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WO2018237369A2 - Administration médiée par des nanoparticules lipidiques (lnp) d'un adn plasmidique exprimant crispr pour le traitement d'une infection chronique par le virus de l'hépatite b - Google Patents

Administration médiée par des nanoparticules lipidiques (lnp) d'un adn plasmidique exprimant crispr pour le traitement d'une infection chronique par le virus de l'hépatite b Download PDF

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Publication number
WO2018237369A2
WO2018237369A2 PCT/US2018/039179 US2018039179W WO2018237369A2 WO 2018237369 A2 WO2018237369 A2 WO 2018237369A2 US 2018039179 W US2018039179 W US 2018039179W WO 2018237369 A2 WO2018237369 A2 WO 2018237369A2
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sequence
vector
seq
grna
sequences
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PCT/US2018/039179
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WO2018237369A3 (fr
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Sean M. Sullivan
Larry Smith
Brenda CLEMENTE
Qun Wei
Ming Ye
Yasuhiro Hayashi
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Vical Incorporated
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Publication of WO2018237369A3 publication Critical patent/WO2018237369A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates generally to hepatitis B infection and more specifically to delivery, engineering, optimization and therapeutic applications of a polynucleotide capable of gene expression and gene editing.
  • Hepatitis B is one of the world's most prevalent diseases. Although most individuals seem to resolve acute infection, approximately 30% of cases become chronic. According to current estimates, 350-400 million people worldwide have chronic hepatitis B, leading to 500,000-1,000,000 deaths per year due largely to the development of
  • hepatocellular carcinoma cirrhosis, and other complications.
  • immunoglobulin therapy e.g., interferon, and antiviral drugs
  • hepatitis B remains a major global health problem.
  • the hepatitis B virus is a double-stranded hepatotropic virus that infects only humans and non-human primates. Viral replication takes place predominantly in the liver and, to a lesser extent, in the kidneys, pancreas, bone marrow and spleen (Hepatitis B virus biology. Microbiol Mol Biol Rev . 64: 2000; 51-68.). Viral and immune markers are detectable in blood and characteristic antigen-antibody patterns evolve over time. The first detectable viral marker in blood is hepatitis B surface antigen (HBsAg), followed by hepatitis B e-antigen (HBeAg) and HBV DNA.
  • HBsAg hepatitis B surface antigen
  • HBeAg hepatitis B e-antigen
  • HBeAg is a viral marker detectable in blood and correlates with active viral replication, and therefore high viral load and infectivity (Hepatitis B e antigen—the dangerous end game of hepatitis B. N Engl J Med. 347: 2002; 208-210).
  • the presence of anti-HBsAb and anti-hepatitis B core antibody (HBcAb) IgG indicates recovery and immunity in a previously infected individual.
  • the present invention is based on the finding that a polynucleotide encoding a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) CRISPR-Cas system can be delivered using a lipid envelope engineered to target hepatocytes infected with HBV, resulting in reduction of viral nucleic acid. Accordingly, in one aspect, the invention provides a method of inactivating hepatitis B virus (HBV) nucleic acid in a host cell.
  • HBV hepatitis B virus
  • the method includes transfecting a host cell containing HBV viral nucleic acid with a polycistronic expression cassette, wherein the expression cassette comprises: a RNA polymerase III promoter sequence, a 5 '-untranslated region comprising two or more complexes, each complex consisting of a guide RNA (gRNA) sequence and a scaffold RNA (scRNA) sequence, and a 3 '-untranslated region sequence, wherein CpG content of all sequences has been minimized to reduce immunostimulatory response but maintain maximal gRNA and CRISPR nuclease expression and wherein each gRNA sequence is capable of hybridizing to a target sequence of hepatitis B viral nucleic acid.
  • the expression cassette comprises: a RNA polymerase III promoter sequence, a 5 '-untranslated region comprising two or more complexes, each complex consisting of a guide RNA (gRNA) sequence and a scaffold RNA (scRNA) sequence, and a 3 '-untranslated region
  • the method includes incubating the cell to promote expression of the polycistronic expression cassette, thereby inactivating HBV nucleic acid in the host cell.
  • the method may be performed in vitro or in vivo, and the host cell may be a human cell, such as a hepatocyte.
  • the vector may be administered in a lipid envelope encapsulating the vector, such as a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • the LNP includes one or more cationic lipids of the ssPalm class, such as, but not limited to, ssPalmM, ssPalmE-P4C2, ssPalmE-Paz4-C2, and any combination thereof.
  • the LNP has a ratio of cationic lipid to plasmid DNA of 4: 1 to 16: 1, such as 8: 1 or 12: 1.
  • the LNP may also include one or more lipids selected from the group consisting of cholesterol, phospholipids such as l,2-dimyristoyl-sn-glcero-3- phosphatidylcholine (DMPC), l,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), l,2-distearoyl-sn-glycero-3-phosphatidylcholine(DSPC), l-palmitoyl-2-oleyol-sn-glycero- 3 -phosphatidylcholine (POPC), l,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE),
  • DMPE diole
  • the DOPE when present the DOPE can be substituted for other lipids, such as, but not limited to, dieliadoylphosphatidylethanolamine (DEPE) and lipids with different head groups, such as phosphatidylcholine,
  • DEPE dieliadoylphosphatidylethanolamine
  • lipids with different head groups such as phosphatidylcholine
  • acyl chains can vary from myrsitoyl, palmitoyl, steroyl, oleoyl.
  • the acyl chains can be the same or mixed, such as oleoyl-palmitoyl-phosphatidylethanolamine.
  • Other lipids useful in the LNP include diacyl glycerols.
  • the LNP may also include dimyristroyl glycerol- poly ethylene glycerol 2000 da (DMG-PEG), dipalmitoylglycerol-PEG, distearoylglycerol- PEG.
  • DMG-PEG dimyristroyl glycerol- poly ethylene glycerol 2000 da
  • the LNP includes DMG-PEG at a concentration of about 2 mol% to 3.4 mol%.
  • the polyethylene glycol portion of the DMG-PEG can vary in length from about 1,500 daltons to about 5,000 daltons.
  • the LNP includes cholesterol at about 20 mol% to about 33 mol%.
  • the LNP includes dexamethasone palmitate (DP) at a concentration of about 1.65 mol% to 5 mol%, such as 1.65 mol%, 3.3 mol%, or 5 mol%.
  • the LNP includes ssPalmE-P4C2, DOPE, and cholesterol at mole ratios of about 60: 10:30, 40:30:30, or 50:20:30.
  • the method further includes detecting a decrease in hepatitis B surface antigen (HBsAg) production, a decrease in hepatitis B e-antigen (HBeAg) production, or a decrease in both HBsAg and HBeAg production in the cell.
  • the step of transfecting comprises administering a vector comprising the polycistronic expression cassette to a subject suffering from chronic HBV infection.
  • the gRNA sequence of each complex is selected from the group consisting of SEQ ID NOs: 15-38 and 39.
  • the polycistronic expression cassette is a bicistronic expression cassette, and the gRNA sequences may, for example, be SEQ ID NOs: 16 and 25, SEQ ID NOs: 27 and 28, or SEQ ID NOs: 28 and 39.
  • the bicistronic expression cassette may further include a plurality of transfer RNA (tRNA) sequences, each tRNA sequence flanking one of the two or more complexes.
  • the plurality of tRNA sequences are independently selected from the group consisting of SEQ ID NOs: 1 and 2.
  • the scRNA sequence of each complex is SEQ ID NO: 3.
  • the bicistronic expression cassette may further include a microRNA (miRNA) sequence located between each of the two or more complexes, wherein the miRNA sequence encodes a miRNA molecule that targets HBV nucleic acid, such as a sequence encoding precursor miRNA (pre-miRNA) molecule.
  • miRNA sequences are SEQ ID NOs: 28 and 39 or SEQ ID NOs: 16 and 25.
  • the polycistronic expression cassette may be a tetracistronic expression cassette, and may further include a plurality of transfer RNA (tRNA) sequences, each tRNA sequence flanking one of the two or more complexes.
