid structures can contain PS (which may be partially or entirely lyso-PS), PC, and retinoid acids and / or rapamycin. The amount of retinoic acid may be between 0.1 mol% and 10 mol%, and may be any percentage value between these two values ​​up to 10 decimal places. The amount of rapamycin may be between 0.1 mol% and 10 mol%, and may be any percentage value between these two values ​​up to 10 decimal places.

[0044] Acyl chains can be saturated or unsaturated. The length of the acyl chain of PC may be 12 to 22 carbon atoms. It is preferable that both acyl chains are saturated and of equal length. A chain length of 14 (C14:0, dimyristoyl-sn-glycero-3 phosphatidylcholine (DMPC)) was observed to be particularly effective in providing stability to liposomes. A chain length of 18 (1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)) was found to be less effective than DMPC.

[0045] The lyso-PS acyl chain may be unsaturated. It may be 14 to 22 carbon atoms long. It should have at least one double bond. For example, it may be 18:1 (where 18 represents the number of carbon atoms and 1 represents the number of double bonds). In various examples, the acyl chain may have two or three double bonds, but it has been found that liposome stability is better with a single double bond than with two or three double bonds. However, it has been found that at least one double bond is necessary to increase tolerance.

[0046] At least some of the PS is lyso-PS, but the PC is not lyso-PC. Therefore, all PCs have two acyl chains, and at least some PSs have only one acyl chain. For example, the PC:PS ratio is 90:10 to 60:40, where all PSs are lyso-PS. For example, the PC:lyso-PS ratio may be 85:15 to 65:35. In one example, the PC:lyso-PS ratio may be 70:30. In one example, lyso-PS accounts for 15-50% or 15-35%, and the remaining phospholipids are PCs. Only lyso-PS and PCs may be phospholipids present in the bilayer of the liposome. Since liposomes are unstable when the percentage of lyso-phospholipids is higher than 50%, it is preferable that the total lyso-phospholipids in liposomes be less than 50% of the total phospholipids.

[0047] These liposomes may further contain additional phospholipids. For example, any of the liposomes described herein may contain phosphatidylethanolamine (PE). The amount of PS (which may be partially or entirely lyso-PS) may be 0 to 30 mol%, the amount of PC may be 0 to 30 mol%, and the remainder may be PE. For example, the amount of PS (which may be partially or entirely lyso-PS) may be 1 to 30 mol%, the amount of PC may be 1 to 30 mol%, and the remainder may be PE.

[0048] Liposomes containing lyso-PS as described herein were found to be smaller than liposomes containing only PS (without lyso-PS). For example, after filtration through a 0.2 micron cutoff filter, the size of liposomes containing lyso-PS (liposomes containing both lyso-PS and PC) was approximately 50–150 nm (about 90% of liposomes), while the size of liposomes containing PS (PS+PC) was approximately 200–400 nm (about 90% of the population). It was surprising that the lyso-PS liposomes were not as negatively charged despite being smaller than PS liposomes. The zeta potential of lyso-PS liposomes was -10–-17 (for comparison, the zeta potential of PS liposomes is -24–-33, and that of size-matched PS liposomes is -17–-26). The lower negative charge on lyso-PS liposomes may increase protein loading. For example, in PS liposomes, the typical protein:lipid ratio is 1:10,000 molar ratio, but in lyso-PS liposomes, proteins can be loaded and used in molar ratios of at least 1:5,000. Proteins can be loaded into lyso-liposomes in molar ratios of 1:1,000 to 1:10,000 (and all ratios in between) or 1:100 to 1:10,000 (and all ratios in between).

[0049] In one embodiment, the disclosure provides a composition comprising liposomes. For example, a composition may contain a plurality of liposomes described herein in a suitable carrier. The suitable carrier may be a buffer or other pharmaceutical carrier or additive, excipient, stabilizer, or a combination thereof. For example, liposomes can be formulated with sugars, starch, cetyl alcohol, cellulose, tragacanth powder, malt, gelatin, talc, oil, glycol, glycerol monooleate, polyol, polyethylene glycol, ethyl alcohol, additional emulsifiers, etc. Examples of pharmaceutically acceptable carriers, excipients, and stabilizers are listed in "Remington: The Science and Practice of Pharmacy (2012) 22nd Edition, Philadelphia, PA. Lippincott Williams & Wilkins".

[0050] This priming composition can be formulated for oral delivery. The composition may be delivered directly to a desired location in the gastrointestinal tract using enteral nutrition. Alternatively, they can be formulated as liquids, suspensions, tablets (including enteric-coated tablets), gels, capsules, powders, or any other form that can be ingested. The formulations may include pharmaceutical carriers known to be used in oral formulations. The formulations may be pediatric formulations and may include various flavors, etc.

[0051] This composition is not only useful for inducing immune tolerance, but surprisingly, it is also useful for reducing the antibody titers of existing antibodies.

