CN112646871A

CN112646871A - Composition and screening method for screening therapeutic agent for hepatitis B

Info

Publication number: CN112646871A
Application number: CN201910965986.9A
Authority: CN
Inventors: 瞿镕
Original assignee: China Israel Hyde Artificial Intelligence Drug Research And Development Co ltd
Current assignee: China Israel Hyde Artificial Intelligence Drug Research And Development Co ltd
Priority date: 2019-10-12
Filing date: 2019-10-12
Publication date: 2021-04-13

Abstract

The present invention provides a composition for screening a therapeutic agent for hepatitis b, comprising at least one carrier, the at least one carrier comprising: (a) a unicellular therapeutically effective amount of at least one therapeutic agent to be screened for hepatitis B; (b) one or more nucleic acid barcodes uniquely identifying the at least one therapeutic agent for hepatitis b; and (c) optionally a label. The invention also provides a screening method using the composition.

Description

Composition and screening method for screening therapeutic agent for hepatitis B

Technical Field

The invention relates to the technical field of antiviral drug screening, in particular to a screening method of an anti-human hepatitis B virus drug and a corresponding screening composition.

Background

Human Hepatitis B Virus (HBV) infection is a major public health problem worldwide. After acute hepatitis B virus infection, about 8% of hepatitis B virus still develops into chronic hepatitis B infection, and persistent HBV infection can cause cirrhosis and even liver cancer. China is a big country with hepatitis B, and hepatitis B virus carriers are close to 1.3 hundred million people and account for about 9 percent of the total population. Although the new hepatitis B infection rate is effectively controlled along with the wide popularization of hepatitis B vaccines, the population base of hepatitis B carrying population is large, and the prevention and treatment of hepatitis B become the most important public health problem in China. The hepatitis B transmission pathway is mainly through vertical transmission and horizontal transmission. Vertical transmission refers to mother-to-baby transmission; horizontal transmission is primarily through the blood.

Although there are many anti-HBV drugs on the market today, antiviral therapy is mainly performed by using interferon or nucleoside analogs. Wherein the nucleoside analog inhibits HBV production by inhibiting reverse transcriptase activity during HBV replication. Although reverse transcriptase inhibitors can control hepatitis B virus levels in patients, the problems of drug resistance, high medical costs, and serious side effects of drugs are not insignificant. The research of de novo antiviral drugs against HBV itself is an urgent need. In order to obtain a de novo antiviral drug, a suitable drug screening method must first be established.

The existing hepatitis B drug screening methods comprise a cell level screening method, an animal level screening method and the like.

The cell level screening method can intuitively screen the anti-hepatitis B virus effect of the therapeutic agent by taking the model cell as an object, the most widely applied HBV cell model is a HepG22.2.15 cell model which is obtained by Sells and the like in 1986 by transfecting HepG2 cell with recombinant vector pDOLT-HBV-1 (containing 2 HBV head-to-tail dimers which are connected in series in the tail-to-tail direction), and high-level secretion of HBeAg and HBsAg is obtained by screening G418, and HBV virus can be replicated in the HepG22.2.15 cell for a long time and can be maintained for 1 year by subculture. The evaluation indexes under the model comprise the levels of HBV virus DNA (such as cccDNA, rcDNA and dsNDA) and RNA (such as mRNA and pgRNA) in cells, the levels of hepatitis B antigens (such as HBeAg and HBsAg) in cell culture solution and the like, however, the actual antiviral efficacy of the medicament to be screened cannot be comprehensively embodied in a cell level experiment because the factors such as the actual metabolic process, the liver targeting property, the PK/PD and the like of the medicament in a patient body to be treated are ignored. The superior therapeutic agents obtained by screening at the cellular level are not necessarily capable of being equally effective in the body of the subject.

The animal level screening method includes constructing proper animal model and screening anti-hepatitis B virus medicine through in vivo screening process. Human HBV belongs to the hepadnaviridae and has strong species specificity, and only human, non-human primates (such as chimpanzees and the like) and tree shrews are infected by the HBV under natural conditions. In recent years, due to the advantages of clear genetic and immune background, easy feeding and the like, mice gradually receive attention of researchers, and become the best choice for constructing HBV animal models. Researchers have also established human-mouse liver chimera HBV mouse models, gene-transfected HBV mouse models, and HBV transgenic mouse models, among others, see CN104862336A, CN107080757A, etc. (the disclosures of which are incorporated herein by reference in their entirety). Compared with a cell level screening method, the animal level screening method can more comprehensively reflect the actual efficacy of the hepatitis B drug in a patient body, and provides a powerful basis for clinical hepatitis B drug screening. However, the current animal level screening methods still have a lot of problems, such as difficulty in realizing high-throughput, multi-drug simultaneous screening, and low screening efficiency; after the drug is delivered in vivo, liver targeting (including liver targeting and liver cell targeting) is lacked, the treatment efficiency is low, and a high-sensitivity detection marker is lacked, so that the detection difficulty is high, and the drug screening resolution is low.