  • tRNA sequences are independently selected from the group consisting of SEQ ID NOs: 1, 2, and 3, and the gRNA sequences may be SEQ ID NOs: 16, 25, 27, and 28.
  • the expression cassette further includes a RNA polymerase II promoter sequence selectively active in hepatocytes located upstream of the CRISPR nuclease sequence.
  • the expression cassette further includes one or more additional promoters and/or enhancers such as, but not limited to, human alphal antitrypsin, human phenylalanine hydroxylase, apolipoprotein E/C-l hepatic control region, ou microglobulin/bikunin enhancer, human thyroxine binding globulin (TBG), serum albumin, multimerized hepatocyte nuclear factor-3 (HNF-3) binding site, and an enhancer that can bind an HBV-specific transcription activator.
  • the 3 '-untranslated region sequence comprises a poly (A) tail.
  • the invention provides a vector.
  • the vector includes a polynucleotide that includes: a RNA polymerase III promoter sequence, a 5 '-untranslated region comprising at least one intron, wherein the intron comprises two or more guides, each being flanked by a transfer RNA (tRNA) sequence, wherein each guide comprises a guide RNA (gRNA) sequence and a scaffold RNA (scRNA) sequence, a CRISPR nuclease sequence, and a 3 '-untranslated region sequence, wherein CpG content of all sequences has been minimized to reduce immunostimulatory response but maintain maximal gRNA and CRISPR nuclease expression.
  • tRNA transfer RNA
  • gRNA guide RNA
  • scRNA scaffold RNA
  • the RNA polymerase III promoter is selected from the group consisting of SEQ ID NOs: 12, 13, and 14.
  • the flanking tRNA sequences are independently selected from the group consisting of SEQ ID NOs: 1 and 2.
  • the gRNA sequences are independently selected from the group consisting of SEQ ID NOs: 15-38 and 39.
  • the vector may also include a RNA polymerase II promoter sequence selectively active in hepatocytes located upstream of the CRISPR nuclease sequence.
  • the vector may also include one or more of human alphal antitrypsin, human phenylalanine hydroxylase, apolipoprotein E/C-l hepatic control region, ai microglobulin/bikunin enhancer, human thyroxine binding globulin (TBG), serum albumin, multimerized hepatocyte nuclear factor-3 (HNF-3) binding site, and an enhancer that can bind an HBV- specific transcription activator.
  • the vector may also include a hepatocyte-specific enhancer sequence operably linked to the RNA polymerase II promoter.
  • the intron comprises two guides, wherein the gRNA sequences are SEQ ID NOs: 16 and 25, SEQ ID NOs: 27 and 28, or SEQ ID NOs: 28 and 39. In various embodiments, the intron comprises four guides, wherein the gRNA sequences are SEQ ID NOs: 16, 25, 27, and 28.
  • the vector may include a polynucleotide which comprises: a RNA polymerase III promoter sequence, a 5 '-untranslated region comprising at least one intron, wherein the intron comprises a microRNA (miRNA) molecule flanked by two guides, wherein each guide comprises a guide RNA (gRNA) sequence and a scaffold RNA (scRNA) sequence, an RNA polymerase II enhancer sequence, RNA polymerase II promoter sequence, a CRISPR nuclease sequence, and a 3 '-untranslated region sequence, wherein CpG content of all sequences has been minimized to reduce immunostimulatory response but maintain maximal gRNA and CRISPR nuclease expression and wherein each gRNA sequence is capable of hybridizing to a target sequence of hepatitis B viral nucleic acid.
  • miRNA microRNA
  • gRNA guide RNA
  • scRNA scaffold RNA
  • the RNA polymerase III promoter is selected from the group consisting of SEQ ID NOs: 12, 13, and 14.
  • the miRNA sequence encodes a precursor miRNA molecule (e.g., pre-HBV-miRNA).
  • the gRNA sequences are independently selected from the group consisting of SEQ ID NOs: 15-38 and 39.
  • the gRNA sequences are SEQ ID NOs: 28 and 39 or SEQ ID NOs: 16 and 25.
  • the vector also includes a RNA polymerase II promoter sequence selectively active in hepatocytes located upstream of the CRISPR nuclease sequence.
  • the vector may also include one or more of human alphal antitrypsin, human phenylalanine hydroxylase, apolipoprotein E/C-l hepatic control region, ou microglobulin/bikunin enhancer, human thyroxine binding globulin (TBG), serum albumin, multimerized hepatocyte nuclear factor- 3 (HNF-3) binding site, and an enhancer that can bind an HBV-specific transcription activator.
  • the vector also includes a hepatocyte-specific enhancer sequence operably linked to the RNA polymerase II promoter.
  • the expression plasmid may contain introns at other locations besides the 5'UTR, such as the open reading frame (ORF).
  • ORF open reading frame
  • the target hepatitis B viral nucleic acid is an episomal nucleic acid molecule, such as a cccDNA, and integrated into the genome of the organism.
  • the CRISPR nuclease may be capable of reducing the amount of episomal viral nucleic acid molecule in a cell of the organism compared to the amount of episomal viral nucleic acid molecule in a cell of the organism in the absence of providing the vector.
  • Hepatitis B infected hepatocytes can have viral genomes integrated into the hepatocyte genome.
  • the target may also be the HBV integrated genome as well as the episomal HBV cccDNA.
  • the CRISPR nuclease sequence encodes a CRISPR nuclease selected from the group consisting of Streptococcus pyogenes Cas9 (SpCas9) and variants of SpCas9, such as VRER, VQR, and EQR, high-fidelity SpCas9 (SpCas9-HFl),
  • CRISPR nuclease is CpG-free and human codon-optimized, and may be encoded by the sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 11.
  • the nuclease gene also includes DNA sequence(s) encoding one or more nuclear localization sequences (NLS).
  • the present invention provides a gene delivery system.
  • the gene delivery system includes a lipid envelope encapsulating the vector as described herein.
  • the vector is complexed with a condensing agent, such as protamine, spermine, spermidine, cadaverine, putrescine, histones, and virus capsid proteins.
  • the condensing agent is protamine sulfate.
  • the lipid envelope may form an LNP, or may be a naturally occurring or synthetic exosome.
  • the LNP is formed from one or more cationic lipids of the ssPALM class, such as, but not limited to, ssPalm, ssPalmE-P4C2, ssPalmE-Paz4-C2, and any combination thereof.
  • the LNP also includes one or more lipids selected from the group consisting of cholesterol, DOPE, DMG-PEG, distearoyl glycerol, SOPC, DEPE, DMPC, DPPC, DSPC, DOPC, POPC, DOPS, DMPS, DOPA, DMPA, and DPPA.
  • the lipid envelope includes one or more ligands that bind to hepatocyte receptors displayed thereon.
  • ligands may be adsorbed to the surface of the lipid envelope or are covalently derivatized to specific components of the lipid envelope, such as a functionalized DMG-PEG-maleimide or simply a DMG-PEG ending with a primary amine or thiol.
  • a phospholipid can be functionalized, such as phosphatidylethanolamine with a terminal maleimide or thiol group.
  • the present invention provides a method of treating chronic HBV infection in a subject.
  • the method includes administering an effective amount of the gene delivery system as described herein.
  • Figures 1A and IB are pictorial diagrams showing exemplary HBV gene editing expression plasmids of the invention.
  • Figure 2 is a pictorial diagram showing an exemplary poly-gRNA/tRNA expression cassette for use in the expression plasmids of the invention.
  • Figure 3 is a pictorial diagram showing an exemplary gRNA-miRNA-gRNA expression cassette for use in the expression plasmids of the invention.
  • the present invention is based on the finding that a polynucleotide encoding a CRISPR-Cas system can be delivered using a lipid envelope engineered to target hepatocytes infected with HBV, resulting in reduction of viral nucleic acid. Such reduction in viral nucleic acid reduces the extent of infection, thereby treating HBV infection in the subject.
