Patent Grant
11613750
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled ALIG032SeqListing.TXT, which was created and last modified on Apr. 15, 2021 and is 9,278 bytes in size. The information in the electronic Sequence Listing is hereby incorporated by reference in its entirety.
This application relates to methods of reducing levels of virus molecules and/or treating viral infections by contacting infected cells with a therapeutic agent that targets a cellular host factor that is a target RNA or target protein involved in viral replication.
Hepatitis B virus (HBV) is a DNA virus and a member of the Hepadnaviridae family. HBV infects more than 300 million worldwide and is a causative agent of liver cancer and liver disease such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HBV infection can be acute and/or chronic. Acute HBV infection can be either asymptomatic or present with symptomatic acute hepatitis. Chronic Hepatitis B is characterized by persistent infection of liver cells by HBV that can last years and decades. HBV is classified into ten genotypes, A to J.
HBV has a partially double-stranded circular DNA of about 3.2 kilobase (kb) pairs. The HBV replication pathway has been studied in great detail (1). One part of replication includes the formation of the covalently closed circular DNA (cccDNA) form. The presence of the cccDNA gives rise to the risk of viral reemergence throughout the life of the host organism. HBV carriers can transmit the disease for many years. It is estimated that over 750,000 people worldwide die of hepatitis B each year. In addition, immunosuppressed individuals or individuals undergoing chemotherapy are especially at risk for reactivation of an HBV infection.
HBV can be transmitted by blood, semen, and/or another body fluid that contains virus. This can occur through direct blood-to-blood contact, unprotected sex, sharing of needles, and from an infected mother to her baby during the delivery process. The HBV surface antigen (HBsAg) is most frequently used to screen for the presence of this infection, as the majority of HBsAg is present in HBV virions. Currently available medications do not cure an HBV infection. Rather, the medications suppress replication of the virus.
Coronavirus disease 2019 (COVID-19) (also referred to as novel coronavirus pneumonia or 2019-nCoV acute respiratory disease) is caused by infection by SARS-CoV2, the severe acute respiratory syndrome coronavirus 2 (also referred to as novel coronavirus 2019, or 2019-nCoV). The disease was first identified in December 2019 and spread globally, causing a pandemic. Symptoms of COVID-19 include fever, cough, shortness of breath, fatigue, headache, loss of smell, nasal congestion, sore throat, sputum formation, pain in muscles or joints, chills, nausea, vomiting, and diarrhea. In severe cases, symptoms can include difficulty walking, confusion, blueish complexion, coughing up blood, decreased white blood cell count, and organ failure. Complications can include pneumonia, viral sepsis, acute respiratory distress syndrome, and kidney failure.
SARS-CoV-2 is not the only coronavirus that causes disease. It is a 3-coronavirus, a genus of coronaviruses that includes other human pathogens, including the SARS-CoV (the causative agent of Severe Acute Respiratory Syndrome), MERS-CoV (the causative agent of Middle East Respiratory Syndrome), and HCoV-OC43 (one causative agent of the common cold). The infectivity of these viruses, and the severity of the diseases they cause, varies widely. β-coronavirus can also manifest as zoonotic infections, spread to and from humans and animals. Additionally, non-human species such as camels, bats, tigers, non-human primates, and rabbits can be susceptible to β-coronavirus. There is a pressing need for treatments or cures for multiple coronaviruses.
The present disclosure provides molecules useful against viruses that utilize cellular host factors for viral replication, including HBV and respiratory viruses such as coronaviruses. Accordingly, the present disclosure fulfills the need in the art for compounds that can be used to safely and effectively treat or prevent such infections in humans.
Various cellular factors have now been identified as having roles in the production of certain molecules utilized by HBV and other viruses in order to replicate their genome and/or regulate the outcome of the viral infection. Oligonucleotides and siRNAs have been developed to target these cellular factors and thereby reduce production of the corresponding virus molecule(s) by the infected cell.
An embodiment provides a method of reducing a level of virus molecule produced by an infected cell, comprising:
contacting the infected cell with an effective amount of an oligonucleotide inhibitor that targets a cellular host factor to thereby inhibit production of the cellular host factor by the infected cell and reduce the amount of the virus molecule produced by the infected cell;
wherein the infected cell produces (a) at least one cellular host factor that is a target RNA or a target protein selected from a GRP78/BIP RNA, a GRP78/BIP protein, a SRSF1 RNA, a SRSF1 protein, a HNRNPA2B1 RNA, a HNRNPA2B1 protein, a RPLP1 RNA, a RPLP1 protein, a RPLP2 RNA, and a RPLP2 protein; and (b) an amount of at least one virus molecule that the virus utilizes for replication, wherein the virus molecule is a viral protein, a viral DNA or a viral RNA. In an embodiment, the virus is HBV. In another embodiment, the virus is a coronavirus. In an embodiment, the oligonucleotide inhibitor has an EC50 value that is less than 100 nM as determined by an assay for the virus molecule.
Another embodiment provides a method of treating a viral infection, comprising:
administering a therapeutically effective amount of an oligonucleotide inhibitor to a subject in need thereof, wherein the oligonucleotide inhibitor targets a cellular host factor to thereby reduce production of the cellular host factor by infected cells of the subject;
wherein the infected cells produce at least one cellular host factor that is a target RNA or a target protein selected from a GRP78/BIP RNA, a GRP78/BIP protein, a SRSF1 RNA, a SRSF1 protein, a HNRNPA2B1 RNA, a HNRNPA2B1 protein, a RPLP1 RNA, a RPLP1 protein, a RPLP2 RNA, and a RPLP2 protein. In an embodiment, the virus causing the infection is HBV. In another embodiment, the virus causing the infection is a coronavirus. In an embodiment, the oligonucleotide inhibitor has an EC50 value that is less than 100 nM as determined by an assay for the virus molecule.