[0052] In some embodiments, the method comprises administering to an individual a first composition (also referred to herein as a priming composition) (which may comprise one or more doses) comprising a target protein (such as a viral vector protein) complexed with liposomes containing PS and PC (referred to herein as PS liposomes), wherein at least a portion of the PS may exist as lyso-PS. In some embodiments, all of the PS may exist as lyso-PS. The administration(s) of the priming composition(s) may be followed by gene therapy, CRISPR-mediated gene editing, or vector vaccine, wherein the gene therapy, CRISPR-mediated gene editing, or vector vaccine comprises administering a viral vector containing a gene encoding a viral protein and a therapeutic or immunoprotein gene. The priming composition(s) may be administered once or more times per week. The priming composition(s) may be administered, for example, once or two to six times per week, or daily. Administration may take place for one, two, three, four, five, six weeks, or more, and then be discontinued. The therapeutic or immunological composition may be delivered before the priming composition administration regimen, during a period overlapping with the administration regimen, or after the discontinuation of the administration regimen. In one embodiment, the gene therapy / vector vaccine composition may be administered to the individual after a period suitable for inducing immune tolerance (e.g., at least 4 days after the last primer).

[0053] In several embodiments, the priming composition contains only proteins or antigenic fragments thereof from a viral vector and does not contain therapeutic or immunological proteins.

[0054] In one embodiment, the priming composition may be administered for up to several weeks. For example, the priming composition may be administered for four weeks. The frequency of oral administration of the priming composition (such as protein-lysoPS liposomes) may be once a week or more for four weeks. Higher doses of protein-lysoPS may also be used.

[0055] The priming composition may be administered once or multiple times, or continuously (sequentially). Similarly, the second composition (therapeutic composition or vaccine composition) may be administered once or multiple times, or continuously (sequentially), or intermittently as needed. The second composition may be administered together with the first composition, or after the administration of the first composition has begun (e.g., 1 to 6 weeks after the start of administration of the first composition), or simultaneously with or at the start of administration of the first composition, or within 1 to 6 weeks after the start of administration of the first composition. In one embodiment, the priming composition may be administered on a continuous schedule, and the second composition (also referred to herein as the therapeutic composition) may be administered with a time lag from the start of administration of the priming composition, but may overlap with the administration schedule of the first composition. Thus, the administration of the second composition may be simultaneous with the administration of the first composition, or after the initial administration period of the first composition alone.

[0056] In several embodiments, one or more doses of the priming composition may be administered sequentially over a period of time to reduce existing antibodies or to reduce the production of antibodies against the vector-delivery vehicle protein. Therefore, the administration regimen may include substantially regular administrations, which may be, for example, daily or weekly, or more frequently or less frequently, and may be administered via any route. Such an administration regimen may be carried out over several weeks, months, or years. The intervals between consecutive administrations may be the same or different, but generally, periodicity of administration will be maintained.

[0057] This composition can be used in any individual (e.g., human or non-human mammal). The individual may or may not show recent signs of immune intolerance. It is also useful for administration to naive individuals (i.e., individuals who have not previously been administered a protein or peptide). As used herein, immune intolerance means that an individual has antibody production that can be measured (by standard methods such as ELISA or activity assays). Conversely, the absence of immune intolerance (or a state of immune tolerance) means that an individual does not have measurable antibodies. Antibodies can be measured after exposure to the free protein or administration of the free protein after the individual has been "tolerized". As used herein, the term "tolerized" means administering one or more doses of a priming composition and giving the individual an appropriate length of time (e.g., 4 to 30 days, etc.) to develop immune tolerance. The development of immune tolerance to a target protein also involves down-regulation of the expression of co-stimulatory signals in dendritic cells, CD4 + It can also be identified by measuring a decrease in T cell proliferation and induction of the secretion of the immunomodulatory (regulatory) cytokines TGF-β and IL-10. One or more of these identifiers can be evaluated under culture conditions. The expression of immune tolerance is specific to the protein for which tolerance is induced (i.e., the protein complexed with the liposome of the priming composition).

[0058] In one embodiment, a priming composition can be administered and then the induction of immune tolerance can be determined by evaluating one or more of the following: down-regulation of the expression of co-stimulatory signals in dendritic cells, CD4 + A decrease in T cell proliferation, induction of the secretion of the immunomodulatory cytokines TGF-β and IL-10, or a decrease in antibody titer compared to before administration of the priming composition. After confirming the induction of immune tolerance to the viral vector protein, gene therapy or vaccination can be performed.