  • compositions and methods are inclusive of open-ended language and does not exclude additional, unrecited elements or method steps.
  • the present disclosure contemplates embodiments of the invention compositions and methods corresponding to the scope of each of these phrases.
  • a composition or method comprising recited elements or steps contemplates particular embodiments in which the composition or method consists essentially of or consists of those elements or steps.
  • subject or "host organism,” as used herein, refers to any individual or patient to which the subject methods are performed. Generally the subject is human, although as will be appreciated by those in the art, the subject may be an animal.
  • mammals including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject.
  • rodents including mice, rats, hamsters and guinea pigs
  • cats dogs, rabbits
  • farm animals including cows, horses, goats, sheep, pigs, etc.
  • primates including monkeys, chimpanzees, orangutans and gorillas
  • terapéuticaally effective amount or “effective amount” means the amount of a compound or pharmaceutical composition that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.
  • therapeutically effective amount is used herein to denote any amount of a formulation that causes a substantial improvement in a disease condition when applied to the affected areas after a administration or multiple administrations over a period of time. The amount will vary with the condition being treated, the stage of advancement of the condition, and the type and concentration of formulation applied. Appropriate amounts in any given instance will be readily apparent to those skilled in the art or capable of determination by routine experimentation.
  • a "therapeutic effect,” as used herein, encompasses a therapeutic benefit and/or a prophylactic benefit as described herein.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral (usually orally) and topical administration, or by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and infrasternal injection and infusion.
  • systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the subject's system and, thus, is subject to metabolism and other like processes, for example, intravenous administration.
  • the terms “reduce” and “inhibit” are used together because it is recognized that, in some cases, a decrease can be reduced below the level of detection of a particular assay. As such, it may not always be clear whether the expression level or activity is “reduced” below a level of detection of an assay, or is completely “inhibited.”
  • treatment means to administer a composition to a subj ect or a system with an undesired condition.
  • the condition can include a disease or disorder.
  • prevention means to administer a composition to a subject or a system at risk for the condition, and therefore includes preventing disease progression in symptomatic or asymptomatic subjects.
  • the condition can include a predisposition to a disease or disorder.
  • the effect of the administration of the composition to the subject can be, but is not limited to, the cessation of one or more symptoms of the condition, a reduction or prevention of one or more symptoms of the condition, a reduction in the severity of the condition, the complete ablation of the condition, a stabilization or delay of the development or progression of a particular event or characteristic, or minimization of the chances that a particular event or characteristic will occur.
  • pharmaceutically acceptable carrier encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water and emulsions such as an oil/water or water/oil emulsion, cryopreservatives for lyophilization and various types of wetting agents.
  • Acute hepatitis B infection results when a person is exposed to the hepatitis B virus, but may or may not have begun to develop the signs and symptoms of viral hepatitis. This period of time from infection to the presentations of symptoms, called the incubation period, is an average of 90 days, but could be as short as 45 days or as long as 6 months. For most people this infection will cause mild to moderate discomfort, but will go away by itself because of the body's immune response succeeds in fighting the virus.
  • Chronic hepatitis B infection occurs when a person infected with HBV is unable to eliminate the virus; this is clinically defined as having detectable HBsAg in blood for over 6 months. Whether the disease becomes chronic or completely resolves depends mostly on the age when the person becomes infected. About 90% of infants infected at birth will develop to chronic HBV infection. However, as a person ages, the risk of developing chronic infection decreases such that between 20%-50% of children and less than 10% of older children or adults will develop chronic infection.
  • polypeptide As used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, a- carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e.
  • R group e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
  • Such analogs have modified R groups (e.g. , norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid.
  • a "protein coding sequence” or a sequence that encodes a particular protein or polypeptide is a nucleic acid sequence that is transcribed into mRNA (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • the boundaries of the coding sequence are determined by a start codon at the 5' terminus (N-terminus) and a translation stop nonsense codon at the 3' terminus (C -terminus).
  • a coding sequence can include, but is not limited to, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic DNA, viral DNA, and synthetic nucleic acids.
  • a transcription termination sequence will usually be located 3' to the coding sequence.
  • an "expression cassette” refers to a portion of vector DNA that includes one or more genes and one or more regulatory sequences controlling their expression. In each successful transformation, the expression cassette directs the cell's machinery to make RNA and/or protein(s).
  • the term “gene” means the deoxyribonucleotide sequences that codes for a molecule that has a function.
  • a "structural gene” refers to a gene that codes for an RNA or protein other than a regulatory factor, but is nonetheless encompassed within the definition of “gene.”
  • a “gene” may also include non-translated sequences located adjacent to the coding region on both the 5' and 3' ends such that the gene corresponds to the length of the full-length mRNA. The sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non-translated sequences.
  • genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns” or “intervening regions” or “intervening sequences.”
  • Introns are segments of a gene which are transcribed into heterogenous nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript.
  • mRNA messenger RNA
  • polycistronic mRNA carries several open reading frames (ORFs), each of which is translated into a polypeptide.
  • ORFs open reading frames
  • These polypeptides usually have a related function (they often are the subunits composing a final complex protein) and their coding sequence is grouped and regulated together in a regulatory region, containing a promoter and an operator.
  • transfer RNA or "tRNA” refers to an adaptor molecule composed of RNA, typically 76 to 90 nucleotides in length that serves as the physical link between the mRNA and the amino acid sequence of proteins. Without being bound by theory, the role of tRNA is to specify which sequence from the genetic code corresponds to which amino acid during protein synthesis.
  • exemplary tRNA sequences useful in the invention include, but are not limited to:
  • cccDNA refers to a partially double-stranded DNA that is ligated by means of DNA ligase to a covalently closed ring. cccDNA arises during propagation of some viruses, such as HBV, in the cell nucleus.
  • microRNA refers to a small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals and some viruses that functions in RNA silencing and post-transcriptional regulation of gene expression. While the majority of miRNAs are located within the cell, some miRNAs, commonly known as circulating miRNAs or extracellular miRNAs, have also been found in extracellular environment, including various biological fluids and cell culture media.
  • miRNAs resemble the small interfering RNAs (siRNAs) of the RNA interference (RNAi) pathway, except miRNAs derive from regions of RNA transcripts that fold back on themselves to form short hairpins, whereas siRNAs derive from longer regions of double-stranded RNA.
  • siRNAs small interfering RNAs
  • RNAi RNA interference
  • HBV capsid protein plays essential functions during the viral life cycle.
  • HBV capsid/core proteins form metastable viral particles or protein shells that protect the viral genome during intercellular passage, and also play a central role in viral replication processes, including genome encapsidation, genome replication, and virion morphogenesis and egress. Capsid structures also respond to environmental cues to allow uncoating after viral entry. Proper capsid assembly has consistently been found to be critical for viral infectivity.
  • HBV capsid proteins The crucial function of HBV capsid proteins imposes stringent evolutionary constraints on the viral capsid protein sequence, leading to the observed low sequence variability and high conservation. Consistently, mutations in HBV capsid that disrupt its assembly are lethal with regard to negatively impacting virus replication, and mutations that perturb capsid stability severely attenuate viral replication.
  • HBV replication centers on the establishment of a cccDNA form of its genome in the host cell nucleus. This episomal form is established from conversion of the partially double stranded circular DNA (relaxed circular, or rcDNA) genome upon initial infection, and functions as the template for transcribing all HBV mRNAs. As indicated above, HBV DNA synthesis is coupled to assembly of its capsid, and most copies of the encapsidated genome then efficiently associate with the envelope proteins for virion assembly and secretion; a minority of these genomes are shunted to the nucleus where they are converted to cccDNA, thus amplifying levels of the episome.
  • rcDNA partially double stranded circular, or rcDNA
  • HBV cccDNA should impair these processes within the infected subject.
  • the optimal outcome of cccDNA inactivation would be to cure a subject of chronic HBV infection and consequently mitigate the risk of developing cirrhosis and hepatocellular carcinoma.
