Patent Application
20240050547
The contents of the electronic sequence listing (0431.54.Sequence listing.092223_ST25.txt; Size: 6,772 bytes; and Date of Creation: Sep. 22, 2023) is herein incorporated by reference in its entirety.
The present invention relates to methods and/or treatments for preventing viral infections in animals (non-human). More specifically, the present invention relates to methods of treating and preventing viral infections in swine and other animals.
African swine fever virus (ASFV) is a large double stranded DNA virus that primarily infects domestic pigs, wild boars, warthogs, and bush pigs. It also resides in soft ticks, thereby acting as an infectious vector. ASFV primarily infects the monocytes and macrophages, although, at acute infection many other cell types can be infected. ASFV causes high fever, hemorrhagic lesions, cyanosis, anorexia, and fatalities in these animals. There is no vaccine or treatment for this virus, and the only way to currently prevent its spread is culling animals.
U.S. Provisional Patent Application No. 62/871,949 to Applicants discloses a gene drive for eliminating or neutralizing virus carriers such as soft ticks that carry ASFV. In the gene drive, an allele is altered so that it always shows up as the dominant allele in all offspring (not just 50%).
There have been many attempts to develop vaccines for ASFV over several including the intravenous injection of: 1) attenuated viruses (PMID: 31106218, PMID: 29609966, PMID: 27795430, PMID: 28814514, PMID: 33371460, PMID: 32124180, PMID: 31969432, PMID: 33327488, PMID: 34643433), 2) DNA/RNA vaccines, 3) antibody stimulating proteins that are derived from the outer membrane or capsid of the virus, (PMID: 8615037, PMID: 14980493, PMID: 28241999, PMID: 31275307, PMID: 34715492) 4) outer membrane or capsid neutralizing antibodies, (PMID: 28241999, PMID: 33562314), and 5) gene editing (PMID: 29362418, PMID: 34395778, PMID: 32587905).
Live naturally occurring and/or recombinant attenuated virus approaches have been shown to promote robust protection in swine against ASFV (PMID: 31969432), and to date represent the only protective approaches to ASF viral infection in swine. These approaches are meant for temporary relief only against viral infection due to the risk of reversion into more virulent strains of ASFV. Therefore, these technologies are less desirable than more advanced and safer subunit-based vaccines and therapeutics that are currently being sought by multiple laboratories.
DNA/RNA vaccines are highly potent and have been shown to be very effective in protecting hosts from viruses such as SARS-CoV-2. However, few attempts using DNA/RNA vaccine approaches for ASFV infection have been developed due to a lack of targeting and strategy knowledge that is confounded by viral structure and genomic complexities. ASFV is a multi-layered viral particle with at least two mechanisms of infection. Further, the ASFV multi-layered viral particle encompasses a viral genome with more than 150 open reading frames (PMID: 30185597, PMID: 31787524), most that have yet to be characterized.
Current subunit protein vaccine approaches that are designed to stimulate the immune system have fallen short of prolonged protection against the virus for a number of reasons. First, (as above) a lack of knowledge of protein function, genomic properties and viral infection cycles has impaired meaningful translation into a robust vaccine and/or therapeutic. Second, a rapid-fire combination of subunit protein injections that emulate multiple viral antigens has had minimally lasting protective effects likely due to inaccurate timing of treatment (per protein antigen), incorrect concentrations, and over-burdening of the immune system to produce a functional and lasting counter offensive, and most importantly, not considering the viral method of replication (as defined in
Neutralizing antibody approaches have also been met with limited protective quality against primary infections of ASFV in swine due to the limited knowledge related to the minimal structural, genomic, and replication cycles of the virus. Additional antibody-based therapeutic prophylactic approaches include the use of convalescent serum or selected/engineered monoclonal antibodies. Convalescent serum consisting of protective polyclonal pools of antibodies are likely not strong or stable enough to recognize viral antigen to elicit a prolonged immune response in the swine. Further, engineered, screened, or in silico designed monoclonal antibodies or monoclonal equivalents such as camelid antibodies may likely be better candidates than polyclonal antibodies (pooled either in vitro or derived and expressed in vivo). Additionally, antibody therapeutic approaches must take into account the lysogenic (outer membrane containing virus) and lytic (capsid-based virus) cycles and the timing of treatment (as previously described by Chen and Malcolm in PCT/US20/50939). For example, if the lysogenic outer membrane containing virions are not neutralized and the cycle is allowed to proceed (hidden from antibody therapies) to a lytic stage, the immune (and therapeutic advantage) will be overcome by virus flooding the body of the swine (as in
CRISPR gene editing has been shown to ablate the virus and prevent its spread in culture (PMID: 29362418). This powerful technique holds promise to cure ASFV in swine, but such therapies will never make it to market due to their high cost, especially when taking into consideration the low cost of swine per head.