[0059] The following statements illustrate various embodiments of the present disclosure. statement 1 A method for reducing an existing antibody titer against a viral vector protein, comprising administering a composition comprising liposomes to an individual in need of treatment, wherein the liposomes comprise phosphatidylcholine (PC) and lysophosphatidylserine (lyso-PS), the PC:lyso-PS ratio being 90:10 to 60:40, some or all of the PS existing as lyso-PS, and the liposomes being complexed with the protein or its antigenic fragment. statement 2 The method according to Statement 1, wherein the acyl chain of lyso-PS is oleic acid. statement 3 The method described in Statement 1 or Statement 2, wherein all PSs exist as lyso-PSs. statement 4 The method described in any one of the aforementioned statements, wherein the PC to lyso-PS ratio is between 85:15 and 70:30. statement 5 The method according to Statement 1, wherein the PC exists as dimyristoyl-sn-glycero-3 phosphatidylcholine (DMPC). statement 6 The method according to Statement 1, wherein the peptide is a capsid protein or a Cas protein. statement 7 The method according to Statement 1, wherein the sequence of the peptide is IVADNLQQNTAPQIG (Sequence ID 1). statement 8 A method for reducing the production of antibodies against a viral vector protein, comprising administering a composition comprising liposomes to an individual in need of treatment, wherein the liposomes comprise phosphatidylcholine (PC) and lysophosphatidylserine (lyso-PS), the ratio of PC to lyso-PS being 90:10 to 60:40, some or all of the PS being present as lyso-PS, and the liposomes being complexed with the protein or its antigenic fragment. statement 9 A composition comprising liposomes, wherein the liposomes comprise phosphatidylcholine (PC) and lysophosphatidylserine (lyso-PS), the ratio of PC to lyso-PS being 90:10 to 60:40, and the liposomes are complexed with a protein encoded by a viral vector or an antigenic fragment thereof. statement 10 The composition according to Statement 9, wherein the acyl chain of lyso-PS is oleic acid. statement 11 The composition according to Statement 9 or Statement 10, wherein the ratio of PC to lyso-PS is 85:15 to 70:30. statement 12 The composition according to any one of statements 9 to 11, wherein PC is present as dimyristoyl-sn-glycero-3 phosphatidylcholine (DMPC). statement 13 The composition according to any one of statements 9 to 12, wherein the protein or peptide is a viral capsid protein or peptide. statement 14 The composition according to any one of statements 9 to 13, wherein the protein is a Cas protein. statement 15 The composition according to any one of statements 9 to 13, wherein the sequence of the protein or peptide is IVADNLQQNTAPQIG (Sequence ID 1). statement 16 The composition according to any one of statements 9 to 13, wherein the protein or peptide is Ad5. Statement 17 The composition according to any one of statements 9 to 14, wherein the Cas protein is Cas9.

[0060] The following examples are provided for illustrative purposes only and are not intended to limit you. [Example 1]

[0061] The synthesis and use of the liposomes and liposome complexes of this disclosure are described below.

[0062] Materials and methods

[0063] Lysophosphatidylserine (Lyso-PS) and dimyristoylphosphatidylcholine (DMPC) were purchased from Avanti Polar Lipids (Alabaster, AL) and stored in chloroform at -80°C. AAV8 and AAV5 capsids were obtained from Vigene Biosciences (Rockville, MD). AAV8 581-596 The following were purchased from GenScript and reconstituted with sterile water (Piscataway Township, NJ). Tween 20, hydrogen peroxide, and phosphorus standard solution were obtained from Sigma (Saint Louis, MO). Fetal bovine serum was purchased from Biowest (Riverside, MN). Rhodamine PE was purchased from Avanti Polar Lipids (Alabaster, AL). Alkaline phosphatase-conjugated goat anti-mouse Ig antibody was purchased from Southern Biotech (Birmingham, AL). Horseradish peroxidase-conjugated goat anti-mouse IgG antibody and 3,3',5,5'-tetramethylbenzidine substrate (TMB) were purchased from Sigma Aldrich (St. Louis, Missouri). Endosafe Endochrome-K (登録商標) The kit was purchased from Charles River Laboratories (Charleston, SC). The NUNC MaxiSorp 96-well plate was purchased from Thermo Fisher Scientific (Rochester, NY). The CD21 / CD35 monoclonal antibody (8D9), PE conjugate [12-0211-82], and the CD23 monoclonal antibody (B3B4), FITC conjugate [11-0232-81] were purchased from eBiosciences. TMI obtained them from Charles River Laboratories (Charleston, SC). The Swiss-Webster mouse was obtained from Charles River Laboratories (Charleston, SC).

[0064] animal

[0065] Eight-week-old male Swiss-Webster mice were purchased from Charles River Laboratory (Kingston, NY) and maintained in situ. All animal experiments were approved and followed the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the State University of New York at Buffalo. At the end of the experiments, the mice were sacrificed via cardiac puncture under isoflurane anesthesia.

[0066] Preparation of Lyso-PS nanoparticles

[0067] Lyso-PS liposomes were prepared with a lyso-PS:DMPC molar ratio of 30:70. A rotary evaporator (Buchi-R200, Fisher Scientific) was used to form a lipid film by evaporation of chloroform solvent. Next, the lipid film was rehydrated in Tris-calcium chloride buffer (1x, pH 7.4) and incubated at 37°C. Liposomes were extruded several times through a 200 nm pore size polycarbonate membrane (double-layered) using a nitrogen-pressurized extruder (Mico, Inc., North Mankato, WI). The liposomes were then sterilized by filtration using a 0.2 μm pore size syringe filter (Corning, NY). Tris-calcium chloride buffer was tested for endotoxin, and it was confirmed to be endotoxin-negative. In the treatment group administered AAV8 capsid, the protein-to-lipid molar ratio was set to 1:10,000. 581-596In the treatment group administered the peptide, the molar ratio of protein to lipid was set to 1:1,000. Association of AAV8 capsid to lyso-PS liposomes and AAV8 to lyso-PS liposomes 581-596 Peptide association was achieved by incubating the two complexes at 37°C for 30 minutes, which triggered the loading of the protein and peptide into the liposomes. Phosphate assays were performed to confirm the lipid content of the final liposomal formulation, and the size distribution of the liposomes was measured using a NICOMP Model CW380 particle size analyzer (Particle Sizing Systems (Port Richey, FL)).