  • the term "genetic modification” is used to refer to any manipulation of an organism's genetic material in a way that does not occur under natural conditions. Methods of performing such manipulations are known to those of ordinary skill in the art and include, but are not limited to, techniques that make use of vectors for transforming cells with a nucleic acid sequence of interest. Included in the definition are various forms of gene editing in which DNA is inserted, deleted or replaced in the genome of a living organism using engineered nucleases, or "molecular scissors.” These nucleases create site-specific double-strand breaks (DSBs) at desired locations in the genome. The induced double-strand breaks are repaired through nonhomologous end-joining (NHEJ) or homologous recombination (HR), resulting in targeted mutations (i.e., edits).
  • NHEJ nonhomologous end-joining
  • HR homologous recombination
  • CRISPR is an acronym for DNA loci that contain multiple, short, direct repetitions of base sequences.
  • the prokaryotic CRISPR/Cas system has been adapted for use as gene editing (i.e., silencing, enhancing or changing specific genes) in eukaryotes (see, for example, Cong, Science, 15:339(6121):819-823 (2013) and Jinek, et al., Science, 337(6096):816-21 (2012)).
  • gene editing i.e., silencing, enhancing or changing specific genes
  • eukaryotes see, for example, Cong, Science, 15:339(6121):819-823 (2013) and Jinek, et al., Science, 337(6096):816-21 (2012).
  • nucleic acid sequences can be cut and modified at any desired location.
  • CRISPR system refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-mate sequence
  • tracrRNA encompassing a "direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system
  • a guide sequence (“guide RNA” or “gRNA” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus.
  • guide RNA or “gRNA” in the context of an endogenous CRISPR system
  • gRNA guide sequences and transcripts from a CRISPR locus.
  • tracr-mate sequences operably linked to a guide sequence e.g., direct repeat-spacer-direct repeat
  • pre-crRNA pre- CRISPR RNA
  • a tracrRNA and crRNA are linked and form a chimeric crRNA-tracrRNA hybrid where a mature crRNA is fused to a partial tracrRNA via a synthetic stem loop to mimic the natural crRNA:tracrRNA duplex as described in Cong, Science, 15:339(6121):819-823 (2013) and Jinek, et al, Science, 337(6096):816-21 (2012)).
  • a single fused crRNA-tracrRNA construct can also be referred to as a guide RNA or gRNA (or single-guide RNA (sgRNA)).
  • the crRNA portion can be identified as the 'target sequence' and the tracrRNA is often referred to as the 'scaffold' RNA (scRNA).
  • the sgRNA expression plasmid therefore contains the target sequence (about 20 nucleotides), a form of the tracrRNA sequence (i.e., the scRNA) as well as a suitable promoter and necessary elements for proper processing in eukaryotic cells.
  • a suitable promoter and necessary elements for proper processing in eukaryotic cells are commercially available (see, for example, Addgene).
  • the CRISPR complex of the invention when introduced into a cell, creates a break (e.g., a single or a double strand break) in the target DNA sequence.
  • a break e.g., a single or a double strand break
  • the method can be used to cleave a disease gene in a cell.
  • the break created by the CRISPR complex can be repaired by repair processes such as the error prone nonhomologous end joining (NHEJ) pathway or the high fidelity homology-directed repair (HDR).
  • NHEJ error prone nonhomologous end joining
  • HDR high fidelity homology-directed repair
  • the HDR process is used to modify a genome sequence.
  • an exogenous polynucleotide template comprising a sequence to be integrated flanked by an upstream sequence and a downstream sequence is introduced into a cell.
  • the upstream and downstream sequences share sequence similarity with either side of the site of integration in the chromosome.
  • a donor polynucleotide can be DNA, e.g., a plasmid DNA (pDNA), a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear piece of DNA, a PCR fragment, a naked nucleic acid, or a nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.
  • pDNA plasmid DNA
  • BAC bacterial artificial chromosome
  • YAC yeast artificial chromosome
  • viral vector e.g., a viral vector, a linear piece of DNA, a PCR fragment, a naked nucleic acid, or a nucleic acid
  • the present invention provides an expression system for delivering a CRISPR system to cells (e.g., hepatocytes) harboring HBV cccDNA, such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at the target site, which leads to inactivation of the HBV cccDNA.
  • cells e.g., hepatocytes
  • HBV cccDNA e.g., hepatocytes
  • a "vector” is a tool that allows or facilitates the transfer of an entity from one environment to another. It is a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment in the appropriate prokaryotic or eukaryotic cell. Generally, a vector is capable of replication when associated with the proper control elements. In general, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Vectors include, but are not limited to, nucleic acid molecules that are single- stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art.
  • One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • the vector can be made synthetically using appropriate primers and a high fidelity proof reading DNA polymerase.
  • viral vector Another type of vector is a "viral vector,” wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses, AAVs).
  • viruses e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses, AAVs.
  • viruses e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses, AAVs.
  • Viral vectors also include
  • polynucleotides carried by a virus for transfection into a host cell are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively -linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression (e.g., transcription and translation) of the nucleotide sequence in a host cell when the vector is introduced into the host cell.
  • regulatory element is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences).
  • promoters e.g., promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences).
  • IRES internal ribosomal entry sites
  • regulatory elements e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences.
  • Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).
  • the invention provides a therapeutic expression plasmid for inactivating HBV cccDNA, thereby treating or preventing chronic HBV infection in a subject and mitigating the risk of developing cirrhosis and/or hepatocellular carcinoma.
  • the expression system is a HBV gene editing expression plasmid containing that includes at least one a promoter, at least one enhancer, a 5' untranslated region (5'-UTR), a nuclease spaced apart from the 5'-UTR by a spacer or intron, and a 3' untranslated region (3'-UTR), all of which will be explained in detail below.
  • a "promoter” is defined as a regulatory DNA sequence generally located upstream of a gene that mediates the initiation of transcription by directing RNA polymerase to bind to DNA and initiating RNA synthesis.
  • a promoter can be a
  • constitutively active promoter i.e., a promoter that is constitutively in an active/"ON” state
  • it may be an inducible promoter (i.e., a promoter whose state, active/"ON” or inactive/"OFF", is controlled by an external stimulus, e.g., the presence of a particular compound or protein)
  • it may be a spatially restricted promoter (i. e., transcriptional control element, enhancer, etc.) (e.g., tissue specific promoter, cell type specific promoter, etc.)
  • it may be a temporally restricted promoter (i.e., the promoter is in the "ON" state or "OFF” state during specific stages of embryonic development or during specific stages of a biological process.
  • a tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as a specific organ (e.g., liver), or particular cell types (e.g., hepatocytes).
  • a desired tissue of interest such as a specific organ (e.g., liver), or particular cell types (e.g., hepatocytes).
  • the plasmid of the invention includes a promoter that is selectively active in hepatocytes that will transcribe CRISPR RNA only in liver cells where HBV replication occurs.
  • the plasmid may express the gRNAs under a RNA polymerase II (pol II) promoter along with the nuclease.
  • An exemplary promoter useful in the plasmid of the invention includes, but is not limited to, the elongation factor- 1 alpha (EF-la) promoter.
  • the gRNAs can be expressed using a RNA polymerase III (pol III) promoter, such as the U6 promoter that is commonly used for driving small hairpin RNA (shRNA) expression.
  • pol III promoters useful in the invention include, but are not limited to, U6 promoter, 7SK promoter, and HI promoter.
  • the pol III promoter will not restrict gRNA expression to hepatocytes whereas the pol II promoter will not only restrict expression to hepatocytes but can be engineered to restrict expression to hepatocytes infected with HBV.
  • Exemplary hepatocyte- specific promoters useful in the plasmid of the invention include, but are not limited to, human alpha-1 antitrypsin (Hafenrichter, et al. 1994 Blood 84:3394-3404) and human phenylalanine hydroxylase (Chatterjee, et al. 1996 PNAS 93 :728-733).
  • an “enhancer” is a short (50-1500 bp) region of DNA that can be bound by proteins (activators) to increase the likelihood that transcription of a particular gene will occur.