Liposomes (liposomes) are an artificial membrane. The hydrophilic head of phospholipid molecule is inserted into water, the hydrophobic tail of liposome extends to air, and spherical liposome with double-layer lipid molecule with diameter of 25-1000nm is formed after stirring. The liposome can be used in transgenic technology or used for preparing medicine, and the medicine is delivered into cells by utilizing the characteristic that the liposome can be fused with cell membranes. In addition, by utilizing the passive targeting of the liver and spleen reticuloendothelial system, the liposome can target the liver, such as the hepatic leishmania drug meglumine antimonate liposome, and the concentration in the liver is improved by 200-700 times compared with that of a common preparation. The liposome is used as a carrier to encapsulate the anti-hepatitis B drug, so that the liver targeting property of the anti-hepatitis B drug can be potentially improved, and the similarity between a lipid bilayer and a lipophilic cell membrane is utilized to assist in loading hydrophilic drugs, charged DNA and the like into liver cells. However, the liposome technology is directly applied to high-throughput in vivo screening of anti-hepatitis B drugs, and no literature report exists.

In view of the foregoing, there is an urgent need in the art to develop an in vivo screening method capable of screening anti-hepatitis b drugs at high throughput and a corresponding screening composition.

Disclosure of Invention

The present invention solves the above problems by designing a screening composition based on liposome carriers.

In one aspect, the present invention provides a composition for screening a therapeutic agent for hepatitis b, comprising at least one carrier, the at least one carrier comprising: (a) a unicellular therapeutically effective amount of at least one therapeutic agent to be screened for hepatitis B; (b) one or more nucleic acid barcodes uniquely identifying the at least one therapeutic agent for hepatitis b; and (c) optionally a label.

In some embodiments, the at least one vector is a 1-5000 type of vector. In some embodiments, the at least one carrier is a 1-4000 type of carrier. In some embodiments, the at least one vector is a vector of type 1 to 2000. In some embodiments, the at least one vector is a 1-500 type of vector. In other embodiments, the at least one carrier is a 1-200 type of carrier. In other embodiments, the at least one carrier is 1-10 types of carriers. In other embodiments, the at least one carrier is 1-5 types of carriers. In other embodiments, the at least one carrier is 1-4 types of carriers.

In another embodiment, the composition further comprises at least one control vector that does not contain a hepatitis b therapeutic agent and comprises one or more nucleic acid barcodes that uniquely identify the control vector.

In some embodiments, the therapeutically effective amount is substantially a single cell therapeutically effective amount. In another embodiment, the therapeutically effective amount is sufficient to modulate the state of a single cell. In another embodiment, the condition that modulates a single cell is selected from the group consisting of a condition that modulates cell activity such as proliferation, differentiation, senescence or apoptosis of an infected hepatocyte, and a function or activity of hepatitis b virus in an infected hepatocyte. In one embodiment, assessing the function or activity of said hepatitis B virus comprises the following indicators: HBV DNA, HBV RNA, HBeAg, HBsAg, cccDNA, etc.

In another embodiment, the nucleic acid barcode molecule is 5 to 1000 nucleotides in length. In another embodiment, the nucleic acid barcode molecule is 5 to 500 nucleotides in length. In another embodiment, the nucleic acid barcode molecule is 5 to 400 nucleotides in length. In another embodiment, the nucleic acid barcode molecule is 5 to 300 nucleotides in length. In another embodiment, the nucleic acid barcode molecule is 5 to 250 nucleotides in length. In another embodiment, the nucleic acid barcode molecule is 15 to 200 nucleotides in length.

In another embodiment, the nucleic acid molecule comprises a sequence that is not substantially identical or complementary to a nucleic acid of the cell. In another embodiment, the nucleic acid molecule does not enter the nucleus of the cell. In another embodiment, the nucleic acid molecule does not contain a component capable of entering the nucleus (such as a Nuclear Localization Signal (NLS) or nuclear carrier).

In another embodiment, the optional label (e.g., tracer) is selected from the group consisting of a fluorophore, a chromophore, a chemiluminescent molecule, a magnetic particle or dye, a metal, a rare earth, and a radioisotope. In some embodiments, the marker is used to screen and/or detect hepatocytes that are successfully targeted by the vector. In some embodiments, the label is further used to detect the activity of the therapeutic agent for hepatitis b.

In another embodiment, the at least one support has a diameter of at most 250nm (nanometers). In another embodiment, the at least one support has a diameter of at least 50 nm. In another embodiment, the at least one carrier is a lipid-based particle. In another embodiment, the at least one carrier is selected from liposomes.

In another embodiment, the composition is formulated for systemic administration.