Due to these challenges, there is a need to develop novel strategies for the treatment of ASFV based on newly discovered structural (PMID: 31624094), genomic, replication cycle, and immune interactions with viral antigens (
Structural Discoveries and Corresponding Inventions.
ASFV is a multi-layered and extremely stable virus. ASFV has 5 layers: 1) a nucleoid, 2) a core shell, 3) an inner membrane 4) a capsid and 5) an outer membrane (
ASFV goes through two infectious cycles, a rapid lysogenic cycle followed by an overwhelming lytic cycle (
Once ASFV has entered the macrophage through an unknown mechanism that is likely receptor-mediated (either by EP402R or I177L, or a combined mechanism from either receptor), the virus is released into the cytoplasm, and trafficked to viral factories (via p54 (E183L)/dynein interactions) in the ER where it begins to replicate (PMID: 15047843) (
Once the lytic cycle begins, capsid-based virions explode from the cell and spread quickly through the organism (
This model, based off recent structural and viral replication data (PMID: 31787524, PMID: 34643433), allows for a strategic plan to be implemented to fulfill the need for meaningful, safe and long-lasting vaccines and/or therapeutics (
The three-fold strategy depending on the temporal properties of the lysogenic and lytic cycles of ASFV:
Specific to blocking of the early lysogenic cycle:
By combining blocking strategies for each of these mechanisms of action, the lysogenic cycle can be halted, the immune system unsuppressed (by recoupling between MHC Class I antigen presentation and T-cell responses), and downstream B-cell mediated neutralization of outer membrane containing virions (if facilitated by antibody therapeutic blocking/neutralizing methods).
Specific to blocking of the early lytic cycle:
The blocking/neutralizing methods mentioned herein can be used to target any of the viral proteins associated with the lysogenic and lytic replication cycles to achieve cycle disruption, but at least one target for each cycle must be used simultaneously to effectively achieve that disruption. (
By inhibiting each of these outer membrane and/or capsid-based proteins in defined combinations (but not limited to these proteins) from interacting with a) RBCs and T-cell receptors, ASFV's primary MOA of entry into macrophage (which has yet to be defined) b) MHC Class I molecules on macrophage (and other antigen presenting cells), c) signal transduction proteins involved the inhibition of anti-viral cytokine expression and d) other receptor-mediated functionalities, the virus is: 1) blocked from other cellular interactions, 2) the T-cell response is no longer inhibited, 3) the macrophage MHC Class 1 complexes continue to be expressed thereby aiding to suppress the early lytic cycle from taking root (FIG. 5D1) and, 4) anti-viral cytokine inhibition will be prevented, respectively. Any combination of CD2v (EP402R), EP153R, p1177L, and p12 (063L), can be used to treat the swine and block the viral lysogenic cycle from taking root (
(2) By creating antibodies that neutralize the capsid proteins on the virions that do not contain an outer membrane, early infection through capsid-related endo/pinocytosis can be inhibited. Capsid proteins p14.5 (E120R), p49 (B438L), p72 (B646L), p30 (CP204L), p54 (E183L), and p12 (063R), represent formidable targets for vaccines and/or therapeutic regimen approaches (
(3) Additional protection can be implemented by engineering each of the targeted antibodies (toward CD2v (EP402R), EP153R, p72 (B646L), p54 (E183L), p30 (CP204L), and p12 (061R), p49 (B438L), and I177L, but not limited to these proteins) with a swine equivalent of human CD47 tag (
Strong antibody responses can be triggered in a number of ways. Therapeutics and Vaccines:
Therapeutic. The antibodies (for each desired target) can be selected and engineered using a platform screening approach or designed using artificial intelligence software such as Alphafold or Rosetta. Once the very strong, robust, highly selective, precise, specific, high affinity and high avidity antibodies are selected and/or A.I. designed, these antibodies (monoclonal) can be used as a therapeutic for direct injection, post biochemical and immunological pre-clinical testing. The therapeutic can be used to treat infected swine or as an antibody vaccine to protect swine against infection—prophylactic.