  • an enhancer may be used to increase promoter strength with regard to expression of the open reading frame for gene expression.
  • the enhancer may be paired with an endogenous hepatocyte promoter or it can be a non-specific enhancer that increases the promoter strength of an endogenous hepatocyte promoter.
  • liver-specific enhancers useful in the plasmid of the invention include, but are not limited to, albumin or hepatitis B enhancers (Kramer, et al., 2003 Mol Ther 7: 375-385), multimerized HNF-3 binding site (Hafenrichter, et al., 1994 J Surgical Research 56: 510-717), multimerized ai-microglobulin enhancer + two copies of hepatocyte control region 1 (Jacobs, et al., 2008 Gene Therapy 15: 594-603) and the cytomegalovirus (CMV) enhancer.
  • albumin or hepatitis B enhancers Karlin, et al., 2003 Mol Ther 7: 375-385
  • multimerized HNF-3 binding site Hefenrichter, et al., 1994 J Surgical Research 56: 510-717
  • multimerized ai-microglobulin enhancer + two copies of hepatocyte control region 1 Jacobs, et
  • the enhancer can bind an HBV-specific transcription activator that is expressed during HBV replication, namely the X protein.
  • the enhancer element can bind a transcription activator that is part of the hepatocyte genome but is preferentially upregulated during expression of hepatitis virus proteins.
  • the expression cassette further includes one or more additional promoters and/or enhancers such as, but not limited to, human alphal antitrypsin, human phenylalanine hydroxylase, apolipoprotein E/C-l hepatic control region, ai microglobulin/bikunin enhancer, human thyroxine binding globulin (TBG), serum albumin, multimerized hepatocyte nuclear factor-3 (HNF-3) binding site, CMV enhancer, and an enhancer that can bind an HBV-specific transcription activator.
  • additional promoters and/or enhancers such as, but not limited to, human alphal antitrypsin, human phenylalanine hydroxylase, apolipoprotein E/C-l hepatic control region, ai microglobulin/bikunin enhancer, human thyroxine binding globulin (TBG), serum albumin, multimerized hepatocyte nuclear factor-3 (HNF-3) binding site, CMV enhancer
  • 5' untranslated region or “5' -UTR” (also known as a leader sequence or leader RNA) refers to a region of mRNA that is directly upstream from the initiation codon and important for the regulation of translation of a transcript. While called untranslated, the 5' UTR or a portion of it is sometimes translated into a protein product. This product can then regulate the translation of the main coding sequence of the mRNA.
  • the 5' UTR contains introns that are spliced out prior to translocation of the mRNA from the nucleus into the cytoplasm.
  • the intron RNAs remain in the nucleus.
  • the gRNAs can be incorporated into the intron and flanked on the 5' and 3' ends with processing ribozymes (i.e., catalytic RNAs) to remove excess bases.
  • ribozyme is an RNA molecule capable of catalyzing specific biochemical reactions.
  • the intron includes one or more (i.e., 1, 2, 3, 4, 5, or more) gRNAs, each flanked with 5' and 3' processing ribozymes.
  • the intron may include three gRNAs, each being flanked by 5' and 3' processing ribozymes.
  • multiple gRNAs can be processed from the intron using tRNA or miRNA endogenous processing enzymes as in the polycistronic and ternary gRNAs expressed under the Pol III promoter, discussed in further detail below.
  • the expression system may include two or more gRNA sequences to maximize HBV inactivation.
  • expression of more than one gRNA can be accomplished in several ways, including, for example, administering multiple expression plasmids, each including a RNA Pol III promoter driving the respective gRNA. While inactivation of HBV may be accomplished utilizing a smaller dose of each plasmid, this strategy requires that all administered plasmids express the gRNA in the same cell at the same time.
  • a single plasmid may include two or more gRNA sequences, along with a RNA Pol III promoter for each gRNA sequence.
  • this strategy can result in competition between the various RNA Pol III promoters resulting in expression of different amounts of each of the gRNA sequences.
  • propagating the pDNAs in bacteria can be challenging because bacteria are known to sometimes delete DNA segments from pDNAs that contain repetitive sequences.
  • the expression system described herein expresses all gRNA sequences in a single transcript and utilizes the mammalian cell machinery to process the individual guides.
  • the expression system includes a polycistronic tRNA expression cassette that places a tRNA between each gRNA/scRNA and utilizes endogenous ribonuclease P (RNase P) and endogenous ribonuclease Z (RNase Z) to specifically cleave the RNA sequences on either side of the tRNA yielding individual gRNA/scRNAs.
  • Figure 2 shows a diagram of an exemplary polycistronic tRNA expression cassette driven by the RNA Pol III promoter.
  • An exemplary scRNA sequence useful in the invention includes, but is not limited to:
  • an exemplary polycistronic tRNA expression cassette may have the following structure:
  • polycistronic tRNA expression cassette may, for example, be set forth as follows:
  • polycistronic tRNA expression cassette may be set forth as follows:
  • the expression system includes a gRNA-miRNA-gRNA expression cassette, where the miRNA, Pre-HBV-miRNA, is positioned between two gRNA/scRNAs, as described by Wang, et al. (Wang, et al. The gRNA-miRNA-gRNA temary cassette combining CRISPR/Cas9 with RNAi approach strongly inhibits hepatitis B virus Replication Theranostics (2017) 7: 3090-3105, incorporated herein by reference).
  • a nuclear endonuclease, Drosha specifically cleaves the RNA transcript on either side of the short hair pin RNA to release the short hair pin RNA.
  • the stem-loop precursor is thereafter exported from the nucleus and subsequently processed by Dicer into microRNA, which is released in the cytoplasm.
  • the miRNA is further processed for the siRNA strand to enter the RNA-induced silencing complex (RISC) (see, Petersen, et al. Short RNAs repress translation after initiation in mammalian cells Mol Cell (2006) 21 :533-542, incorporated herein by reference).
  • RISC RNA-induced silencing complex
  • FIG. 3 A diagram of an exemplary bicistronic miRNA processing cassette is shown in Figure 3.
  • an exemplary polycistronic miRNA expression cassette may have the following structure:
  • the Pre-HBV -miRNA molecule will include one or more of:
  • AGGGATGGTATTGCTCCTGTAACTCGGAACTGGAGAGG (SEQ ID NO: 5; spacer RNA);
  • GGTGAAGCGAAGTGCACACGG SEQ ID NO: 6; miRNA (minus strand)
  • GTTGAACTGGGAACG SEQ ID NO: 7; loop
  • ACGTGTGCACATCGATTCACGGC SEQ ID NO: 8; miRNA (plus strand)
  • polycistronic miRNA expression cassette may, for example, be set forth as follows:
  • a plurality of both types of poly-gRNA expression cassettes were constructed and tested for inhibition of HBeAg and HBsAg.
  • gRNA selection was based on >20% inhibition of HBeAg production in HepAD38 cells and where the gRNA had 100% homology HBV genotypes A, B, C and D.
  • exemplary poly-gRNA expression cassettes useful in the expression system are set forth in
  • each of the eight poly -gRNA (polycistronic gRNA) expression cassettes demonstrated >20% inhibition of HBeAg production in HepAD38 cells.
  • the polycistronic gRNAs ranged from two gRNAs (bicistronic) to four gRNAs (tetracistronic), with guide numbers corresponding to those listed in Table 2, below.
  • the first listed plasmid, PTGl was a tetracistronic guide with a gRNA to luciferase (LUC).
  • the last two plasmids incorporate a miRNA into bicistronic guides using the gRNA sequences from PTG4 and PTG6, to form gRNA-miRNA-gRNA expression cassettes.
  • the miRNA may be targeted to the n-terminus of any of the structural HBV proteins eliminating the 7-methyl guanosine cap, which is required for mRNA export from the nucleus into the cytoplasm and for efficient translation.
  • the miRNA may be targeted to the AUG start site sequence of any of the HBV structural proteins or the common poly(A) tail sequence.