Another embodiment provides a pharmaceutical composition for treating a viral infection in a subject, comprising:
a pharmaceutically acceptable carrier, diluent, excipient or combination thereof; and
an therapeutically effective amount of an oligonucleotide inhibitor that targets at least one of a target RNA or a target protein selected from a GRP78/BIP RNA, a GRP78/BIP protein, a SRSF1 RNA, a SRSF1 protein, a HNRNPA2B1 RNA, a HNRNPA2B1 protein, a RPLP1 RNA, a RPLP1 protein, a RPLP2 RNA, and a RPLP2 protein. In an embodiment, the infecting virus is Hepatitis B virus. In another embodiment, the viral infection is COVID-19. In an embodiment, the oligonucleotide inhibitor has an EC50 value that is less than 100 nM as determined by an assay for a virus molecule produced by the infected cell.
These and other embodiments are described in greater detail below.
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications referenced herein are incorporated by reference in their entirety unless stated otherwise. In the event that there are a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.
As used herein the term “oligonucleotide” (or “oligo”) has its usual meaning as understood by those skilled in the art and thus refers to a class of compounds that includes oligodeoxynucleotides, oligodeoxyribonucleotides and oligoribonucleotides. Thus, “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof, including reference to oligonucleotides composed of naturally-occurring nucleobases, sugars and phosphodiester (PO) internucleoside (backbone) linkages as well as “modified” or substituted oligonucleotides having non-naturally-occurring portions which function similarly, and/or including modifications such as GalNAc that increase circulation half-life and/or reduce degradation of the oligonucleotide, e.g., degradation by endonucleases, and increase cellular uptake. An “oligonucleotide inhibitor” is an oligonucleotide that at least partially inhibits production by a cell of an RNA or protein to which it is targeted.
An “antisense” oligonucleotide (ASO) is a synthetic oligonucleotide that recognizes or specifically anneals to a target RNA in a sequence-specific manner. The sequence of the target RNA recognized by the siRNA or ASO is defined by the nucleotide number of the protein-coding sequence of the mRNA, where the adenylate of the initiation codon (AUG) is designated nucleotide 1. The sequences used in this application are of a length and sequence that can recognize the target with high specificity within a cell. The specificity can be expressed by the calculated melting temperature of the duplex of 1 nM of a duplex of the siRNA and the target RNA sequence set at physiological salt (155 mM) and using the nearest neighbor parameters. The specificity can also be expressed by a thermodynamic constant for the annealed duplex, the change in Gibbs Free Energy (ΔG). A computer program to calculate Tm and thermodynamic constants for the siRNA/target duplex is available as The OligoCalc (19).
As used herein the term “siRNA” has its usual meaning as understood by those skilled in the art and thus refers to a class of oligonucleotides that are referred to as small interfering RNA, short interfering RNA and/or silencing RNA. These compounds are non-coding double-stranded RNA molecules, typically 19-25 base pairs in length, that operate within the RNA interference (RNAi) pathway and interfere with the expression of specific genes that contain complementary nucleotide sequences to the anti-sense strand of the siRNA and mediate degradation of the mRNA. The reduced level of the mRNA will decrease translation and the amount of the target protein. Reference herein to siRNA shall be understood to include reference to siRNA composed of naturally-occurring nucleobases, sugars and phosphodiester (PO) internucleoside (backbone) linkages as well as “modified” or substituted siRNA having non-naturally-occurring portions which function similarly, and/or including modifications such as GalNAc that increase circulation half-life and/or reduce degradation of the oligonucleotide, e.g., degradation by endonucleases, and increase cellular uptake.
As used herein the terms “target”, “targeting” and similar terms, as used in the context of oligonucleotide inhibitor recognizing a target RNA through base pairing and have their usual meaning as understood by those skilled in the art to refer to a process by which the oligonucleotide inhibitor hybridizes to the target RNA and at least partially inhibits production of the RNA or protein to which it is targeted. For example, siRNA may cause silencing of a gene that encodes an mRNA by reducing the mRNA and thus decrease the products synthesized, including proteins, from that target RNA. Various assay techniques may be used to determine the degree to which the oligonucleotide inhibitor at least partially inhibits production of the RNA or protein to which it is targeted. Non-limiting examples of such siRNA and various RNA they target are set forth in Tables 1 and 2.
As used herein the term “virus molecule” has its usual meaning as understood by those skilled in the art and thus refers to a class of molecules made from the genome of a virus. A virus molecule is typically a viral protein, a viral DNA or a viral RNA. These molecules can be utilized or required by the virus to replicate their genome or regulate the outcome of the viral infection. For example, the term “HBV molecule” refers to a class of molecules made from the HBV genome. Examples of HBV molecules include S-antigen, E-antigen, Core-antigen, HBV RNA, and HBV DNA. In an embodiment, the virus molecule is a coronavirus molecule, such as coronavirus RNA.
As used herein the terms “cellular host factor”, “cellular factor”, “host factor” and similar terms have their usual meaning as understood by those skilled in the art and thus refer to an RNA or protein produced by a cell and utilized by the virus for replication. As noted above, various cellular factors have now been identified as having roles in the production of certain virus molecules utilized by HBV and other viruses in order to replicate.