[0068] AAV8 into Lyso-PS liposomes 581-596 Measurement of peptide association efficiency

[0069] The association efficiency of the amount of protein associated with PS liposomes was pre-evaluated using size exclusion column chromatography. A size exclusion column was prepared using G-50 Sephadex beads. The liposomes were fluorescently labeled with rhodamine-PE so that they could be detected upon elution from the column. 1 mol% of rhodamine phosphatidylethanolamine (PE) was incorporated into this lipid mixture before evaporation, and then liposomes were prepared as described in the previous section. LysoPS-rhodamine liposomes were processed using AAV8. 581-596 Combined with (500 μg / ml AAV8 581-596 (A protein-to-lipid ratio of 1:50 was used.) The eluent fraction was recovered from the column after the run. Liposome content was determined by measuring the fluorescence of rhodamine by excitation at 560 nm and emission at 583 nm. Protein content in each fraction was quantified using a Micro-BCA assay kit (ThermoFisher, Waltham, MA).

[0070] Measurement of the association efficiency of AAV8 capsids to Lyso-PS liposomes

[0071] The association efficiency of proteins associated with PS liposomes was pre-evaluated using a Nanosep 30K centrifuge (Pall Laboratories, Port Washington, NY). Liposomes were prepared as described in the previous section. LysoPS-AAV8 complexes were prepared using 30 μg / ml AAV8 (protein-to-lipid molar ratio of 1:10,000). These complexes were incubated at 37°C for 30 minutes. The LysoPS-AAV8 complex samples were placed in the Nanosep instrument and centrifuged at 9,300xg at 25°C for 5 minutes. The pellet obtained after centrifugation was collected and analyzed using a Micro BCA protein assay kit (ThermoFisher, Waltham, MA). Inclusion efficiency was reported as the average after performing this experiment three times.

[0072] Induction of anti-AAV8 antibody titers in Swiss Webster mice

[0073] Fifty naive, untreated 8-week-old Swiss Webster male mice were first divided into two distinct groups (Group 1, containing 20 mice, and Group 2, containing 30 mice). Both groups received weekly subcutaneous injections to induce anti-AAV8 antibody titers specific to AAV8 peptide and AAV8 capsid. In Group 1, a dose of 1 μg of free AAV8 was administered. 581-596 Peptides were administered subcutaneously for six consecutive weeks. In Group 2, a dose of 1 μg of free AAV8 capsid was administered subcutaneously for six consecutive weeks (Figure 1). All subcutaneous injections were performed on anesthetized animals, and fluids were administered after each injection according to the IACUC protocol. Blood was collected from the saphenous vein one week after each subcutaneous injection. Blood samples were rotated at 5000 g for 5 minutes to collect plasma, which was then stored at -80°C. Total anti-AAV8 antibody titer levels were measured over the period of subcutaneous injections from week one to week six. Free antigen (AAV8 capsid and AAV8) 581-596Peptide administration was discontinued after animals in Group 1 and Group 2 reached equivalent antibody titer levels. Animals that did not produce equivalent titers were excluded from the study, and a reversal study was conducted using the remaining animals. Sixteen mice from Group 1 and 24 mice from Group 2 were selected for the reversal study after antibody titer induction, and the rest were sacrificed. Figure 2 shows the classification of the animals used.

[0074] Reversal of pre-induced anti-AAV8 titer in Swiss Webster mice

[0075] A total of 40 animals already possessing anti-AAV8 antibody titers were used for this study. Animals in Group 1 (Figure 2) were divided into two treatment groups: "Lyso-peptide" and "Lyso-capsid 1". Mice in Group 2 (Figure 2) were divided into three different treatment groups (each group containing 8 animals). The treatment groups were: "Lyso-capsid 2", "free capsid", and "buffer". Animals in the lyso-peptide group received a 1 μg dose of lyso-PS-AAV8. 581-596 A peptide preparation was administered (protein to lipid molar ratio of 1:1,000). AAV8 581-596 Mice in the lyso-capsid 1 group with pre-established antibody titers against the peptide were administered a 1 μg dose, which is approximately 2 × 10⁶. 6 This corresponds to AAV8 capsids conjugated with lyso-PS liposomes at a concentration of gc / kg (protein-to-lipid molar ratio: 1:10,000). Animals in the lyso-capsid 2 group, which had pre-induced antibody titers against complete AAV8 capsids, were administered the same formulation as the lyso-capsid 1 group. This formulation contained 1 μg (approximately 2 × 10⁻¹⁶) of AAV8 capsid conjugated with lyso-PS liposomes. 6 The sample consists of gc / kg (the molar ratio of protein to lipid is 1:10,000). The positive control group received 1 μg of free AAV8 capsid (2 × 10⁻¹⁶). 6The animals were administered gc / kg of the drug, while the negative control group received only Tris-Calcium Chloride Buffer. All animals received each formulation via forced oral administration for 9 consecutive weeks. The amount of oral injection was 100 μL each week. Blood was collected before each weekly administration, and weekly at weeks 7, 9, 11, 13, and 15, and anti-AAV8 capsid and peptide-specific antibody titers were analyzed. Terminal blood samples were collected via cardiac puncture for titer analysis, and the spleen was collected and analyzed by flow cytometry.