  • nuclease refers to an enzyme capable of cleaving the phosphodiester bonds between monomers of nucleic acids. As described above, the CRISPR system includes a nuclease that is guided to the target DNA for cleavage. Thus, the terms “Cas”, “CRISPR enzyme”, and “nuclease” are generally used herein
  • the CRISPR enzyme is a type I, II, or III CRISPR enzyme from any species of microbe.
  • a preferred Cas enzyme may be identified as "Cas9,” as this can refer to the general class of enzymes that share homology to the biggest nuclease with multiple nuclease domains from the type II CRISPR system.
  • An exemplary Cas9 enzyme useful in the CRISPR system may be from the type II CRISPR locus in Streptococcus pyogenes (SpCas9).
  • SpCas9 Streptococcus pyogenes
  • this invention includes many more Cas9s from other species of microbes, such as but not limited to, Staphylococcus aureus (SaCas9), Streptococcus thermophilus (StCas9), and so forth.
  • the Cas9 enzyme may be codon-optimized, for example optimized for humans (i.e., being optimized for expression in humans), such as human codon-optimized CpG-free Streptococcus pyogenes (Sp) Cas9 as set forth in SEQ ID NOs: 10 and 11.
  • the Cas9 enzyme may include one or more alterations designed to reduce non-specific DNA contacts, such as high-fidelity SpCas9 (SpCas9-HFl), as described in Kleinstiver, et al. 2016, Nature, 529,490-495, incorporated herein by reference.
  • the nuclease may be derived from Prevotella and Francisella bacteria, such as Cpfl, which is a smaller and simpler endonuclease than Cas9, and nucleases derived from Neisseria meningitides, Treponema denticola, and any biologically active fragments or derivatives of the nucleases.
  • the plasmid may further include gene sequence(s) encoding one or more nuclear localization sequences (NLS).
  • NLS nuclear localization sequences
  • nuclear localization sequence refers to an amino acid sequence that 'tags' a protein for import into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface.
  • the NLS may be located at the 3' or 5' end or both, depending on the nuclease being used.
  • 3' Untranslated Region refers to the section of messenger RNA (mRNA) that immediately follows the translation termination codon.
  • mRNA messenger RNA
  • An mRNA molecule is transcribed from the DNA sequence and is later translated into protein. Several regions of the mRNA molecule are not translated into protein including the 5' cap, 5' untranslated region, 3' untranslated region, and the poly(A) tail.
  • the 3'-UTR often contains regulatory regions that post-transcriptionally influence gene expression.
  • the 3 '-UTR may be synthetic or derived from naturally occurring genes.
  • the 3 '-UTR may or may not include short hairpin RNA (shRNA) target sequences.
  • the 3'-UTR may also contain a sequence that directs addition of multiple adenine residues called the poly(A) tail to the end of the mRNA transcript.
  • Poly(A) binding protein (PABP) binds to this tail, contributing to regulation of mRNA translation, stability, and export.
  • PABP Poly(A) binding protein
  • poly(A) tail bound PABP interacts with proteins associated with the 5' end of the transcript, causing a circularization of the mRNA that promotes translation.
  • the presence of a poly(A) tail usually aids in triggering translation, the absence or removal of one often leads to exonuclease-mediated degradation of the mRNA.
  • the poly(A) tail protects the mRNA molecule from enzymatic degradation in the cytoplasm and aids in transcription termination, export of the mRNA from the nucleus, and translation.
  • Polyadenylation itself is regulated by sequences within the 3'-UTR of the transcript. These sequences include cytoplasmic polyadenylation elements (CPEs), which are uridine-rich sequences that contribute to both polyadenylation activation and repression. CPE-binding protein (CPEB) binds to CPEs in conjunction with a variety of other proteins in order to elicit different responses.
  • CPEs cytoplasmic polyadenylation elements
  • CPEB CPE-binding protein
  • polyadenylate polymerase builds the poly(A) tail by adding adenosine monophosphate units from adenosine triphosphate to the RNA, cleaving off pyrophosphate.
  • Another protein, PAB2 binds to the new, short poly(A) tail and increases the affinity of polyadenylate polymerase for the RNA.
  • the poly(A) tail is approximately 250 nucleotides long the enzyme can no longer bind to CPSF and polyadenylation stops, thus determining the length of the poly(A) tail.
  • the poly(A) tail of most mRNAs in the cytoplasm gradually get shorter, and mRNAs with shorter poly(A) tail are translated less and degraded sooner, thereby influencing expression levels. This deadenylation and degradation process can be accelerated by microRNAs complementary to the 3' untranslated region of an mRNA.
  • the 3'-UTR includes a rabbit beta globin poly (A) sequence.
  • the invention provides a gene delivery system that includes the plasmid DNA of the invention complexed with a condensing agent and encapsulated by a lipid envelope.
  • the invention provides methods comprising delivering one or more polynucleotides, such as one or more plasmids of the invention and/or one or more transcripts thereof to a host cell, such as a hepatocyte.
  • a CRISPR enzyme in combination with (and optionally complexed with) one or more guide sequences is delivered to a cell.
  • Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues.
  • Non-viral vector delivery systems include plasmid DNA, RNA (e.g., a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. See, e.g., US Pub. No.
  • DNA condensation refers to the process of compacting DNA molecules in vitro or in vivo.
  • DNA diameter is about 2 nm, while the length of a stretched single molecule may be up to several dozens of centimeters depending on the organism.
  • the physical properties of the DNA double helix such as the sugar-phosphate backbone, electrostatic repulsion between phosphates, stacking interactions between the bases of each individual strand, and strand-strand interactions contribute to the overall stiffness of the molecule.
  • DNA can pack itself in the appropriate solution conditions with the help of ions and other molecules (i.e., DNA condensation agents).
  • DNA condensation can be induced in vitro either by applying external force to bring the double helices together, or by inducing attractive interactions between the DNA segments.
  • the former can be achieved with the help of the osmotic pressure exerted by crowding neutral polymers in the presence of monovalent salts.
  • the forces pushing the double helices together are coming from entropic random collisions with the crowding polymers surrounding DNA condensates, and salt is required to neutralize DNA charges and decrease DNA-DNA repulsion.
  • DNA condensation may also be realized by inducing attractive interactions between the DNA segments by multivalent cationic charged ligands (multivalent metal ions, inorganic cations, polyamines, protamines, peptides, lipids, liposomes and proteins).
  • a condensing agent useful in the delivery system of the invention can be naturally occurring, such as a DNA binding protein or molecules capable of binding to DNA either through ionic interactions, hydrogen bonding or combination of both types of bonding.
  • the DNA condensing agent may have an endogenous domain or may be derivatized from an exogenous domain that traffics the plasmid DNA to the nucleus.
  • Exemplary plasmid DNA condensing agents include, but are not limited to, protamine (e.g., protamine sulfate), spermine, spermidine, cadaverine, putrescine, histones, and virus capsid proteins.
  • the gene delivery system incorporates an encapsulating envelope that may be composed of lipids, such as cationic lipids, that are both naturally occurring and synthetic. While the encapsulating lipids can protect the plasmid DNA from degradation prior to target cell entry, the lipids may also function as condensing agents themselves, either alone or in combination with other condensing agents, as described above. As such, upon binding to the DNA, the lipids compact the DNA structure to facilitate packaging into lipid particles of less than about 100 nm in diameter.
  • the lipid composition can be made up of naturally occurring lipids, synthetic lipids or synthetic amphiphiles that trigger intracellular release into the cytoplasm of the target cell.
  • encapsulating lipids may be formulated to include the expression cassette and the CRISPR Cas system of the present invention to form lipid nanoparticles (LNPs).
  • LNPs are typically spherical with an average diameter of between 10 nanometers to less than 1 ⁇ .
  • lipid nanoparticles useful in the gene delivery system may be formed from, for example, triglycerides (e.g., tristearin), diglycerides (e.g., glycerol bahenate), monoglycerides (e.g., glycerol monostearate), fatty acids (e.g., stearic acid), steroids (e.g., cholesterol), and waxes (e.g., cetyl palmitate), and may further include emulsifiers to stabilize the lipid dispersion.