As used herein the terms “virus assay” and “viral assay” have their usual meaning as understood by those skilled in the art and thus refer to an assay that quantitatively assesses a virus molecule. For example, “HBV assay” refers to an assay that quantitatively assesses an HBV molecule or an effect of the HBV infection on the infected cell. Examples of HBV assays include HBsAg assay, HBeAg assay, HBV Core assay, HBV RNA assay, and HBV DNA assay. Likewise, a “coronavirus assay” is an example of a respiratory virus assay that quantitatively assesses a molecule or molecules of the respiratory virus or an effect of the viral infection on the infected cell. Examples of Coronavirus assays include quantitative polymerase chain reaction (qPCR) assay and a Branched DNA assay. A suitable virus assay can be used to identify a cell and/or a subject that is infected with the virus.
As used herein, a “carrier” refers to a compound or particle that facilitates the passage, delivery and/or incorporation of a compound to cells, tissues and/or bodily organs. For example, without limitation, a lipid carrier of the siRNA that can deliver the siRNA to the cytoplasm of cells. Another example is nanoparticle (LNP), a type of carrier that can encapsulate an oligonucleotide and/or siRNA to thereby protect the oligonucleotide inhibitor from degradation during passage through the bloodstream and/or to facilitate delivery to a desired organ, such as to the liver.
As used herein, a “diluent” refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent may be used to increase the bulk of a potent drug whose mass is too small for manufacture and/or administration. It may also be a liquid for the dissolution of a drug to be administered by injection, ingestion or inhalation. A common form of diluent in the art is a buffered aqueous solution such as, without limitation, phosphate buffered saline that mimics the composition of human blood.
As used herein, an “excipient” refers to an inert substance that is added to a pharmaceutical composition to provide, without limitation, bulk, consistency, stability, binding ability, lubrication, disintegrating ability etc., to the composition. A “diluent” is a type of excipient.
Methods of Reducing Virus Molecules and Treating Viral Infections
As demonstrated in the working examples below, the cellular proteins GRP78/BIP, SRSF1, HNRNPA2B1, RPLP1, and RPLP2 have now been identified as cellular host factors having roles in viral replication and infection. Various methods have now been developed for reducing the levels of virus molecules by targeting such host factors. For example, an embodiment provides a method of reducing the quantity of a virus molecule produced by an infected cell, comprising contacting the infected cell with an effective amount of an oligonucleotide inhibitor that targets at least one of a target RNA or target the reduction of the protein as described herein. Such contacting is effective to inhibit production by the infected cell of the cellular host factor and reduce the amount of the virus molecule produced by the infected cell. Those skilled in the art recognize that the methods described herein also provide descriptions of corresponding uses of the oligonucleotide inhibitors. For example, various embodiments provide an oligonucleotide inhibitor for use in a method as described herein, e.g., a method of reducing a virus level and/or treating a viral infection. Other embodiments provide an oligonucleotide inhibitor for use in preparing a medicament that is administrable to a subject by a method as described herein, e.g., a method of reducing the amount of virus produced and/or treating a viral infection.
An embodiment provides a method of reducing a level of a virus molecule produced by an infected cell, comprising:
In an embodiment, the cell infected with the virus is a mammalian cell. For example, in an embodiment, the mammalian cell is a human cell. For an HBV infection, the infected cell is typically a cell from the liver, such as hepatocytes.
In an embodiment, the virus is HBV and the virus molecule is an HBV molecule. For example, in various embodiments the HBV molecule is an S-antigen (HBsAg), an E-antigen (HBeAg), a Core-antigen, an HBV RNA, or an HBV DNA. In an embodiment, the HBV molecule is an S-antigen (HBsAg).
In another embodiment, the virus is a respiratory virus and the virus molecule is a respiratory virus molecule. In an embodiment, the respiratory virus is a coronavirus, e.g., a coronavirus such as SARS-CoV, MERS-CoV, HCoV-OC43 (aka OC43), or SARS-CoV2 (the virus that causes Covid-19). In an embodiment, the coronavirus molecule is coronavirus RNA or the viral genome.
Various methods have also been developed for treating viral infections. For example, an embodiment provides a method of treating a viral infection, comprising:
Unless the context indicates otherwise, descriptions herein of various inventive features or aspects shall be understood to apply to both such embodiments of methods of reducing levels of virus molecules produced by infected cells and methods of treating viral infections. In an embodiment, the subject infected with the virus is a mammalian subject, in which case the infected cells are mammalian cells. For example, in an embodiment, the mammalian subject is a human subject and the infected cells are human cells. In various embodiments, the methods further comprise identifying the cell and/or subject as being infected by the virus prior to contacting the infected cell with the effective amount of the oligonucleotide inhibitor and/or administering the therapeutically effective amount of oligonucleotide inhibitor to the subject.
In various embodiments, the cellular host factor produced by the infected cells of the subject is an RNA or protein as described herein. In various embodiments, the reduction in the cellular host factor by the infected cells of the subject is determined by determining a reduction in the virus molecule produced by the infected cells. In an embodiment, the oligonucleotide inhibitor reduces the level of a virus molecule produced by the infected cells and thereby reduces the viral load of the subject. In an embodiment, the virus infecting the subject is HBV and the reduction in production of the cellular host factor is indicated by a reduced level of an HBV molecule. For example, in various embodiments the HBV molecule is an S-antigen (HBsAg), an E-antigen (HBeAg), a Core-antigen, an HBV RNA, or an HBV DNA. In an embodiment, the HBV molecule is an S-antigen (HBsAg). Thus, in an embodiment, the reduction in production of the cellular host factor is indicated by a reduced level of HBsAg as determined by an HBsAg assay.
In another embodiment, the virus infecting the subject is a respiratory virus and the virus molecule is a respiratory virus molecule. In an embodiment, the respiratory virus is a coronavirus, e.g., a human coronavirus, and the respiratory virus molecule is a human coronavirus molecule. For example, in an embodiment, the coronavirus is a β-coronavirus such as SARS-CoV, SARS-CoV2, MERS-CoV, or HCoV-OC43.