[0076] Determination of anti-AAV8 antibody titer

[0077] Antibody titers were determined by ELISA using the Frey method for titer analysis. According to this method, the endpoint titer is defined as the reciprocal of the highest sample dilution that yields an absorbance value above the cutoff value. A formula is used to determine the cutoff value or "upper predictive limit," where the upper predictive limit is expressed as the standard deviation multiplied by a coefficient based on the number of negative controls and the confidence level (1-α). Unlike other methods that arbitrarily set the cutoff value, this method allows for the calculation of the upper predictive limit using the Student t-distribution. A 96-well Maxisorb plate was used for this immunoassay. Anti-AAV8 581-596 Antibody titer determination is performed using 5 μg / mL of AAV8 in sodium carbonate-bicarbonate buffer. 581-596 This was performed by coating the plate with 50 μL of peptide and incubating it overnight at 4°C. For the determination of anti-AAV8 capsid antibody titers, the plate was coated with 8 × 10⁶ of peptide in a 100 μL volume. 7The genomes were coated with the AAV8 capsid vector. The plates were then washed six times with phosphate buffer containing 0.05% w / v Tween20. Nonspecific binding was blocked with 200 μL of 0.1% w / v solution of 1% casein in phosphate buffer and incubated at room temperature for 2 hours. Plasma samples were diluted 3-fold. After incubation, the plates were washed and 50 μL of each sample was added to the plates (double). To account for plate variability, three control samples were used from naive, untreated animals, and blocking buffer was added in triples along with the sample runs. Controls were added to the plates in triples. Samples were incubated at 37°C for 2 hours and washed again. 100 μL of goat anti-mouse immunoglobulin peroxidase diluted 1:5,000 with blocking buffer was added to each well and incubated at 37°C for 2 hours. The plates were washed again, and 100 μL of 3,3',5,5'-tetramethylbenzidine was added to each well. The mixture was incubated at room temperature for 45 minutes without light. After 45 minutes, 100 μL of 1N hydrochloric acid was added to each well to stop the reaction, and the color intensity at 450 nm was read using a Spectramax plate reader. After 45 minutes, 100 pL of IN hydrochloric acid was added to each well to stop the reaction, and the color intensity at 450 nm was read using a Spectramax plate reader. Total antibody titers were measured using plasma samples obtained from weekly blood collections and analyzed for comparison between different treatment groups.

[0078] statistical analysis

[0079] All statistical analyses were performed using GraphPad Prism 8 (La Jolla, CA). One-way ANOVA and Tukey post-hoc analysis were performed as instructed. Nonparametric Kruskal-Wallis tests and Dunn post-hoc analyses were also performed. A p-value < 0.05 was considered statistically significant, and significant results are indicated with an asterisk (*).

[0080] AAV8 581-596 Association efficiency of peptides and AAV8 capsids to Lyso-PS nanoparticles

[0081] Size exclusion chromatography was performed in Tris-calcium chloride buffer (pH 7.4) at 37°C, and AAV8 was obtained. 581-596 The association efficiency of peptides to Lyso-PS nanoparticles was measured. Lyso-PS nanoparticles with a diameter of approximately 200 nm were eluted using a Sephadex column containing G-50 beads, and AAV8 associated with the nanoparticles was measured. 581-596 Free peptides were separated from the peptides. In Tris-calcium chloride buffer at 37°C, the mean association efficiency was 38.3% with a standard deviation of 9%. The association efficiency was reproducibly achieved with an acceptable protein recovery rate of 19.1 μg. The association efficiency of the capsid protein to the nanoparticles was confirmed using a nanosep 30K centrifuge, and the protein content was analyzed using a micro-BCA assay. The mean encapsulation efficiency of AAV8 capsids into lyso-PS nanoparticles (from three independent operations) was 40.7% with a standard deviation of 8.8%. These results are relevant to AAV8. 581-596 This demonstrates that a viable lyso-PS associated AAV8 capsid has been achieved, similar to peptide nanoparticles. 581-596 Table 1 shows all the data demonstrating the association efficiency of peptides and AAV8 capsids to lyso-PS nanoparticles. Results from three different size exclusion chromatography experiments using Sephadex G-50 beads are shown. The encapsulation efficiency of AAV8 capsids into lyso-PS nanoparticles was measured using a nanosep 30K centrifuge, and micro-BCA assays were used to quantify the free and encapsulated proteins. The results from three independent measurements are shown in Table 1. Mean ± SD association efficiency is shown as the result.

[0082] [Table 1]

[0083] Orally administered Lyso-PS-AAV8 581-596 The complex is AAV8 581-596 To induce tolerance towards

[0084] Swiss Webster Mouse, lyso-PS-AAV8 581-596 The peptide complex was administered for 9 weeks. At week 6, the free peptide was administered subcutaneously to the animals for challenge. If Lyso-PS induces tolerance, mice pre-exposed to the Lyso-PS-AAV8 peptide would show a lower response to re-challenge compared to animals pre-exposed to the free antigen or those treated as shams. Saphenous blood samples were collected at weeks 9 and 10. Anti-AAV8 581-596 Antibody titer levels were measured. At week 10, Lyso-PS-AAV8 581-596 Compared to the control buffer treatment group, AAV8 581-596 The antibody titer level was significantly lower (p-value = 0.0162). LysoPS-AAV8 581-596 The group had an average SEM of 75.713.3 and free AAV8 581-596 The ratio was 164.922.5 for the individual group and 187.737.4 for the buffered treatment group. These data suggest that Lyso-PS induced tolerance.