  • triglycerides e.g., tristearin
  • diglycerides e.g., glycerol bahenate
  • monoglycerides e.g., glycerol monostearate
  • fatty acids e.g., stearic acid
  • steroids e.g., cholesterol
  • waxes e.g., cetyl palmitate
  • the LNPs may be formed from one or more cationic lipids of the ssPalm class.
  • the ssPalm class of cationic lipids were selected for evaluation based on the following parameters: (1) all are biodegradeable in that the polar head groups can be hydrolyzed from the hydrophobic domain by acid labile ester bond and the dual amphiphile can be dissociated into single amphiphiles by reduction of the disulfide bond; (2) formulation of these lipids with helper lipids (e.g., cholesterol,
  • phosphatidylethanolamine or phosphatidylcholine create a surface to which ApoE, an endogenous apolipoprotein can bind.
  • This association results in liver uptake of the nanoparticles through the low density lipoprotein (LDL) receptor, which is highly expressed on hepatocytes; and (3) the cationic lipids have a head group that when formulated with the other helper lipids has a pK at 6.0 ⁇ 0.5 (Ukawa, et al. Neutralized nanoparticle composed of SS-Cleavable and pH-activated lipid-like material as a long lasting and liver -specific gene delivery system Adv, Healthcare Materials (2014) 3: 1222-1229, incorporated herein by reference).
  • the lipid formulation includes disulfide-cleavable, pH-responsive amphiphiles, dioleoylphosphatidylethanolamine (e.g., l,2-dioleoyl-sn-glycero-3- phosphoethanolamine; DOPE), cholesterol and polyethylene glycol ( ⁇ MW2,000)- dimyristoyl glycerol (e.g., l,2-Dimyristoyl-rac-glycero-3-methylpolyoxy ethylene; DMG- PEG 2000) with an actual molar ratio of 58.3:9.71 :29.1 :2.91, respectively.
  • dioleoylphosphatidylethanolamine e.g., l,2-dioleoyl-sn-glycero-3- phosphoethanolamine; DOPE
  • DOPE dioleoylphosphatidylethanolamine
  • DOPE dioleoylphosphatidylethanolamine
  • DOPE dioleoylphosphati
  • Exemplary cationic lipids from which the LNPs may be formed include, but are not limited to, ssPalm, ssPalmE-P4C2, ssPalmE-Paz4-C2.
  • the LNP may further include one or more lipids selected from the group consisting of cholesterol, l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-Dimyristoyl-rac- glycero-3-methylpolyoxyethylene (DMG-PEG), distearoyl glycerol, stearoy- oleoylphosphatidylcholine (SOPC), dipalmitoylphosphatidylcholine (DMPC),
  • DOPE dioleoyl-sn-glycero-3-phosphoethanolamine
  • DMG-PEG 1 ,2-Dimyristoyl-rac- glycero-3-methylpolyoxyethylene
  • SOPC stearoy- oleoylphosphatidylcholine
  • DMPC dipalmitoylphosphatidylcholine
  • dipalmitoylphosphatidylcholine DPPC
  • DSPC dipalmitoylphosphatidylcholine
  • DOPC dipalmitoylphosphatidylcholine
  • POPC dipalmitoylphosphatidylcholine
  • DOPS dimyristoylphosphatidylserine
  • DOPA dioleoylphosphatydic acid
  • DOPA 1,2- Dimyristoyl-sn-glycero-3-phosphate
  • DPP A dipalmitoylphosphatidylcholine
  • DOPC dipalmitoylphosphatidylcholine
  • DOPC dipalmitoylphosphatidylcholine
  • DOPC dipalmitoylphosphatidylcholine
  • DOPC dipalmitoylphosphatidylcholine
  • DOPC dipalmitoylphosphatidylcholine
  • DOPC dipalmitoylphosphatidylcholine
  • DOPC dipalmitoylphosphati
  • the disulfide cleavable, pH-responsive amphiphile includes a vitamin E (a-tocopherol) hydrophobic chain covalently bonded to a piperidine.
  • Vitamin E a-tocopherol
  • Chemical structures of exemplary cationic lipids useful in the invention are as follo
  • the lipid composition of the gene delivery system may further include a mechanism for releasing the pDNA into the cytoplasm.
  • disulfide- linked dimyristoyl amphiphiles containing a quaternary amine with a pk ⁇ 6 are formulated with phospholipid, cholesterol and a diglycerol-polyetheylene glycol.
  • the release mechanism takes advantages of a pH gradient as intracellular vesicles, primary and secondary endosomes, bud off from the plasma membrane and migrate to the lysosome.
  • the pH of each endosomal membrane transitions from about a pH of 6.0 at the plasma membrane to about a pH of 4.0 in the lysosome.
  • the pDNA thereby gets released from the endosome into the cytoplasm and is then trafficked to the nucleus where it exists episomally for expression.
  • the lipid envelope may be in the form of naturally occurring secreted vesicles, such as exosomes, or may be synthetic versions thereof (e.g., synthetic exosomes).
  • Exosomes are endogenous nano-vesicles that transport RNAs and proteins that can deliver short interfering (si)RNA to target organs. Composed of cellular membranes with multiple adhesive proteins on their surface, exosomes are known to specialize in cell-cell communications and provide the ability to deliver various therapeutic agents to target cells.
  • the lipid envelope of the gene delivery system may further include one or more ligands that bind to hepatocyte receptors disposed thereon.
  • the ligands may be either adsorbed to the surface of the lipid envelope or may be covalently derivatized to specific components of the lipid envelope.
  • the surface chemistry thereof may be engineered to adsorb a protein ligand on the surface while maintaining exposure of the hepatocyte receptor binding domain, thereby facilitating docking of the gene delivery system specifically to a hepatocyte.
  • Exemplary protein ligands useful in the gene delivery system include, but are not limited to, ligands that bind to the LDL receptor, i.e., ApoE, viral glycoproteins either inserted into the membrane or derivatized to lipid. Peptides derived from viral glycoproteins may also either be adsorbed to the surface or derivatized to lipid components of the lipid envelope. Likewise, peptides that bind to hepatocyte receptors, such as those derived from malaria sporozoite proteins, may also be covalently derivatized to the phospholipids or to the ends of the polyethylene glycol derivatized diacylglycerols of the lipid envelope.
  • LDL receptor i.e., ApoE
  • Peptides derived from viral glycoproteins may also either be adsorbed to the surface or derivatized to lipid components of the lipid envelope.
  • peptides that bind to hepatocyte receptors such as those derived from malaria sporozoite proteins
  • non-protein ligands such as carbohydrates or vitamins, may be incorporated into the lipid envelope for hepatocyte targeting.
  • Such non-protein ligands may be covalently attached to various lipid components of the lipid envelope.
  • incorporation of a specific glycolipid may contain the appropriate carbohydrate configuration to trigger endocytosis of the delivery system into the hepatocyte.
  • synthetic peptides useful for hepatocyte targeting may either be adsorbed to the surface or chemically derivatized to one of the components of the lipid envelope.
  • RNA Polymerase III Promoter 7SK (SEQ ID NO: 12):
  • RNA Polymerase III Promoter U6 (SEQ ID NO: 13):
  • the invention provides a method of treating chronic hepatitis B viral infection in a subject by administering the plasmid of the invention using the gene delivery system described herein.
  • such treatment may be useful in subjects having viral infection wherein target viral sequences are comprised in an episomal nucleic acid molecule which is not integrated into the genome of the organism, such as a cccDNA.
  • target viral sequences are comprised in an episomal nucleic acid molecule which is not integrated into the genome of the organism, such as a cccDNA.
  • the HepAD38 cell line is derived from a HepG2 cell line in which a 1.3X hepatitis B virus genotype D is integrated into the HepG2 genome (Ladner, et al. Inducible expression of human hepatitis B virus in stable transfected heptoblastoma cells: a novel system for screening potential inhibitors of HBV replication Antimicro Agnts Chemother (1997) 41 : 1715-1720, incorporated herein by reference).