In another aspect, a method for screening for efficacy of a therapeutic agent for hepatitis b in a subject having a disease is provided, comprising the steps of:

(a) administering to the subject a composition for screening for a therapeutic agent for hepatitis b comprising at least one carrier comprising: a single cell therapeutically effective amount of at least one therapeutic agent for hepatitis b to be screened, one or more nucleic acid barcodes uniquely identifying said at least one therapeutic agent for hepatitis b, and optional indicia;

(b) obtaining a sample comprising hepatocytes from the subject; and

(c) identifying the efficacy of the at least one therapeutic agent in hepatocytes comprising a plurality of types of carriers by a unique barcode; thereby screening the diseased subject for the efficacy of a therapeutic agent for hepatitis B.

In some embodiments, the administering is intravenous and the composition comprises 1-200 vectors per single cell of the liver of the subject.

In another embodiment, the method further comprises the step of sequencing the nucleic acid molecule. In another embodiment, the method further comprises the step of amplifying the nucleic acid molecule.

In another embodiment, the sample is broken down into a single cell suspension prior to step (c). In another embodiment, the cells of the sample are sorted using a method selected from the group consisting of: FACS, magnetic bead sorting, enzyme-linked immunosorbent assay (ELISA), microfluidic-based sorting, cell tension-based sorting, and the like.

In a third aspect, there is provided the use of a composition according to the first and/or second aspects in the manufacture of a screening kit for a therapeutic agent for hepatitis b.

The technical scheme of the invention has the following beneficial technical effects:

1. the liver cell targeting of liposome and other carrier is utilized to load the screened treating agent and nucleic acid bar code molecule into liver cell in high efficiency and avoid the metabolism damage of the circulating system to the molecule.

2. The nucleic acid barcode molecules and optional marker molecules are utilized to carry out unique marking on different therapeutic agents to be screened, and the molecules are utilized to establish the correlation between the concentration (quantity) of the specific therapeutic agent in the cells and the treatment effect (antiviral evaluation index), so that the trace and high-sensitivity therapeutic agent efficacy screening on the single cell level is realized.

3. By introducing a plurality of different therapeutic agents or combinations of the therapeutic agents into the composition, simultaneous and high-throughput screening of the plurality of different therapeutic agents can be realized, and the screening efficiency is greatly improved.

4. Depending on the existing in vivo models such as animal models, in vivo screening can be realized, and the screening result can better reflect the actual anti-hepatitis B virus efficacy of the therapeutic agent.

Further embodiments and the full scope of the invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Detailed Description

In some embodiments, each carrier contains a very low dose of a therapeutic agent for hepatitis b, which is sufficient to treat only one hepatocyte and well below the systemic therapeutic threshold.

In some embodiments, the at least one therapeutic agent for hepatitis b and the nucleic acid barcode molecule are encapsulated within at least one carrier. In embodiments wherein the vector further comprises a label, the at least one therapeutic agent for hepatitis b, the nucleic acid barcode molecule, and the label are encapsulated within the at least one vector.

Therapeutic agents

In some embodiments, the therapeutically effective amount is a substantially single cell therapeutically effective amount.

As used herein, "therapeutically effective amount" or "effective amount" refers to an amount that is effective at a dose and for a period of time required to achieve the desired therapeutic result. A therapeutically effective amount of a therapeutic agent for hepatitis b will depend on the nature of the disorder or condition and on the particular agent, and can be determined by standard clinical techniques known to those skilled in the art.

As used herein, "single cell therapeutically effective amount" refers to an effective amount sufficient to achieve the desired therapeutic result within a single cell. In particular embodiments, the single-cell therapeutically effective amount of the therapeutic agent is a substantially lower dose of the agent as compared to the Minimum Effective Dose (MED) of the therapeutic agent. In some embodiments, the single cell therapeutically effective amount is at most 0.1%, 0.01%, 0.001%, or 0.0001% of the agent as compared to the Minimum Effective Dose (MED) of the therapeutic agent. In some embodiments, a "single cell therapeutically effective amount" refers to the minimum effective amount sufficient to achieve the desired therapeutic result within a single cell. The amount may vary depending on the cell type, drug type and/or duration of treatment.

In another embodiment, the single cell therapeutically effective amount is substantially sufficient to modulate the state of the single cell or achieve a desired therapeutic result within the single cell. In another embodiment, the therapeutically effective amount is no greater than 50%, 40%, 30%, 20%, or 10% of the amount sufficient to modulate the state of a single cell or achieve a desired therapeutic result within a single cell.

The therapeutic outcome may be, for example, alleviation of symptoms, prolongation of survival, increased mobility, and the like. The therapeutic result need not be a "cure". The therapeutic outcome may also be prophylactic.

In another embodiment, the state of the regulatory single cell is selected from the group consisting of a state that modulates cell activity such as proliferation, differentiation, senescence or apoptosis of an infected hepatocyte, and a function or activity of hepatitis b virus in an infected hepatocyte. In one embodiment, assessing the function or activity of said hepatitis B virus comprises the following indicators: HBV DNA, HBV RNA, HBeAg, HBsAg, cccDNA, etc.