In various embodiments, the oligonucleotide inhibitor targets at least one of a target RNA selected from a GRP78/BIP RNA, a SRSF1 RNA, a HNRNPA2B1 RNA, a RPLP1 RNA, and a RPLP2 RNA. In other embodiments, the oligonucleotide inhibitor targets at least one of a target protein selected from a GRP78/BIP protein, a SRSF1 protein, a HNRNPA2B1 protein, a RPLP1 protein, and a RPLP2 protein. In an embodiment, the oligonucleotide inhibitor is an anti-sense oligonucleotide (ASO) that recognizes the target RNA, wherein the target RNA includes mRNA. In another embodiment, the oligonucleotide inhibitor is a silencing RNA (siRNA) that recognizes the target RNA, wherein the target RNA includes mRNA. In various embodiments, the oligonucleotide inhibitor comprises one or more chemically-modified nucleotides that enhance binding of the oligonucleotide inhibitor to the target RNA or target protein.
Oligonucleotides and siRNAs that target at least one of a target RNA or target protein can be obtained from commercial sources, prepared by known methods or modifications thereof, and/or prepared as described elsewhere herein. Non-limiting examples of siRNAs that target GRP78/BIP, HNRNPA2B1, RPLP1, RPLP2 and SRSF1 are listed in Table 1 and Table 2 and described in the Examples below. In an embodiment, the oligonucleotide inhibitor is a siRNA as described in Table 1 and Table 2, or a modified version thereof. In various embodiments, the oligonucleotide inhibitor is modified to reduce degradation of the oligonucleotide by nucleases. In another embodiment, the oligonucleotide inhibitor is modified to enhance binding of the oligonucleotide inhibitor to the target RNA or target protein. In another embodiment, the oligonucleotide inhibitor is conjugated to amino sugars or lipids.
In an embodiment, the oligonucleotide inhibitor targets at least one of the GRP78/BIP RNA and the GRP78/BIP protein. In another embodiment, the oligonucleotide inhibitor targets at least one of the SRSF1 RNA and the SRSF1 protein. In another embodiment, the oligonucleotide inhibitor targets at least one of the HNRNPA2B1 RNA and the HNRNPA2B1 protein. In another embodiment, the oligonucleotide inhibitor targets at least one of the RPLP1 RNA and the RPLP1 protein. In another embodiment, the oligonucleotide inhibitor targets at least one of the RPLP2 RNA and the RPLP2 protein.
In an embodiment, an oligonucleotide inhibitor as described herein is administered to a subject in an amount that is effective to reduce the level of a virus molecule produced by infected cells and thereby reduce the viral load of the subject. Oligonucleotide inhibitors that target at least one of a target RNA or target protein can have an EC50 value that is less than 100 nM, less than 50 nM, less than 30 nM, or less than 10 nM, as determined by a suitable viral assay, e.g., an HBV assay or an assay for a respiratory virus. Various suitable viral assays are known to those skilled in the art and may be selected based on the virus molecule that is selected for reduction. For example, a reduction in the level of a coronavirus RNA can be determined by measuring a reduction in virion production by the infected cell. Examples of HBV molecules include an S-antigen (HBsAg), an E-antigen (HBeAg), a Core-antigen, an HBV RNA, and an HBV DNA. In an embodiment, the HBV assay quantitatively assesses HBsAg, HBeAg, HBV Core, HBV RNA, or HBV DNA. In some situations, the selection of an HBV assay is not necessarily made on the basis of an HBV molecule that is selected for reduction, including for example situations in which the HBV molecule is not known, multiple HBV molecules are present and/or a subject is being treated for HBV. In such situations, an HBsAg assay is selected as a default HBV assay. For example, in an embodiment of a method of treating an HBV infection as described herein, the HBV assay is an HBsAg assay.
In various embodiments, levels of virus molecules can be reduced significantly by contacting a virus-infected cell with an effective amount of an oligonucleotide inhibitor as described herein. For example, the contacting of the virus-infected cell with the effective amount of the oligonucleotide inhibitor may reduce the level of the virus molecule by at least 20%, at least 30%, at least 40%, or at least 50%. In an embodiment, the cell is infected with HBV and the virus molecule is an HBV molecule. In another embodiment, the cell is infected with a coronavirus (such as a β-coronavirus) and the virus molecule is a coronavirus molecule. The reduction in the level of the virus molecule may be determined using a suitable viral assay as described elsewhere herein. Such an assay may be selected on the basis of the virus molecule for which the reduction in level is determined, again with HBsAg assay being a default HBV assay.
Some embodiments described herein relate to a method of treating a viral infection (e.g., HBV infection or respiratory virus infection such as coronavirus infection) that can include administering to a subject identified as suffering from the viral infection an effective amount of an oligonucleotide inhibitor as described herein, or a pharmaceutical composition that includes an effective amount of an oligonucleotide inhibitor as described herein. Other embodiments described herein relate to using an oligonucleotide inhibitor as described herein in the manufacture of a medicament for treating a viral infection (e.g., HBV infection or respiratory virus infection such as coronavirus infection). Still other embodiments described herein relate to the use of an oligonucleotide inhibitor as described herein or a pharmaceutical composition that includes an oligonucleotide inhibitor as described herein for treating a viral infection (e.g., an HBV infection or respiratory virus infection such as coronavirus infection).