[0085] Orally administered Lyso-PS-AAV8 capsid and Lyso-PS-AAV8 581-596 Peptide nanoparticles may reverse the titer of existing anti-AAV8 antibodies.

[0086] The mice were divided into two groups, and Group 1 (n=20) was given 1 μg of free AAV8. 581-596Group 2 (n=30) received 1 μg of free AAV8 capsid protein (Figure 2). Both groups received subcutaneous administration of the respective proteins for 6 consecutive weeks to ensure that anti-AAV8 antibody titers were sufficiently high (Figure 3). At week 6, abnormal values ​​were excluded from the study. For the reversal test after subcutaneous antibody titer induction, 16 animals were selected from Group 1 and 24 animals from Group 2. At the end of subcutaneous antibody titer induction, the 16 animals selected from Group 1 were divided into two treatment groups of 8 animals each (n=8), as shown in Figure 2. Meanwhile, the 24 mice selected from Group 2 at the end of subcutaneous induction were further divided into three treatment groups. Blood was collected weekly from the saphenous vein, and anti-AAV8 capsid antibody titers and anti-AAV8 peptide antibody titers were measured at weeks 7, 9, 11, 13, and 15, when oral administration was performed. Figure 3 shows the anti-AAV8 capsid-specific antibody titers, comparing the Lyso-PS-AAV8 capsid group at week 6 (after antibody titer induction) and week 11 (after 5 oral administrations). At week 6, before oral administration, the average antibody titer was approximately 130 AU. At week 11 (week 5 of oral administration), a clear decrease in anti-AAV8 titers was observed. A similar comparison was made in animals administered free AAV8, where an increase in titer was observed between week 6 and week 11, while the buffer administration group remained similar. This observation demonstrates that oral administration of Lyso-PS-AAV8 capsid reverses existing antibodies against the AAV8 capsid. A similar trend continued between the administration groups as oral administration progressed. 581-596 Peptide titer levels are represented in the form of a bar graph from week 7 to the end of treatment (week 15) (Figure 4). The Lyso-peptide treatment group showed similar results. At the end of 9 weeks of oral administration, the two treatment groups, "lyso-peptide" and "lyso-capsid 1," showed AAV8 581-596 A clear downward trend was observed in peptide-specific titers. [Example 2]

[0087] The uses of the liposomes and liposome complexes described herein are described below.

[0088] Adenovirus (Ad) is a non-enveloped linear double-stranded DNA virus, and 57 human Ad serotypes have been identified. Ad serotypes differ in tropism and are further divided into six subgroups, A to G. The differences between subgroups represent different serotypes. The viral capsid is composed of capsid protein, core protein, and cement protein. Adenovirus virions are 90-100 nm in size and have a unique viral structure as non-enveloped icosahedral particles containing a nucleocapsid. The viral capsid consists of 252 proteins, including three different types: fiber, penton, and hexone-based proteins. There are 240 hexone proteins and 12 penton proteins. Each hexone capsomere is a homotrimer of hexone proteins and is a complex protein of approximately 900 residues. Each hexone monomer has seven flexible serotype-specific loops, called the hypervariable region (HVR), and these HVRs differ subtly depending on the serotype. The adenovirus hexone 5 protein belongs to subgroup C, has a molecular weight of 108 kDa, and an isoelectric point of 5.15.

[0089] Preparation of LysoPS-Ad5

[0090] LysoPS lipid nanoparticles were prepared in chloroform with a molar ratio of LysoPS to DMPC of 30:70. The LysoPS lipid nanoparticles were prepared using a dehydration and rehydration method. The chloroform solvent was removed by rotary evaporation, forming a thin lipid film at the bottom of the test tube, and rehydrated with 1 mL of sterile 5 mM citrate buffer at pH 4.5. The Lyso-PS lipid nanoparticles were extruded through a 200 nm pore size polycarbonate membrane using high-pressure nitrogen extrusion. The average diameter of the lipid nanoparticles was confirmed by dynamic light scattering (Nicomp 380 Particle Sizer, Particle Sizing System, Port Richey, Florida). Lipid concentration was determined by phosphate assay. The molar ratio of Ad5-hexone protein (Bio-rad) to lipid used was 1:10,000. To form adenovirus 5 hexone protein-lyso-PS lipid nanoparticles (Lyso-Ad5), the hexone protein and LysoPS lipid solution were incubated at 37°C for 30 minutes to associate, thereby loading the ad5-hexone protein onto the Lyso-PS lipid nanoparticles using a trigger loading mechanism.

[0091] Biophysical characterization

[0092] Lyso-PS-Ad5 complexes were prepared as described above and separated by ultracentrifugation at 60000 RCF and 4°C for 1 hour. Associated Lyso-PS-Ad5 formed a pellet, while unassociated hexone proteins were contained in the supernatant. Lyso-PS-Ad5 was recovered and digested with 1% Tween20 to disrupt the LysoPS bilayer, and incubated at 37°C for 15 minutes. Samples were quantified using a micro-bca assay. The association efficiency was calculated using the following formula.

number

[0093] The association efficiency of Lyso-PS-Ad5 was 66.1% ± 5.47%, and the particle size increased from (98.5 nm ± 2.55) to (165.8 nm ± 7.53) after association. See Table 2.