In some embodiments, the at least one therapeutic agent for hepatitis b is unable to cross the hepatocyte membrane. In such embodiments, the therapeutically effective amount may be at a higher concentration as compared to the concentration of the treated single cells. Examples of therapeutic agents for hepatitis B that are unable to cross cell membranes include, but are not limited to, RNA (e.g., RNAi, etc.) and hydrophilic drugs.

Nucleic acid barcode

In one embodiment, the nucleic acid barcode molecules (such as DNA strands) exhibit an unlimited number of barcoded selections. As used throughout the present invention, "barcode", "DNA barcode" are all interchangeable with each other and have the same meaning. The nucleic acid molecules of the invention that are DNA barcodes are polymers of deoxyribonucleic acid or ribonucleic acid, or both, and may be single-stranded or double-stranded, optionally containing synthetic, non-natural or modified nucleotide bases. In some embodiments, the nucleic acid molecule is labeled, for example, with biotin, a radiolabel, or a fluorescent label.

As will be appreciated by those skilled in the art, the incorporation of unique DNA barcodes into the therapeutic agent carrier (encapsulation) allows for the identification of individual hepatitis b therapeutics using assays including, but not limited to, microarray systems, PCR, nucleic acid hybridization (including "blotting"), or high throughput sequencing.

In some embodiments, permeation of the negatively charged DNA barcode through the negatively charged lipid bilayer of the cell is achieved by encapsulating the DNA barcode within a vector (e.g., liposome) of the present invention.

In some embodiments, the sequence of the nucleic acid molecule does not include sequences, patterns, features, or any other nucleic acid sequences associated with materials/substances/particles that naturally occur in the environment or that specifically naturally occur in the cell targeted by the methods and compositions of the invention. In other embodiments, the sequence of the nucleic acid molecule does not contain a nucleotide sequence of more than 10 bases. In another embodiment, the nucleic acid molecule comprises a sequence that is substantially non-identical or complementary to the genomic material of the cell (such as to prevent hybridization of the nucleic acid molecule to the genomic material of the cell, and/or to prevent false positive amplification results).

In some embodiments, the nucleic acid sequence may further serve as a molecular beacon. The nucleic acid sequence may typically be a single-stranded molecule, such as a hairpin shape. The nucleic acid molecules can be used to detect complementary genes in a cell, such as to detect gene mutations inside a cell. In some embodiments, the nucleic acid has a sequence complementary to a sequence with a mutation.

After performing the screening methods of the present invention, the unique nucleic acid barcodes are suitable for use in identifying the corresponding at least one therapeutic agent for hepatitis b in a vector. Methods for detecting the presence and identification of nucleic acid sequences are known to the skilled artisan and include sequencing and array (e.g., microarray) systems (e.g., commercially available from Ilumina Inc.) capable of enhancing the presence of multiple barcodes.

In some embodiments, such as when increased accuracy is desired, the nucleic acid molecule has a length suitable for sequencing and/or amplification assays (e.g., PCR). In another embodiment, the nucleic acid molecule has a length suitable for loading into the vector of the invention, preferably on the nanoscale. It will be appreciated that the length of the nucleic acid molecule will depend on the type and size of the vector used in the compositions and methods of the invention (e.g., shorter sequences are suitable for nanoparticles, while longer sequences may be used for microparticles).

In another embodiment, the nucleic acid molecule has a length of at most 1000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 450, at most 400, at most 350, at most 300, at most 250, at most 200, at most 190, at most 180, at most 170, at most 160, at most 150, at most 140, at most 130 or at most 120 bases, wherein each possibility represents a separate embodiment of the invention. In another embodiment, the nucleic acid molecule has a length of at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 bases, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 bases, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or at least 200, at least 300, at least 400, or at least 500 bases, wherein each likelihood represents a separate embodiment of the invention.

Non-limiting examples of nucleic acid lengths include, but are not limited to, 5-50 bases, 5-40 bases, 5-30 bases, 5-25 bases, 5-24 bases, 5-23 bases, 5-22 bases, 5-21 bases, 5-20 bases, 5-19 bases, 5-18 bases, 5-17 bases, 5-16 bases, or 5-15 bases, 15-50 bases, 15-60 bases, 15-70 bases, 15-80 bases, 15-90 bases, 15-100 bases, 15-200 bases, 15-250 bases, or 15-500 bases.

In another embodiment, the nucleic acid molecules within each vector have a concentration of one or more strands per vector or per target cell. It is well within the ability of the person skilled in the art to determine the amount of nucleic acid molecules. In another embodiment, the one or more nucleic acid molecules is 1-10000 nucleic acid molecules. In another embodiment, the one or more nucleic acid molecules are 1-1000 nucleic acid molecules. In another embodiment, the one or more nucleic acid molecules are 1-5000 nucleic acid molecules. In another embodiment, the one or more nucleic acid molecules are 1-500 nucleic acid molecules. In another embodiment, the one or more nucleic acid molecules are 5-500 nucleic acid molecules. One skilled in the art will appreciate that the amount of nucleic acid molecules in each particle can be predetermined to suit the particular assay being performed, such as a sequencing and/or amplification assay.