Various embodiments provide methods of treating viral infections by administering an effective amount of an oligonucleotide inhibitor to a subject as described herein. Other embodiments provide oligonucleotide inhibitors as described herein for use in the treatment of such viral infections and/or for use in the manufacture of medicaments for such treatments of viral infections. In various embodiments, such methods and uses further comprise administering an effective amount of second therapy to the subject in combination with the oligonucleotide inhibitor. The second therapy may be selected from various therapeutic modalities for treatment of the viral infection. In an embodiment, the second therapy comprises administering an effective amount of a second therapeutic molecule to the subject in combination with the oligonucleotide inhibitor. For example, in various embodiments, the second therapeutic molecule comprises a second oligonucleotide inhibitor as described herein, a nucleoside, a nucleotide, a nucleotide prodrug, an interferon, a capsid assembly modulator, a protease inhibitor, or a combination thereof. For example, in an embodiment, the second therapeutic molecule is remdesivir. In an embodiment, the second therapeutic molecule comprises a second oligonucleotide inhibitor. In an embodiment, the second oligonucleotide inhibitor targets at least one cellular host factor. The cellular host factor targeted by the second oligonucleotide inhibitor may be the same or different from the cellular host factor targeted by the first oligonucleotide inhibitor. In an embodiment, the first oligonucleotide inhibitor targets RPLP1 and the second oligonucleotide inhibitor targets RPLP2. In another embodiment, the first oligonucleotide inhibitor targets GRP78 and the second oligonucleotide inhibitor targets RPLP1. In another embodiment, the first oligonucleotide inhibitor targets GRP78 and the second oligonucleotide inhibitor targets RPLP2.
Various routes may be used to administer an oligonucleotide inhibitor to a subject in need thereof as indicated elsewhere herein. In an embodiment, an effective amount of the oligonucleotide inhibitor, or of a pharmaceutical composition that includes the oligonucleotide inhibitor, is administered to the subject by a parenteral route. For example, in an embodiment, an effective amount of the oligonucleotide inhibitor, or of a pharmaceutical composition that includes the oligonucleotide inhibitor, is administered to the subject intravenously. In another embodiment, an effective amount of the oligonucleotide inhibitor, or of a pharmaceutical composition that includes the oligonucleotide inhibitor, is administered to the subject subcutaneously. In another embodiment, an effective amount of the oligonucleotide inhibitor, or of a pharmaceutical composition that includes the oligonucleotide inhibitor, is administered to the subject by inhalation.
Isolated Synthetic Oligonucleotide Inhibitors
Various embodiments provide an isolated synthetic oligonucleotide inhibitor having an antisense strand that hybridizes with high specificity to a target RNA as described herein. Examples of such oligonucleotide inhibitors are described in Table 2. The specificity of hybridization can be expressed by a calculated melting temperature (Tm) of at least about 48° C., about 49° C., or about 50° C. For example, in an embodiment, the specificity of hybridization is expressed by a calculated Tm of 50° C. or higher. In an embodiment, the specificity of hybridization is expressed by a calculated Tm that is greater than or equal to any of the calculated values for Tm shown in Table 2.
An embodiment provides an isolated synthetic oligonucleotide inhibitor having an antisense strand that hybridizes to a target RNA, wherein:
The nucleobases are numbered according to the positions numbered as shown for the respective target RNA as shown in
In various embodiments the antisense strand of the isolated synthetic oligonucleotide inhibitor is any 15-21 mer, 16-21 mer, 17-21 mer, 18-21 mer, 19-21 mer or 20-21 mer antisense strand within the antisense strand of any one of SEQ ID NOS. 21-52, with the proviso that the antisense strand of the isolated synthetic oligonucleotide is not any one of SEQ ID NOS. 1-20. For example, in an embodiment, the antisense strand of the isolated synthetic oligonucleotide inhibitor is any 15-21 mer antisense strand within the antisense strand of any one of SEQ ID NOS. 21-42, 44-45, 47-50 and 52. In another embodiment, the antisense strand of the isolated synthetic oligonucleotide inhibitor is any 17-21 mer antisense strand within the antisense strand of any one of SEQ ID NOS. 21-42, 44-50 and 52.
Discussion of Methods of Reducing Virus Molecules and Treating Viral Infections
Viruses are intracellular parasites that depend on the host cells for many metabolites and factors needed for their reproduction and propagation. The cellular host factors are potential targets for therapeutic intervention, as long as they are not essential for the health of the cell. Targeting cellular proteins to reduce viral infection may be less likely to select for resistant viruses when compared to direct-acting antivirals, as the production of the cellular molecules is not typically subject to error-prone synthesis that typifies viral nucleic acid synthesis (2).
HBV is among the smallest of the enveloped viruses (1). HBV attacks liver cells and its infection can be either acute and cleared by the host, or chronic. Chronic infections and the resulting inflammatory responses can lead to patients developing hepatitis, liver cirrhosis and hepatocellular carcinoma. Over 180 million people worldwide are chronically infected by HBV. While existing drugs that can inhibit the HBV polymerase are available and are effective in reducing viral load, they do not result in cures of the viral infection.
HBV gain entry to liver cells through the liver-specific bile acid transporter named the sodium taurocholate co-transporting polypeptide (NCTP) (3). The nucleocapsid released into the cells contains a relaxed partial double-stranded DNA genome covalently attached to the viral polymerase that can convert the partial double-stranded DNA to a covalently-closed circle, called the cccDNA. The cccDNA is the template for transcription of a nest of HBV RNAs that can be processed and then translated to form several proteins, or the HBV pre-genomic RNA that can be replicated to form the HBV DNA (1). The first HBV protein to be discovered is the HBV surface antigen (HBsAg), which is secreted to affect the immune response as well as assembled around the nucleocapsid to form the infectious virus. The Core protein (also known as the Core antigen) is the structural protein that assembles around the HBV genome to form the icosahedral nucleocapsid. The E-antigen (HBeAg) is produced from an RNA splice variant that contains a leader peptide fused to Core to enable the secretion of the E-antigen from the infected cell, with a possible consequence of interfering with the host immune response. HBV also produces its own DNA polymerase, a reverse transcriptase that can synthesize DNA from either RNA or DNA templates. The HBV protein referred to as HBX is a multi-functional protein that can regulate cellular signaling as well as contribute to liver pathology.