Therapeutic. Further, the epitope sequences can be derived from the monoclonal antibody selection process. These sequences can be used to engineer an antibody that contains the serotype-2 CD47 (mCD47) extracellular domain fused to the Fc region of the antibody. These engineered antibodies (against any desired target) can be used as a therapeutic to neutralize ASFV and prevent infection into macrophage.
Vaccine. The antibodies can be naturally stimulated by injecting the proteins of each target into the swine model. The protein antigen subunit vaccine will then stimulate the B-cells to create antibodies against them, and therefore the virus. Variants may include any peptide derivation from the protein targets with differences up to 90%). The antibodies produced will depend on the concentration of proteins injected and adjuvant release for prolonged effects.
Vaccine. The proteins (in any combination or concentration/dose—toward CD2v (EP402R), EP153R, p72 (B646L), p54 (E183L), p30 (CP204L), and p12 (061R), p49 (B438L), and I177L, (but not limited to these proteins account for critical targets that fall within the lysogenic/lytic dual treatment model. Each protein or subunit peptide related to the proteins can be expressed by delivering RNA/DNA transcripts in targeted liponanoparticles, exosomes, nanovesicles, biomimetic exosomes, AAVs, Anellovirus or Clews to B-cells to produce the protein antigens and elicit a more robust and lasting antibody effect. This approach will induce naturally structured (without a CD47 Fc region tag) antibodies. The delivery vehicle can also be targeted to B-cells using ligands that recognize CD19 receptors on B-cells (for example), but not limited to the CD19 target.
The receptor for ASFV on macrophage is unknown. Endo/pinocytosis has been shown to be one method of entry for ASFV. Due to the inefficient process of this method of infection, it is likely to occur in very early infection to start lysogenic replication processes and become more prominent during the lytic replication stage when ASFV virions are in very high concentrations. A two-hybrid system can be used to determine the viral ligand (bait) to cellular receptor (prey) interaction to define this MOA. The viral proteins p12 (061R), p54 (E183L), p49 (B438L), EP153R, EP402R (potential dual function, infection into host cell cytoplasm due to the presence of cell membrane penetrating peptides sequences and RBC binding), and I177L each have been predicted as potential viral ligands for cellular receptor-mediated infection.
p12 (061L) has been shown to exist in the outer membrane (lysogenic) and between the inner membrane and capsid (lytic) (PMID: 8219802, PMID: 9213394).
p54 (E183L) is an inner envelope protein component with a capsid exposed peptide segment. Antibodies raised against p54 (E183L) have been shown to slow the infection of the ASFV but remain insufficient to prevent infection (PMID: 14980493).
p49 (B438L) exists between the inner membrane and the capsid (lytic) and has a predicted receptor domain (PMID: 31624094).
EP153R, an outer membrane protein reduces the expression of MHC Class 1 surface molecules, suggesting it has role in direct macrophage contact at the cellular surface and a possible MOA for viral entry into the cell (PMID: 29609966, PMID: 21069396).
I177L has been predicted to be an outer membrane (lysogenic) and inner membrane (lytic) protein, containing a transmembrane domain. Its deletion significantly reduces infection (PMID: 33925435).
EP402R is an outer membrane protein that has been shown to facilitate hemadsorption between the outer membrane containing ASFV virion and RBCs. EP402R has also been shown to contain cell penetrating peptides.
By defining the viral ligand and cellular receptor interacting proteins and interaction MOA, the receptor can be blocked with small molecules, or antibodies, or nanobodies, or mutated virus that competes for the receptor, or nucleic acid competitors/binders.
Further, GMO swine or engineered macrophage (for replacement/substitution therapy) can be engineered to have receptors that have been altered in a manner to prevent the binding of ASFV. Similar approaches have been attempted to engineer human cells to be resistant to viruses such SARS-CoV-2 (ACE receptors) and HIV (CCRS delta mutations).
However, methods of treating the virus itself are still needed.
To date, the cellular receptor(s) for ASFV has not been identified, but there is evidence that the virus enters through a dynamin-dependent and clathrin-mediated endo/pinocytosis process in monocyte or macrophage cells (PMID: 29978065). Attempts to create strong antibody responses against viral antigens of ASFV have been met with poor results.