According to some embodiments, each particle comprises a unique barcode. One skilled in the art will appreciate that the use of a unique barcode to barcode each particle (i.e., carrier) can indicate the amount of particles that enter a single cell. In some embodiments, the present invention provides a method of determining a therapeutic dose for treating a hepatitis b disease comprising administering to a subject a composition comprising a plurality of types of carriers, wherein each type of carrier comprises one or more therapeutic agents, wherein each type of carrier differs in the dose of the one or more therapeutic agents.

In another embodiment, the vector within the composition further comprises an additional agent (such as a chemical or biological agent) for operatively using the nucleic acid molecule as a barcode within a cell. Non-limiting examples of such agents include dnases or rnases, such as to prevent degradation by endogenous or exogenous enzymes of DNA or RNA barcodes, respectively.

In one embodiment, there are 1-5 types of vectors (e.g., barcoded liposomes)/cell per cell. In another embodiment, the presence of 1-5 types of carriers per cell provides sufficient data and a high signal-to-noise ratio (SNR). In another embodiment, cells are analyzed that include 1-5 types of vectors per cell. In another embodiment, cells are analyzed that include 1-4 types of vectors per cell.

In one embodiment, 1-200 particles per hepatocyte are administered via intravenous injection. In another embodiment, 1-300 particles per hepatocyte are administered via intravenous injection. In another embodiment, 1-400 particles per hepatocyte are administered via intravenous injection. In another embodiment, 1-500 particles per hepatocyte are administered via intravenous injection. In another embodiment, 1-1000 particles per hepatocyte are administered via intravenous injection.

Marking

In another embodiment, one or more vectors of the invention optionally comprise at least one label or detectable moiety. In some embodiments, the same label is used for all vectors. In other embodiments, unique markers are used for a subset of vectors.

Labels that may be used in the compositions and methods of the present invention include, but are not limited to, fluorophores, chromophores, chemiluminescent molecules, radioactive labels, metals, rare earth elements, magnetic particles, or dyes.

In some embodiments, the label or detectable moiety is a label useful in an assay, including but not limited to immunoassays, such as ELISA, bead-based, chip-based, or plate-based multiplex immunoassays, mass spectrometry, electrophoresis, immunoturbidimetry, enzymatic assays, colorimetric or fluorescent assays, as evaluable by photometers, and fluorescence-related cell sorting (FACS) -based assays or by other clinically established assays. All these methods are known to the person skilled in the art and are described in the literature.

Generally, the amount of label will depend on the assay to be performed and can be determined by and well within the abilities of those skilled in the art. In some embodiments, the vector comprises one molecule or a plurality of molecules of the label.

Carrier

In some embodiments, the at least one carrier is a lipid-based particle. In another embodiment, the lipid-based particle is a liposome.

In one embodiment, the carrier (e.g., liposome) has a diameter of less than 500nm to facilitate its entry into cells through the extracellular matrix. In one embodiment, the carrier (e.g., liposome) has a diameter of less than 400nm to facilitate its entry into cells through the extracellular matrix.

In another embodiment, the carrier (e.g., liposome) has a diameter of at least 1nm, at least 5nm, at least 10nm, at least 20nm, at least 30nm, at least 40nm, at least 50nm, at least 60nm, at least 70nm, at least 80nm, at least 90nm, at least 100nm, at least 1500nm, at least 200nm, at least 250nm, or at least 300 nm. Each possibility represents a separate embodiment of the invention.

In some embodiments, the carrier for intravenous administration has a diameter of 5-250 nm. In some embodiments, wherein the carrier for intravenous administration is a liposome, the diameter of the carrier is 40-250 nm.

In one embodiment, vectors can be selected and/or prepared to optimize delivery of therapeutic agents and nucleic acid molecules (DNA barcodes) to target cells. For example, where the target cell of the invention is a hepatocyte, the properties of the transfer vector (e.g., size, charge and/or pH) can be optimized to efficiently deliver such vector to the target cell.

The liposome-incorporated therapeutic agent, nucleic acid, and/or label can be located entirely or partially within the interior space of the liposome, within the bilayer membrane of the liposome. Incorporation of the agent into the liposome is also referred to herein as "encapsulation," wherein the agent is completely contained within the interior space of the liposome. The purpose of incorporating an agent into a carrier, such as a liposome, is often to protect the agent from the environment, which may contain enzymes or chemical agents that degrade the agent (e.g., nucleic acids) and/or systems or receptors that cause rapid excretion of the agent. Thus, in a preferred embodiment of the invention, the transfer vector is selected to enhance the stability of the hepatitis B therapeutic agent and the nucleic acid barcode molecule, and optionally the label contained therein. Liposomes can allow the encapsulated agent to reach the target cell and/or can preferentially allow the encapsulated agent to reach the target cell, or alternatively limit delivery of the agent to other undesired target sites or cells.