[0094] [Table 2] [Example 3]

[0095] The uses of the liposomes and liposome complexes described herein are described below.

[0096] A rational design for Cas9 associated with lyso-PS liposomes:

[0097] The Cas9 protein successfully associated with Lyso-PS nanoparticles for CRISPR-based therapy. This condition was identified based on conformational and folding analyses of Cas9 under stress conditions. Based on these biophysical studies, the association conditions were confirmed to be HEPES buffer, 37°C, and approximately 40 minutes. The association efficiency with Lyso-PS liposomes was 42.8 ± 13.5 (mean ± SEM) (n=3). The diameter of the Lyso-PS nanoparticles increased from 98.7 nm ± 2.3 to 233.5 nm ± 43.1 after association. The Lyso-PS-Cas9 complex was characterized using far-ultraviolet circular dichroism (CD) (Figure 5). These data indicated that association of Cas9 with Lyso-PS did not alter the secondary structure of Cas9. Furthermore, unfolding studies using far-ultraviolet CD were performed to investigate whether Lyso-PS association altered protein stability (Figure 6). Spectral analysis revealed that Lyso-PS improves the stability of Cas9. Cas9 was purchased from Sigma Aldrich (St. Louis, MO).

[0098] Reversal of existing Cas9 titers using Lyso-PS-Cas9 nanoparticles

[0099] Male Swiss Webster mice were subcutaneously immunized with 1 μg of Cas9 protein (derived from Streptococcus pyogenes) weekly for a total of 4 weeks to stimulate and produce anti-Cas9 antibodies, followed by a 3-week washout period. Host immunity to Cas9 protein was measured by anti-Cas9 antibody ELISA.

[0100] Mice were grouped according to their anti-Cas9 antibody titer (tites were equivalent between the buffer administration group and the Lyso-PS-Cas9 complex administration group). Oral administration was started at week 7, with 100 μL of either 200 mM HEPES buffer or Lyso-PS-Cas9 complex administered. In the LysoPS-Cas9 complex group, Cas9-containing Lyso-PS lipid nanoparticles (protein-to-lipid ratio of 1:10,000) were prepared using 1 μg of protein, and Cas9 was trigger-loaded into the nanoparticles by incubation at 37°C for 30 minutes in 200 mM HEPES buffer at pH 7.4. These data showed that mice administered with the Lyso-PS-Cas9 complex exhibited a decrease in anti-Cas9 antibody titer; 5 out of 6 mice showed a decrease, while 1 showed an increase (Figure 7) (likely due to visible injury). In the group treated with HEPES buffer, only 2 out of 7 mice showed a decrease in anti-Cas9 antibody titer. [Example 4]

[0101] The uses of the liposomes and liposome complexes described herein are described below.

[0102] Rational design of AAV5 associated with lyso-PS liposomes

[0103] Lyso-PS liposomes were prepared in PBS (pH 6) containing 1 mM CaCl2. The liposomes were incubated with AAV5 at a protein-to-lipid ratio of 1:10,000 at 45°C for 30 minutes. Free and associated proteins were separated by ultracentrifugation, and the liposomes were pelleted by rotating at 60,000 g, leaving the free AAV5 in the supernatant. After rotation, the pellet was rehydrated with 300 μL of buffer, and the protein amount was measured by a microBCA assay. The amount obtained from BCA was compared to the initial protein amount added to the ultracentrifuge. Under these conditions, 43.2% of the AAV5 was found to be associated. The complex was analyzed by dynamic light scattering (DLS) to determine its size. The size of the liposomes before association was 121.8 nm (SD=1), and it increased to 220.9 nm (SD=9.8 nm) after association with AAV5.

[0104] [Table 3] [Example 5]

[0105] The uses of the liposomes and liposome complexes described herein are described below.

[0106] Inversion tests were performed in mice using Lyso-PS-AAV8.

[0107] Initial anti-AAV8 titers were induced in all mice by subcutaneous administration of 5e9VP / mouse AAV8 prepared in endotoxin-free PBS (pH=7.28). All mice received subcutaneous induction administration seven times a week. Titers were analyzed using an in-house developed anti-AAV8 ELISA assay, and the mice were divided into three groups (n=16) with similar mean titers and titer distributions. The treatment groups were buffer only, LysoPS liposomes only, or an aggregate of Lyso-PS and AAV8 (Lyso-PS-AAV8). Pre-formed Lyso-PS liposomes were able to associate with AAV8 particles by incubation in citrate buffer (pH=4.5) at 37°C for 30 minutes. Starting at week 10, mice were orally administered weekly for 10 weeks, and titers were monitored by blood collection from the saphenous vein every two weeks. The oral dose of Lyso-PS-AAV8 was 2.5e9VP / mouse, and the Lyso-PS liposomes were approximately 0.0177 μmol / mouse. Two weeks after the final oral dose, half of the mice (n=8) from each group were sacrificed, and the spleen and mesenteric lymph nodes (MLNs) were collected. The samples were stained and fixed, and flow cytometry was performed the following day. The remaining half of the animals (n=8) from each group were intravenously challenged with 5e9VP / mouse free AAV8 to simulate intravenous gene therapy. These mice were sacrificed two weeks after the intravenous challenge, and the spleen and MLNs were collected the following day for flow cytometry.