Gene editing allows DNA or RNA to be inserted, deleted, or replaced in an organism's genome by the use of nucleases. There are several types of nucleases currently used, including meganucleases, zinc finger nucleases, transcription activator-like effector-based nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas nucleases. These nucleases can create site-specific double (or single) strand breaks of the DNA in order to edit the DNA. Targeting the genome of receptors requires precise cuts to the viral genome and no off-target effects that could be harmful to the subject.
US Patent Application Publication No. 2014/0357530 discloses compositions, methods applications and screens used in functional genomics that focus on gene function in a cell and that use vector systems and other aspects related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas systems and components thereof. The application discloses modification of short portions of DNA, creating a 5′ overhang that is at most 200 base pairs, preferably at most 100 base pairs, or more preferably at most 50 base pairs.
U.S. Pat. No. 10,266,850 discloses DNA-targeting RNA that comprises a targeting sequence and, together with a modifying polypeptide, provides for site specific modification of a target DNA and/or a polypeptide associated with the target DNA. Also disclosed are methods of modulating transcription of a target nucleic acid in a target cell, generally involving contacting the target nucleic acid with an enzymatically inactive Cas9 polypeptide and a DNA-targeting RNA.
Gene editing has also been used to create point mutations. PMID: 30323312 teaches base editing, a newer genome-editing approach that uses components from CRISPR systems together with other enzymes to directly install point mutations into cellular DNA or RNA without making double-stranded DNA breaks. DNA base editors comprise a catalytically disabled nuclease fused to a nucleobase deaminase enzyme and, in some cases, a DNA glycosylase inhibitor. RNA base editors achieve analogous changes using components that target RNA. Base editors directly convert one base or base pair into another, enabling the efficient installation of point mutations in non-dividing cells without generating excess undesired editing byproducts.
Cas/deaminase fusion proteins have also been used to make point mutations. PMID: 30271918 used a nickase Cas9-cytidine deaminase fusion protein to direct the conversion of cytosine to thymine within prokaryotic cells, resulting in high mutagenesis frequencies in Escherichia coli and Brucella melitensis. US Patent Application Publication No. 20160304846 also discloses fusion proteins of Cas9 and nucleic acid editing enzymes or enzyme domains, e.g., deaminase domains, for editing a single site within the genome of a cell or subject.
There remains a need for treating and preventing viral infections (such as ASFV) that undergo a combination of early lysogenic viral replication, followed by late-stage lytic viral replication by interrupting their interactions with cellular receptors and cellular internalizing pathways (such as phagocytosis, macropinocytosis and endocytosis).
The present invention provides for a method of preventing and treating viral infections in animals (and preferably ASFV in porcine). Specifically, the invention couples the inhibition/blocking/neutralization of viral proteins associated with the lysogenic stage of replication, to the inhibition/blocking/neutralization of viral proteins associated with the lytic stage of replication in a simultaneous manner to disrupt and prevent both cycles from occurring. The result is the prevention of viral infection.
In more detail, the present invention targets viral ligand interactions associated with cellular receptors involved in hemadsorption, receptor-mediated viral infection of host cells, the inhibition of immune cell suppression, and the inhibition of anti-viral cytokine suppression that are integral with virions produced from a lysogenic replication cycle containing an outer membrane, and by simultaneously neutralizing structural capsid proteins in virions produced from a lytic replication cycle that lack an outer membrane.
Treatment can be accomplished through either 1) the (non-) or competitive inhibition of the viral ligand-cellular receptor interactions through engineered antibody therapeutics, 2) virus neutralization by engineered antibody therapeutics, 3) virus neutralization by engineered antibody therapeutic that also prevent phagocytosis and macropinocytosis (CD47/mCD47 domain included in the Fc region of the antibody), 4) virus neutralization by engineered antibody therapeutics with bispecific heavy and light chain epitopes, 5) virus neutralization by engineered antibody therapeutics with bispecific heavy and light chain epitopes that also prevent phagocytosis and macropinocytosis (CD47/mCD47 domain included in the Fc region of the antibody), 6) the (non-) or competitive inhibition of the viral ligand-cellular receptor interactions with small molecules, or 7) cellular receptor altering through gene editing methods, so that the viral entry proteins no longer recognize the natural/wildtype receptor.