In some embodiments, the carrier facilitates penetration of the encapsulated therapeutically effective amount of the at least one therapeutic agent, the nucleic acid molecule uniquely identifying the at least one therapeutic agent, and the optional marker into the cell. In some embodiments, a therapeutically effective amount of at least one therapeutic agent, a nucleic acid molecule uniquely identifying the at least one therapeutic agent, and optionally a label, can be permeated into the cell by encapsulation within the carrier.

In another embodiment, the at least one carrier is a nanoliposome. Nanoliposomes are capable of enhancing the solubility and bioavailability of bioactive agents, stability in vitro and in vivo, and avoiding their undesirable interactions with other molecules. Another advantage of nanoliposomes is cell-specific targeting, which is a prerequisite for obtaining optimal therapeutic effect in the target cell while minimizing the concentration of drug required for side effects on healthy cells and tissues.

The liposome carrier used in the composition of the present invention can be prepared by various techniques currently known in the art. Multilamellar vesicles (MLVs) can be prepared by conventional techniques, for example, by dissolving the lipids in a suitable solvent and then evaporating the solvent to leave a film on the interior of the vessel or by spray drying to deposit the selected lipids on the interior walls of a suitable container or vessel. The aqueous phase may then be added to the vessel using a swirling motion, which may result in the formation of MLVs. Monolayer vesicles (ULV) can then be formed by homogenizing, sonicating or extruding multiple layer vesicles. Alternatively, the monolayer of vesicles may be formed by detergent removal techniques.

In certain embodiments, the compositions of the invention may be loaded with diagnostic radionuclides, fluorescent materials, or other materials detectable in both in vitro and in vivo applications.

In some embodiments, the vectors of the present invention comprise 40-70% mol of liposome-forming lipids. In some embodiments, the vectors of the present invention comprise 20 to 50% mol cholesterol. In some embodiments, the carrier of the present invention comprises 4-8% mol of PEG-lipid. In some embodiments, the vectors of the invention comprise 0-3% mol of a functional lipid (e.g., a cationic lipid or a lipid having a targeting moiety). According to a specific embodiment, nanoparticles comprising 40-70% mol of liposome forming lipids, 20-50% mol of cholesterol, 4-8% mol of PEG-lipids and 0-3% mol of functional lipids are suitable for intravenous administration.

Screening application of hepatitis B therapeutic agent

According to some embodiments, there is provided a composition for screening for a therapeutic agent for hepatitis b comprising a plurality of vectors, the plurality of vectors independently comprising a therapeutically effective amount of at least one therapeutic agent for hepatitis b, a nucleic acid barcode molecule uniquely identifying the at least one therapeutic agent for hepatitis b, and optionally a label.

According to some embodiments, there is provided a method for screening for efficacy of a therapeutic agent for hepatitis b in a subject suffering from a disease, comprising the steps of:

(b) obtaining a sample comprising hepatocytes from the subject; and

In another embodiment, the method further comprises the step of sequencing the nucleic acid molecule. In another embodiment, the method further comprises the step of amplifying the nucleic acid molecule. Methods for amplifying and sequencing nucleic acids are well known to those skilled in the art.

In another embodiment, the sample is broken down into a single cell suspension prior to determining efficacy. Methods of breaking up cell samples into single cell suspensions are known in the art and may include, for example, genetlemecs acs^TMAn organization processor (dispatcher).

In another embodiment, the method further comprises a cell sorting step. Methods of cell sorting are well known in the art and include, but are not limited to, FACS, magnetic bead sorting, ELISA, microfluidic-based sorting, cell tension-based sorting, and the like. According to a preferred embodiment, the activity of each hepatitis b therapeutic is analyzed by sorting liver target cells and correlating the cell status to DNA barcodes. In additional embodiments, the method enables screening of multiple drugs for their patient-specific and cell-specific activities.

In embodiments, the methods disclosed herein comprise providing a therapeutic agent for an amount of time sufficient to perform its therapeutic activity. The amount of time that lasts before the target cells are sampled will depend on the nature of the disorder or condition, and on the particular agent, and can be determined by standard clinical techniques known to those skilled in the art. By way of non-limiting example, the amount of time that lasts includes about 24, about 30, about 48 hours, or more than 24 hours after administration of the composition. Each possibility represents a separate embodiment of the invention. In some embodiments, the amount of time that lasts is less than 72 hours in order to avoid dnase degradation of the barcode inside the cell.

In another embodiment, the composition is formulated for systemic administration. In another embodiment, the systemic administration is intravenous injection.