HBV replication has been extensively studied in cultured cells. The human hepatoblastoma cell line, HepG2 cells, can be infected with HBV virions, especially when they overexpress the NCTP receptor (4). HepG.2.2.15 cells derived from HepG2 cells containing integrated copies of the HBV genome have also been used to produce HBV molecules and form infectious virions (5).
It is believed that cellular proteins play important roles throughout the HBV replication/infection process. Several of these host factors have been identified, including ones that play roles in the synthesis, folding, modification, and trafficking of the HBV proteins (6). Host factors that interact with the processing of HBV nucleic acids have also been identified, although their roles in HBV infection are less well-studied and understood. Host proteins are known to interact with the epsilon element, an RNA motif present at the two termini of the HBV pre-genomic RNA and at the 3′ terminus of the HBV mRNAs (7). The Epsilon element is required for HBV nucleic acid synthesis and encapsidation (8).
The RNAs and proteins known as GRP78/BIP RNA, GRP78/BIP protein, SRSF1 RNA, SRSF1 protein, HNRNPA2B1 RNA, HNRNPA2B1 protein, RPLP1 RNA, RPLP1 protein, RPLP2 RNA, and RPLP2 protein are known but oligonucleotide inhibitors have not been previously identified as candidates for targeting to reduce HBV or respiratory virus molecule levels and/or for treating HBV infection or respiratory virus infection. The GRP78/BIP protein, which may be referred to as GRP78 or as BIP, is believed to be a protein chaperone that regulates the recognition of mis-folded proteins that can affect cell survival, protein synthesis, and protein degradation (9). GRP78 protein levels may be elevated during HBV infection, and may associate with over-expressed HBsAg (10). SRSF1 protein is an RNA-binding protein that impacts RNA splicing, RNA transport and translatability, and can regulate nonsense-mediated mRNA decay (11). HNRNPA2B1 protein recognizes RNA, including those with N6-methylated adenylates to regulate RNA stability, processing, and translatability (12). HBV RNAs have been shown to possess N6-methyladenylates at the base of the epsilon element implicated in HBV RNA stability, replication and translatability (13). HNRNPA2B1 may also be implicated in the regulation of proinflammatory cytokine production that can activate host innate immune response to viral infection (14). RPLP2 protein is a ribosome-associated protein that is thought to regulate the efficiency of translational elongation (15). RPLP2 protein functions in complex with two additional proteins, RPLP0 and RPLP1 to comprise a structure named the acidic ribosome stalk that can interact with translation elongation factors to enhance the translation of some transmembrane proteins (16). Both RPLP1 and RPLP2 are implicated in the production of wild-type levels of the envelope proteins from several positive-strand RNA viruses, and both affect infectivity (17, 18).
Pharmaceutical Compositions
Some embodiments described herein relate to a pharmaceutical composition that can include an oligonucleotide inhibitor as described herein, and a pharmaceutically acceptable carrier, diluent, excipient or combination thereof. For example, an embodiment provides a pharmaceutical composition for treating a viral infection (e.g., an HBV infection or respiratory virus infection such as coronavirus infection), comprising:
In an embodiment, the oligonucleotide inhibitor has an EC50 value that is less than 100 nM as determined by a suitable assay (e.g., an HBV assay or an assay for a respiratory virus such as a coronavirus assay).
Those skilled in the art will understand that the oligonucleotide inhibitor that are included in the pharmaceutical composition can be any one or more of the various oligonucleotide inhibitors that target at least one of a target RNA or a target protein as described elsewhere herein. For example, in an embodiment, the siRNA in the pharmaceutical composition is an siRNA as described in Table 1 or a modified version thereof. In another embodiment, the siRNA in the pharmaceutical composition is an siRNA as described in Table 2 or a modified version thereof.
Proper formulation of the pharmaceutical composition is dependent upon the route of administration chosen. Techniques for formulation and administration of the oligonucleotide inhibitors described herein are known to those skilled in the art. Multiple techniques of administering oligonucleotide inhibitors exist in the art including but not limited to parenteral delivery, such as by inhalation, intravenous or subcutaneous delivery. Pharmaceutical compositions will generally be tailored to the specific intended route of administration. For example, in an embodiment, the pharmaceutical composition is in a form suitable for administration to a subject via parenteral delivery. In various embodiments such a parenteral delivery form includes a carrier that enhances delivery. For example, in an embodiment the oligonucleotide inhibitor is encapsulated in lipids that protect the oligonucleotide inhibitor from degradation during passage through the bloodstream and/or facilitates delivery to a desired organ, such as to the liver.
Additional embodiments are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the claims.
A series of siRNA that target cellular host factors to reduce HBV molecule levels were prepared or obtained from commercial sources. Table 1 describes the sequence for each siRNA and the corresponding target RNA.
1The first adenylate in the initiation codon (AUG) of the target mRNA is designated as nt. 1.
2Tm was determined for 1 nM double-stranded RNA in 155 mM salt (Na+) and using Nearest Neighbor parameters.
3ΔG was calculated for 1 nM of annealed dsRNA at thermodynamic constant conditions: 1M NaCl at 25° C., pH 7.0.