Prevention (vaccine) can be accomplished through either 1) immune stimulation (B-cell) through the injection of viral proteins (or domains of the proteins) that are involved with ligand-cellular receptor interactions, 2) immune stimulation (T-cell) through the injection of viral T-cell antigens, 3) immune stimulation (B-cell and T-cell simultaneously) through the injection of viral proteins (or domains of the proteins) that are involved in the ligand-cellular receptor interaction or T-cell antigens, respectively, 4) the delivery (via exosomes, biomimetic exosomes, nanoparticles, AAV, Anellovirus, clews, liposomes) of mRNA encoding viral proteins or domains of the proteins that are involved in ligand-cellular receptor interactions such as to elicit an immune response from B-cells to produce neutralizing antibodies or any one of the combinations above that can be used in a pre-infective/prophylactic manner.
The present invention provides for a method of treating a viral infection in an individual with a virus that is both lysogenic and lytic, by administering a viral antigen that targets protein on an outer membrane of a lysogenic phase of the virus, administering a viral antigen that targets protein on a capsid of a lytic phase of the virus, and treating the viral infection.
The present invention also provides for a composition for treating a viral infection in an individual with a virus that is both lysogenic and lytic including a viral antigen that targets protein on an outer membrane of a lysogenic phase of the virus and a viral antigen that targets protein on a capsid of a lytic phase of the virus.
The present invention also provides for a vaccine for preventing viral infection, including whole and/or partial domains of proteins of both a lysogenic and lytic phase of a virus.
Other advantages of the present invention are readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein:
The present invention provides for a method of preventing and treating viral infections (and preferably ASFV in porcine), by simultaneously using disruptive combinations of therapeutics by, i) inhibiting the viral entry protein-to-cellular receptor interaction, ii) inhibiting the suppression of immune responses caused by viral proteins, iii) prevention of proximity mechanisms of infection by disrupting hemadsorption caused by viral proteins and, iv) by neutralizing structural proteins to mount an immune response against the virus. Treatment can be accomplished through either 1) the (non-) or competitive inhibition of the viral ligand-cellular receptor interactions through engineered antibody therapeutics, 2) virus neutralization by engineered antibody therapeutics, 3) virus neutralization by engineered antibody therapeutic that also prevent phagocytosis and macropinocytosis (CD47/mCD47 domain included in the Fc region of the antibody), 4) virus neutralization by engineered antibody therapeutics with bispecific heavy and light chain epitopes, 5) virus neutralization by engineered antibody therapeutics with bispecific heavy and light chain epitopes that also prevent phagocytosis and macropinocytosis (CD47/mCD47 domain included in the Fc region of the antibody), 6) the (non-) or competitive inhibition of the viral ligand-cellular receptor interactions with small molecules, or 7) cellular receptor altering through gene editing methods, so that the viral entry proteins no longer recognize the natural/wildtype receptor.
Prevention (vaccine) can be accomplished through either 1) immune stimulation (B-cell) through the injection of viral proteins (or domains of the proteins) that are involved with ligand-cellular receptor interactions, 2) immune stimulation (T-cell) through the injection of viral T-cell antigens (ref), 3) immune stimulation (B-cell and T-cell simultaneously) through the injection of viral proteins (or domains of the proteins) that are involved in the ligand-cellular receptor interaction or T-cell antigens, respectively, 4) the delivery (via exosomes, biomimetic exosomes, nanoparticles, AAV, Anellovirus, clews, liposomes, or any other suitable delivery methods) of mRNA encoding viral proteins or domains of the proteins that are involved in ligand-cellular receptor interactions such as to elicit an immune response from B-cells to produce neutralizing antibodies or anyone of the combinations above that can be used in a pre-infective/prophylactic manner.
“Animal” as used herein refers to any non-human species of animal.
“Porcine” or “swine” as used herein, can be a domestic pig, wild boar, warthog, or bush pig.
The term “vector” includes cloning and expression vectors, as well as viral vectors and integrating vectors. An “expression vector” is a vector that includes a regulatory region. Vectors are also further described below.
The term “antibody” as used herein refers to a blood protein produced in response to and counteracting a specific antigen. Antibodies combine chemically with substances which the body recognizes as alien, such as bacteria, viruses, and foreign substances in the blood.