As used herein, the term "subject" refers to any animal (e.g., a mammal) to which the compositions and methods of the present invention are administered, including but not limited to humans, non-human primates, rodents, and the like. In some embodiments, the terms "subject" and "patient" are used interchangeably herein with respect to a human subject. In other embodiments, the terms "subject" and "patient" are used interchangeably herein with respect to a non-human subject. In certain embodiments, the subject comprises an animal model.

As used herein, the term "about" when combined with a value refers to plus or minus 10% of the referenced value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000nm + -100 nm.

Additional objects, advantages and novel features of the present invention will become apparent to one of ordinary skill in the art upon examination of the following examples, which are not intended to be limiting.

Examples

Example 1 construction of humanized hepatitis B murine model

The humanized hepatitis B mouse model is prepared by the following specific steps:

first, obtaining human stem cell

1. Isolated culture of human stem cells

1) Purified human stem cells were obtained.

2) And (4) culturing stem cells and carrying out passage.

3) Culturing at 20-40 deg.C and 2-10% CO₂In an incubator.

2. Commercial isolated or cryopreserved human stem cells or cell lines are obtained.

Second, the mice with liver injury transplanted with stem cells

1. Obtaining experimental mice of different strains, wherein the experimental mice comprise normal mice, immunodeficient mice, normal rats and immunodeficient rats.

2. Liver damage drugs are applied by means of intraperitoneal, intramuscular and peripheral intravenous injection, oral administration or intragastric administration, or surgical partial hepatectomy is applied, and a liver damage mouse model is established.

3. Transplantation of 1X 10 by peripheral vein, portal vein, spleen or liver injection^4-8A stem cell.

Third, HBV infects human mouse

Each mouse was injected with hepatitis b virus via peripheral vein, subcutaneous, intramuscular, or intraperitoneal injection.

Example 2 Liposome Synthesis/preparation/transfection

Liposomes are prepared using methods known in the art. HSPC, PEG-DSPE and cholesterol 58%, 2%, 40% were dissolved in pure ethanol and heated at 65 ℃ until complete dissolution.

Synthesis of blank liposomes: the liposome comprisesThe lipid composition of the following: 58% mol Hydrogenated Soy Phosphatidylcholine (HSPC), Mw 762.1; 2 mol% polyethylene glycol distearoyl-phosphoethanolamine (m2000PEG DSPE), which acts to reduce aggregation/fusion of liposomes due to steric effects, Mw 2805.54; and 40 mol% cholesterol Mw 386.65. The working concentration of total lipid in the solution was 50 mM. The medium was 10% PBS/5% glucose in deionized water. First, the lipids were dissolved in pure ethanol, warmed to 65 ℃ and added to 1ml of medium (also warmed to the same temperature). After direct injection and pipetting, more medium was added to reach the final lipid concentration. Hydrogenated soy phosphatidylcholine is contributed by Lipoid (Ludwigshafen, Germany); m2000PEG-DSPE was purchased from Avanti (Alabaster, Alabama, USA) and cholesterol (catalog number: C8667-500MG) was obtained from Sigma (Rehovot, Israel).

Synthetic therapeutic agent-loaded liposomes: referring to the above synthesis method of blank liposome, the difference lies in that serial dilutions with different concentrations of therapeutic agent for hepatitis B to be screened are introduced together in the process of dissolving with ethanol. Heating the lipid component and the therapeutic agent in ethanol until they are completely dissolved, and mixing the heated solution with the lipid component and the therapeutic agentConfocal/cell viewer, etc. to determine the percent encapsulation.

DNA encapsulation:25 nanomolar and desalted ssDNA oligo from Sigma&IDT (Leuven Belgium) is purchased and has a length range of 50-120 base pairs. After annealing of the two ssDNAs, the stock was diluted to 100. mu.M and divided into 100 microliter (μ l) aliquots. For 5ml of lipid solution, 300. mu.l of dsDNA was added to 1ml of medium solution before adding dissolved lipid (for 3 ml-150. mu.l of dsDNA). After encapsulation, an extruder was used to produce 200nm liposomes.

Extrusion process: the lipid solution was extruded 3 times through 400nm and 200nm membranes. The extruder temperature was set to 65 degrees celsius (c).

Particle size determination via particle size of DLS(dynamic light scattering) -after extrusion, sizing was done by DLS. The PDI ranges from 0.003 to 0.08.

In vivo transfection:intravenous injection into humanized hepatitis B mouse model100 μ l of liposome solution. After 36 hours, mice were sacrificed and their livers were removed in ice for further steps.

DNA extraction: cells were detached from the plate using trypsin and transferred into a 1.5ml Eppendorf tube. Cells were centrifuged at 1200rpm for 7min and trypsin was discarded. DNA extraction using Bligh and Dyer assays; 167. mu.l of 1: 2 chloroform: methanol (v/v), 55.5. mu.l chloroform, and 100. mu.l deionized water. After centrifugation at 1000rpm for 5min, the upper aqueous phase was collected.