  • the human HBV genome is under the control of the tetracycline operator modified to be activatable by the tetR/VP16 transactivator by inclusion of the cytomegalovirus early promoter.
  • HBV Dane particles and other HBV replicative intermediates i.e., cccDNA, single stranded DNA, relaxed circular DNA, and other forms.
  • HBV surface antigen contains its own promoter sequences, the HepAD38 cell line is seropositive for the HBV surface antigen in either the repressed or unrepressed states (see, e.g., US Pat. No. 5,723,319, incorporated herein by reference in its entirety).
  • HepAD38 cells were obtained and grown in the absence of tet for 6 days to produce virus. The cells were then plated in a 24-well plate on Day 6 and transfected with CRISPR/Cas9/gRNA expression plasmids using a commercially available transfection reagent (Viafect, Promega Corporation). Media was removed and replaced after 48 hrs, and after an additional 48 hrs the media was removed. The media was then assayed for HBeAg and HBsAg. Reduction in viral antigens was compared to transfection of cells with plasmid DNA expressing CRISPR/Cas9 in the absence of gRNA.
  • gRNAs were screened for in vitro inhibition of HBV production using the HepAD38 cell line. Table 2 provides the list of gRNAs tested. Table 2: gRNAs Tested for Inhibition of HBV Genotype D Production
  • the 14 gRNAs shown in Table 3 were selected based on a showing of greater than 20% inhibition of HBeAg and HBsAg production in HepAD38 cells in vitro. Also shown is the HBV genotype to which the gRNA sequence is 100% identical.
  • gRNA/CRISPR/Cas9 pDNA to the luciferase pDNA resulted in inhibition of luciferase expression to >95% inhibition at a plasmid wt/wt ratio of 15: 1.
  • ternary 1 was constructed for comparison to a PTG (PTG6) expressing identical guide sequences (gl4 and g25, Table 3).
  • a secondary ternary cassette-containing construct was constructed for comparison to its PTG counterpart (PTG4) expressing identical guide sequences (g2 and gl 1, Table 3).
  • Comparison of the inhibition of HBeAg and HBsAg production for the bicistronic PTGs (PTG4 and PTG6) vs the ternaries with the same gRNAs (ternary2 and ternary 1, respectively) showed that additional inhibition of both antigens was obtained by incorporating the miRNA (see Table 1). Furthermore, it was observed that ternary2 strongly inhibited both HBeAg and HBsAg to levels comparable to ternary 1, thereby indicating that guides g2 and gl 1 function effectively in a ternary cassette.
  • the miRNA sequence was targeted to the HBV X protein and was modeled after a naturally occurring pri-miR-31 (Ely, et al. Efficient silencing of gene expression with modular trimeric Pol II expression cassettes comprising microRNA shuttles. Nuc Acids Res (2009) 37:e91, incorporated herein by reference).
  • Alternative naturally occurring short hairpin RNAs include miR-31 (Ely A, Naidoo T, Mufamadi S, et al. Expressed anti-HBV primary microRNA shuttles inhibit viral replication efficiently in vitro and in vivo. Mol Ther. 2008; Ely A, Naidoo T, Arbuthnot P.
  • Dexamethasone palmitate (CAS number, 14899-36-6)
  • PBS Phosphate Buffered Saline
  • dexamethasone palmitate solution was added into the lipid solution at a final concentration of about 0.5 ⁇ / 200 ⁇ .
  • the formulation process was optimized to yield 80 ⁇ 10 nm average diameter particles with a polydispersity index of ⁇ 0.2 and a plasmid DNA trapping efficiency of >80%.
  • the ssPalmM LNPs were prepared with the following formulation: ssPalmM:cholesterol:DOPE:DMG-PEG, 30:40:30:3
  • This example discusses a series of assays performed on different LNP formulations to obtain maximal gene expression and maximal tolerability in mice.
  • CpG free luciferase was expressed from a CpG free plasmid DNA backbone (Invivogen, San Diego, CA) and expression was monitored by bioluminescence imaging.
  • the expression plasmids were formulated using different cationic lipids and helper lipids to form LNPs that were characterized based on average particle size diameters, polydispersity index and plasmid DNA trapping efficiency.
  • a 10 ⁇ g dose of trapped plasmid DNA in an injection volume of 0.2 mL was administered into the tail veins of mice. The mice were then imaged for luciferase expression at 18 to 24 hrs after administration.
  • Imaging was accomplished by first anesthetizing the mice with a 0.2 mL ketamine cocktail administered by intraperitoneal (i.p.) injection, and then given 0.1 mL of 28.5 mg/mL D-luciferin i.p. Animals were then imaged for a duration of 0.5 sec to 5 min using the Xenogen IVIS imaging system. Assessed luciferase activity levels were expressed in photons emitted per second per square centimeter, denoted as p/sec/cm 2 .
  • Table 4 summarize the gene expression obtained with LNPs composed of different cationic lipids, where the amounts of cationic lipids are listed as mol%, and the amount of DMG-PEG is shown as the mol% of the total amount of cationic lipid, DOPE and cholesterol.
  • LNP3 ssPalmE-P4C2 LNP
  • a dose response assay was performed using LNP3 (i.e., the 60: 10 formulation) in which the injection volume was maintained at 0.2 mL, while the amount of trapped DNA was increased.
  • the LNP average diameter was 91 nm with a polydispersity index of 0.15 and a pDNA trapping efficiency of 92%.
  • the average bioluminescence from three mice as a function of pDNA dose is shown in Table 5. As demonstrated herein, maximum expression was obtained with the 10 ⁇ g dose of trapped pDNA.
  • the ratio was further reduced to 4: 1, which significantly reduced the pDNA trapping efficiency to 60% while increasing average particle size diameter to > 100 nm.
  • Physical observations from this study showed that the livers from mice administered Formulations #1 and #2 were pale. It should also be noted that increasing the ratio to 32 and 64 to 1 resulted in all animals dying (results not shown) within 24 hrs after
  • mice were bled 4 hrs after administration of the LNP formulation and assayed for interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-a) and interferon-gamma (INF- ⁇ ). Mice were imaged for luciferase expression 24 hrs after LNP administration. Once mice recovered from anesthesia, blood was collected and analyzed for liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The characterization and luciferase expression results are reported in Table 8A and the cytokine and liver enzyme results are reported in Table 8B. Table 8A: Effect of DP on LNP Characterization Parameters and Luciferase
  • the 60: 10:30 ssPalmE-P4C2:DOPE:Chol formulation with 3.4 mol% DMG-PEG and 3.3 mol% DP yielded the highest in vivo gene expression with reduced inflammatory cytokine levels.
  • Increasing the mol% of DP from 3.3 mol% to 6.6 mol% increased particle size to >100 nm and also reduced trapping efficiency. It is therefore conceivable that any mol% of DP equivalent to 3.3 mol%, or between 3.3 mol% and 6.6 mol%, may be used in the formulations disclosed herein to further reduce inflammatory cytokines and liver enzymes.

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Abstract

L'invention concerne un ADN plasmidique exprimant CRISPR et un système d'administration de gène ciblé sur des hépatocytes infectés par le virus de l'hépatite B. L'invention concerne également des procédés d'orientation de la formation d'un système CRISPR dans ces cellules pour s'assurer d'une spécificité améliorée de la reconnaissance de cible afin de modifier, améliorer ou traiter l'infection chronique par le VHB. En outre, la teneur en CpG de l'ADN plasmidique d'expression a été réduite au minimum pour réduire l'inflammation et maximiser l'expression génique.
PCT/US2018/039179 2017-06-23 2018-06-23 Administration médiée par des nanoparticules lipidiques (lnp) d'un adn plasmidique exprimant crispr pour le traitement d'une infection chronique par le virus de l'hépatite b WO2018237369A2 (fr)

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