A series of siRNA that target cellular factors to reduce virus molecule levels were prepared. Table 2 describes the sequence for each siRNA and the corresponding target RNA.
1The first adenylate in the initiation codon (AUG) of the target mRNA is designated as nt. 1.
2Tm was determined for 1 nM double-stranded RNA in 155 mM salt (Na+) and using Nearest Neighbor parameters.
3ΔG was calculated for 1 nM of annealed dsRNA at thermodynamic constant conditions: 1M NaCl at 25° C., pH 7.0.
The cellular proteins GRP78/BIP, SRSF1, HNRNPA2B1, RPLP1, and RPLP2 were identified as having roles in HBV replication and infection. Various siRNAs and combinations thereof targeting the RNAs encoding these proteins were contacted with HepG2.2.15 cells at a final concentration of 7.5 nM. The introduction of the siRNAs had minimal effects on cell viability (see
The EC50 values of several siRNAs and combinations of siRNAs targeting HNRNPA2B1, GRP78, RPLP1, RPLP2, and/or SRSF1 were determined with respect to the ability to reduce HBsAg levels in HepG2.2.15 cells. See
The ability of siRNAs targeting host factors that then results in the reduced production of multiple HBV molecules was evaluated by quantifying the amount of HBsAg, HBeAg, and Core protein produced by HepG2.2.15 cells after contacting with 10 nM of siRNAs that target HNRNPA2B1, GRP78, RPLP1, RPLP2, and SRSF1. We also contacted the cells with 5 nM of each set of siRNAs that targets both RPLP1 and RPLP2. The level of HBsAg, HBeAg, and Core were all determined using ELISAs. The introduction of the siRNAs had minimal effects on cell viability or cytotoxicity (
Altogether, these results show that the cellular factors can contribute to the levels of multiple HBV molecules in a hepatocyte model for HBV infection and that specific host factor(s) can contribute to the accumulation of an HBV molecule. Importantly, siRNAs and oligonucleotides that target the RNA encoding the host factor can decrease the levels of HBV molecules that are needed for successful infection.
The host factors we identified to affect HBV replication also have roles in coronavirus infection. The human coronavirus OC43 is a member of the β-coronavirus genus, the same as the highly pathogenic SARS-CoV and SARS-CoV2 (20). We established procedures for OC43 to infect HCT8, a human colon epithelial cell line, as coronaviruses can infect human intestinal tract and cause diarrhea. HCT8 cells express GRP78, RPLP1/2, SRSF1, and HNRNPA2B1 and transfection of siRNAs targeting these host factors resulted in the specific knockdown of these host factors. To examine the effects of siRNAs on OC43 infection, HCT8 cells were first infected virus and then transfected with siRNAs and the amount of virus produced from the infection was quantified using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) that had primers specific to the OC43 N gene (
A library of siRNAs that target GRP78, SRSF1, RPLP1 and RPLP2 was designed and synthesized. This library was biased to favor RPLP1 and RPLP2 because these proteins are nonessential in somatic cells and they function upstream of GRP78. That is, viral protein synthesis that failed due to a deficiency in the RPLP1 and RPLP2 proteins cannot be rescued by the chaperon protein GRP78.
The library of siRNAs of Example A5 were tested for the reduction of HBsAg secreted by HepG2.2.15 cells and in HBV virions. As summarized in Table 3, 1 of the siRNAs targeted GRP78, 2 targeted SRSF2, 10 targeted RPLP1, and 19 targeted RPLP2, and all were found to reduce HBsAg levels with EC50 values of less than 10 nM. In fact, the majority of the siRNAs had EC50 values of less than 1 nM. Furthermore, none of the siRNAs had apparent cytotoxicity at the highest concentration tested, 80 nM (Table 3). Finally, the effective siRNAs reduced the levels of another key HBV molecule, HBeAg. Examples of the dose responses for HBsAg and HBeAg reduction and the cytotoxicity profile for two siRNAs (SEQ ID NOS 24 and 34) are shown in
The siRNAs identified as described in Example A6 should function by a mechanism that includes being loaded in the RISC complex to cleave the cognate mRNA, leading to the degradation of the mRNA. To examine whether the siRNAs are effective in reducing the target mRNA, we used reverse transcription coupled with quantitative polymerase chain reaction (RT-qPCR) to assess the level of the target mRNA in HepG2.2.15 cells. All 32 siRNAs described in Table 2 reduced the mRNA by more than 75% (Table 3). A sampling of the percent reduction of the target mRNA by siRNAs is shown in
The host factors likely function in viral infection as proteins. Therefore, we determined whether the siRNAs could decrease the amounts of the target protein. Cell transfected with 10 nM of each siRNA for 72 h were lysed and the target proteins identified using Western blots probed with target-specific antibodies. The effective siRNAs described in Table 3 reduced the levels of the proteins by between 28 and 92% of the mock-treated samples. The reduction in the proteins is less than the reduction of the target mRNA, likely due to the proteins have prolonged half-life and that more time is needed for their degradation. Examples of the reduction for RPLP1 and RPLP2 detected using Western blot analysis are shown in
The siRNAs listed in Table 2 were transfected into human colon epithelial HCT8 cells and the amount of OC43 virion produced was quantified. In these assays, the OC43 vRNA was quantified using a Branched DNA assay (QuantiGene gene expression assay: ThermoFisher). As a positive inhibitory control, the infected cells were treated with Remdesivir. Treatment with Remdesivir at 0.9 M resulted in more than 98% reduction in OC43 vRNA. In addition, siRNAs transfected into HCT8 cells at 10 nM final concentration resulted in a reduction of OC43 vRNA by between 36 and 94% (Table 3). Examples of the inhibition of OC43 vRNA are illustrated in
The host factors for HBV replication function in concert and knockdowns of multiple host factors resulted in more significant reductions in HBV replication and HBsAg production (
The results illustrated in Table 3 and
This application claims priority to U.S. Provisional Application Ser. No. 62/931,962, filed Nov. 7, 2019, which is hereby incorporated herein by reference in its entirety.