DNA amplification: dsDNA was amplified in a temperature cycler ("Daniel Biotech") using 50. mu.l of a reaction premix ("Ornat"). The solution was loaded onto a 3% (w/w) agarose gel ("Hy-Labs") and subjected to PCR for 25 min.

RT-PCR: after DNA extraction, strands were amplified and analyzed using TaqMan probes in an RT-PCR cyclothermocycler ("Bio Rad").

Cell sorting: get to have 10⁶15ml of cell suspension at a concentration of/ml to a flow activated cell sorter (FACS-ARIA). After gating the cells, they were sorted into 1,000 cells, 100 cells, 10 cells and single cells. All cells were sorted into conical clear 96 wells of polypropylene. After sorting, B is carried out&D and PCR.

Cell dilution: cells were suspended in 1ml of medium and counted. The next step was a serial dilution in 900 μ l of PBSX1, up to 10³Cell-100. mu.l. Each dilution was a further 10-fold dilution, resulting in 10³、10²And 10 cells: 10 to 90. mu.l of deionized water. Finally, to theoretically obtain single cells, 1. mu.l of the diluted cells were placed in 99. mu.l of PBSX 1.

Confocal/cell observation instrument: DNA encapsulation-liposomes containing FAM conjugated DNA were prepared using the methods described above. Liposomes were dialyzed in 10% PBS (membrane type) for 24 hours. Fluorescence of the samples (triplicates) before and after dialysis were measured using DLS (manufacturing) excitation and emission 566-345nm, respectively. After and before the redistributionFluorescence values to give percent encapsulation.

Determination of therapeutically effective amount of single cells: determination of therapeutically effective amount of single cells can result in apoptosis of infected hepatocytes and the like And (4) indexes. The barcoded liposomes containing different diluted concentrations of hepatitis B therapeutic were intravenously injected into a humanized hepatitis B murine model After 36 hours, mice were sacrificed and their livers were removed in ice. Dissociating liver tissue into single cell suspension, and based on it Viability (apoptotic/non-apoptotic) of these cells were screened and single cell therapy for specific therapeutic agents for hepatitis B was determined based on statistical methods Effective amounts, the above assays were performed in triplicate.

Claims

1. A composition for screening a therapeutic agent for hepatitis b comprising at least one carrier, said at least one carrier comprising: (a) a unicellular therapeutically effective amount of at least one therapeutic agent to be screened for hepatitis B; (b) one or more nucleic acid barcodes uniquely identifying the at least one therapeutic agent for hepatitis b; and (c) optionally a label.

2. The composition of claim 1, wherein the therapeutically effective amount is sufficient for modulating the state of single cells, i.e., the state of cellular activities such as proliferation, differentiation, senescence or apoptosis of infected hepatocytes, and the function or activity of hepatitis B virus in infected hepatocytes.

3. The composition of claim 2, wherein assessing the function or activity of hepatitis b virus comprises the following indicators: HBV DNA, HBV RNA, HBeAg, HBsAg, cccDNA, etc.

4. The composition of any one of claims 1-3, further comprising at least one control vector that does not contain a hepatitis B therapeutic agent and comprising one or more nucleic acid barcodes that uniquely identify the control vector.

5. The composition of any one of claims 1-4, wherein the nucleic acid barcode is 5-400 nucleotides in length.

6. The composition of claim 5, wherein the nucleic acid barcode comprises a sequence that is substantially non-identical or complementary to a nucleic acid of the cell.

7. The composition of any one of claims 1-6, wherein the at least one carrier is a lipid-based particle.

8. The composition of claim 7, wherein the at least one carrier is a liposome.

9. The composition of claim 1, wherein the label is selected from the group consisting of: fluorophores, chromophores, chemiluminescent molecules, magnetic particles, dyes, metals, rare earth metals, and radioisotopes.

10. The composition of any one of claims 1-9, formulated for systemic administration.

11. A method for screening for efficacy of a therapeutic agent for hepatitis b in a subject suffering therefrom, comprising the steps of: (a) administering to the subject the composition of claim 1; (b) obtaining a sample comprising hepatocytes from the subject; and (c) identifying the efficacy of the at least one therapeutic agent in hepatocytes comprising a plurality of types of carriers by the unique barcode; thereby screening the diseased subject for the efficacy of a therapeutic agent for hepatitis B.

12. The method of claim 11, wherein the administration is intravenous and the composition comprises 1-200 vectors per single cell of the liver of the subject.

13. The method of claim 11, further comprising the step of sequencing and/or amplifying the nucleic acid molecule.

14. The method of claim 11, wherein the sample is broken down into a single cell suspension prior to step (c), and wherein the cells of the sample are sorted using a method selected from the group consisting of: FACS, magnetic bead sorting, microfluidic-based sorting, cell tension-based sorting, and ELISA.

15. Use of the composition of claim 1 for the preparation of a screening kit for a therapeutic agent for hepatitis B.