[0108] The data obtained from these experiments is shown in Figure 8. [Example 6]

[0109] The uses of the liposomes and liposome complexes described herein are described below.

[0110] A reversal test using Lyso-PS-AAV8 was conducted in primates.

[0111] This study used a total of three cynomolgus macaques. Blood and plasma were collected the day before the start of the study to obtain baseline values ​​for comparison with post-treatment values. All animals used in the study had neutralizing antibodies greater than 1:20 before treatment. All three primates were force-administered Lyso-PS-AAV8 four times a week. Pre-formed LysoPS liposomes were able to associate with AAV8 particles by incubation in citrate buffer (pH=4.5) at 37°C for 30 minutes. Primates were administered 2.66e10VP / kg on day 1. Mean size and chi-square values ​​were monitored for all formulations to ensure batch-to-batch consistency. Animals were collected weekly for sample measurement. Endpoints included AAV8 neutralizing antibody assessment, total anti-AAV8 titer analysis, blood and serum chemistry, peripheral blood cytokine levels by multiplex assay, and PBMC phenotyping by flow cytometry. The formulation was well-tolerated, and no blood or serum chemical abnormalities were observed. During this short-term oral immunization regimen, neutralizing antibodies remained above 1:20 throughout the entire study period, but total anti-AAV8 antibodies decreased when evaluated at a 1:20 dilution using our proprietary ELISA assay.

[0112] [Table 4]

[0113] In primate peripheral blood, a transient increase in LAP+Treg was observed on day 29, but it decreased to near baseline within two weeks (day 43). Unstained and FMO control values ​​for LAP+Treg were 1.24% and 1.72%, respectively.

[0114] [Table 5]

[0115] The above description provides specific examples of the present invention. Those skilled in the art will recognize that routine modifications to these embodiments are possible and that they fall within the scope of this specification.

Claims

1. A composition for reducing the titer of existing antibodies against viral vector proteins, The composition comprises liposomes, The liposomes contain phosphatidylcholine (PC) and lysophosphatidylserine (lyso-PS), The ratio of PC to lyso-PS is 90:10 to 60:

40. Some or all of the PS exists as lyso-PS, The liposome is complexed with the protein or its antigenic fragment. The protein or its antigenic fragment contains the sequence IVADNLQQNTAPQIG (SEQ ID NO: 1). composition.

2. A composition for reducing the titer of an existing antibody against the Cas9 protein, The composition comprises liposomes, The liposomes contain phosphatidylcholine (PC) and lysophosphatidylserine (lyso-PS), The ratio of PC to lyso-PS is 90:10 to 60:

40. Some or all of the PS exists as lyso-PS, The aforementioned liposomes are complexed with the Cas9 protein. composition.

3. The composition according to claim 1 or 2, wherein the acyl chain of lyso-PS is oleic acid.

4. The composition according to claim 1 or 2, wherein all of the PS exists as lyso-PS.

5. The composition according to claim 1 or 2, wherein the ratio of PC to lyso-PS is 85:15 to 70:

30.

6. The composition according to claim 1 or 2, wherein PC is present as dimyristoyl-sn-glycero-3 phosphatidylcholine (DMPC).

7. A composition for reducing the production of antibodies against viral vector proteins, The composition comprises liposomes, The liposomes contain phosphatidylcholine (PC) and lysophosphatidylserine (lyso-PS), The ratio of PC to lyso-PS is 90:10 to 60:

40. Some or all of the PS exists as lyso-PS, The liposome is complexed with the protein or its antigenic fragment. The protein or its antigenic fragment contains the sequence IVADNLQQNTAPQIG (SEQ ID NO: 1). composition.

8. A composition for reducing the production of antibodies against Cas9 protein, The composition comprises liposomes, The liposomes contain phosphatidylcholine (PC) and lysophosphatidylserine (lyso-PS), The ratio of PC to lyso-PS is 90:10 to 60:

40. Some or all of the PS exists as lyso-PS, The aforementioned liposomes are complexed with the Cas9 protein. composition.

9. A composition containing liposomes, The liposome comprises phosphatidylcholine (PC) and lysophosphatidylserine (lyso-PS), The ratio of PC to lyso-PS is 90:10 to 60:

40. The liposome is complexed with a protein encoded by a viral vector or its antigenic fragment. The protein or its antigenic fragment contains the sequence IVADNLQQNTAPQIG (SEQ ID NO: 1). composition.

10. A composition comprising liposomes, The liposome comprises phosphatidylcholine (PC) and lysophosphatidylserine (lyso-PS), The ratio of PC to lyso-PS is 90:10 to 60:

40. The aforementioned liposomes are complexed with the Cas9 protein. composition.

11. The composition according to claim 9 or 10, wherein the acyl chain of lyso-PS is oleic acid.

12. The composition according to claim 9 or 10, wherein the ratio of PC to lyso-PS is 85:15 to 70:

30.

13. The composition according to claim 9 or 10, wherein PC is present as dimyristoyl-sn-glycero-3 phosphatidylcholine (DMPC).

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