| Number | Date | Country |
|---|---|---|
| 110393791 | Nov 2019 | CN |
| WO 2009080852 | Jul 2009 | WO |
| WO 2016142948 | Sep 2016 | WO |
| WO 2018026812 | Feb 2018 | WO |
| Entry |
|---|
| Campos et al. (Journal of Virology, 2017, 91, 4, e01706-16, 1-17). |
| Allerson et al. (J. Med. Chem. 2005, 48, 901-904). |
| Huang, K.L. et al. 2009 Involvement of GRP78 in inhibition of HBV secretion by Boehmeria nivea extract in human HepG2 2.2.15 cells. Journal of Viral Hepatitis, 2009, 16:367-375. |
| Spurgers, K.F. et al. 2010 Identification of essential filovirion-associated host factors by serial proteomic analysis and RNAi screen. Molecular and Cellular Proteomics, 2010, 9:2690-2703. |
| PCT Written Opinion of the International Searching Authority dated Feb. 9, 2021 for Application No. PCT/US2020/059385. |
| Alarcon C.R. et al. 2015. HNRNPA2B1 is a mediator of m6A-dependent nuclear RNA processing events. Cell 162: 1299-308. |
| Aznarez, I., et al. 2018. Mechanism of nonsense-mediated mRNA decay stimulation by splicing factor SRSF1. Cell Report 23: 2186-98. |
| Ballesta, J.P., Remacha, M. 1996. The large ribosomal stalk as a regulatory element of the eukaryotic translational machinery. Prog. Nucleic Acid Res. Mol Biol. 55: 157-193. |
| Campos, R.K. et al. 2017. RPLP1 and RPLP2 are essential flavivirus host factors that promote early viral accumulation. J. Virol. 91: e01706-16. |
| Campos R.K. et al., 2019. Ribosomal stalk proteins RPLP1 and RPLP2 promote biogenesis of flaviviral and cellular multi-pass transmembrane proteins. BioRxiv. Doi 10.110/713016. |
| Choi. A.K.H. et al. 2015. Structures of eukaryotic ribosomal stalk proteins and their complex with trichosanthin and their implications in recruiting ribosome-inactivating proteins to the ribosomes. Toxins 7: 638. |
| Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. 2020. The species of Severe acute respiratory syndrome-related coronavirus: classifying the 2019-nCoV and naming it SARS-CoV2. Nature Microbiology 5, 536-544. |
| Imam H. et al. 2018. N6-methyladenosine modification of the Hepatitis B virus RNA differentially regulates the viral life cycle., Proc. Natl. Acad. Sci. USA 115: 8829-34. |
| Kibbe WA. 2007 OligoCalc: an online oligonucleotide properties calculator. Nucl. Acids Res. 35 (webserver issue): May 25. |
| Ko C, Park W.J., Park S, Kim S, Windisch, M.P. and Ryu, W.S. 2015. The FDA approved drug irbesartan inhibits HBV infection in HepG2 cells stably expressing sodium taurocholate co-transporting polypeptide. Antivir. Ther. 20: 835-42. |
| Li, W. 2015. The hepatitis B virus receptor. Annu. Rev. Cell Dev. Biol. 31: 125-47. |
| Li, Y. et al., 2019. Hepatitis B surface antigen activates unfolded protein response in forming ground glass hepatocytes of chronic hepatitis B. Viruses 11: 386. |
| Liang, T.J. 2009. Hepatitis B: The virus and disease. Hepatology 49: S13-S21. |
| Mitra, B., Thapa, R. J., Guo H., and Block, T.M. 2018. Host functions used by hepatitis B virus to complete its life cycle: implications for developing host-targeting agents to treat chronic hepatitis B. Antiviral Res. 158: 185-98. |
| Pollack, J. and Ganem D. 1993. An RNA stem-loop structure directs hepatitis B virus genomic RNA encapsidation. J. Virol. 67: 3254-63. |
| Shin, H.J., Kim, S.S., Cho, Y. H., Lee, S.G. and Rho, H.M. 2002. Host cell proteins binding to the encapsidation signal episilon in the hepatitis B virus RNA. Arch. Virol. 147: 471-91. |
| Wang, M. et al. 2009. Role of the unfolded protein response regulator GRP78/BiP in development, cancer and neurological disorders. Antioxid. & Redox Signal. 11: 2307. |
| Wang, L., Wen, M., and Cao X. 2019. Nuclear HNRNPA2B1 initiates and amplifies the innate immune response to DNA viruses. Science: eaav0758. |
| Wang M. Cao R., Zhang L., Yang X., Liu J. et al. 2020. Remdesivir and chloroquine effectively inhibits the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Research 30, 269-271. |
| Warfield K.L. 2019. Lack of selective resistance of influenza A virus in presence of targeted antiviral UV-4B. Scientific Report. 9:7484. |
| Zhao R. et al. 2011. Hepatoma cell line HepG2.2.15 demonstrate distinct biological features compared with parental HepG2. World J. Gastroenterol. 17(9):1152-1159. |
| Number | Date | Country | |
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| 20210230591 A1 | Jul 2021 | US |
| Number | Date | Country | |
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| 62931962 | Nov 2019 | US |