The term “mRNA” as used herein refers to a type of RNA in cells that carries genetic information required to make proteins.
The terms “CD47 and/or CD47 domain and/or CD47 extra cellular domain” as used herein refer to a transmembrane protein that is present on many different cell types in all tissues. It is involved in cellular processes such as apoptosis, proliferation, adhesion, and migration.
The terms “mCD47 and/or mCD47 domain and/or mCD47 extra cellular domain” as used herein refer to as a modification of the wild type CD47 transmembrane protein that is present on many different cell types in all tissues. This modification/mutant retains the interaction property with SIRP-α receptors to prevent phagocytosis, but no longer binds to TSP1 thereby interrupting micropinocytosis-mediated viral entry.
The term “gRNA” as used herein refers to guide RNA. The gRNAs in the CRISPR Cas9 systems and other CRISPR nucleases herein are used for altering or editing receptors or genes encoding receptors. The gRNA can be a sequence complimentary to a coding or a non-coding sequence and can be tailored to the particular receptor or gene to be targeted. The gRNA can be a sequence complementary to a protein coding sequence, for example, a sequence encoding one or more viral structural proteins, (e.g., in ASFV the CP2475 gene encodes polypeptide 220 which is cut into the proteins p150, p37, p14, and p34). The gRNA sequence can be a sense or anti-sense sequence. It should be understood that when a gene editing composition is administered herein, preferably this includes one or more gRNA.
“Nucleic acid” as used herein, refers to both RNA and DNA, including cDNA, genomic DNA, synthetic DNA, and DNA (or RNA) containing nucleic acid analogs, any of which may encode a polypeptide of the invention and all of which are encompassed by the invention. Polynucleotides can have essentially any three-dimensional structure. A nucleic acid can be double-stranded or single-stranded (i.e., a sense strand or an antisense strand). Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA) and portions thereof, transfer RNA, ribosomal RNA, siRNA, micro-RNA, short hairpin RNA (shRNA), interfering RNA (RNAi), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers, as well as nucleic acid analogs. In the context of the present invention, nucleic acids can encode a fragment of a naturally occurring Cas9 or a biologically active variant thereof and at least two gRNAs where in the gRNAs are complementary to a sequence in a receptor or gene encoding a receptor.
An “isolated” nucleic acid can be, for example, a naturally occurring DNA molecule or a fragment thereof, provided that at least one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule, independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by the polymerase chain reaction (PCR) or restriction endonuclease treatment). An isolated nucleic acid also refers to a DNA molecule that is incorporated into a vector, an autonomously replicating plasmid, a virus, or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid such as a DNA molecule that is part of a hybrid or fusion nucleic acid. A nucleic acid existing among many (e.g., dozens, or hundreds to millions) of other nucleic acids within, for example, cDNA libraries or genomic libraries, or gel slices containing a genomic DNA restriction digest, is not an isolated nucleic acid.
Isolated nucleic acid molecules can be produced by standard techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid containing a nucleotide sequence described herein, including nucleotide sequences encoding a polypeptide described herein. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Various PCR methods are described in, for example, PCR Primer: A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory Press, 1995. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified. Various PCR strategies are also available by which site-specific nucleotide sequence modifications can be introduced into a template nucleic acid.
Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3′ to 5′ direction using phosphoramidite technology) or as a series of oligonucleotides. For example, one or more pairs of long oligonucleotides (e.g., >50-100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector. The isolated nucleic acids of the invention also can be obtained by mutagenesis of, e.g., a naturally occurring portion of a Cas9-encoding DNA (in accordance with, for example, the formula above).
In the methods of the present invention, many different viruses can be treated or prevented in animals, especially porcine. Most preferably, the virus is ASFV. Other animal viruses can include Pseudorabies virus, Bluetongue virus, Foot-and-mouth disease virus (serotypes A, 0, C, SAT1,SAT2, SAT3, Asia1), Japanese encephalitis virus, Rabies virus, Rift Valley fever virus, Rinderpest virus, Vesicular stomatitis virus, West Nile fever virus, BSE prion, Bovine viral diarrhea virus, Bovine leukemia virus, Bovine herpesvirus 1, Lumpy skin disease virus, Caprine arthritis and encephalitis virus, Peste-des-petits-ruminants virus, Scrapie prion, sheeppox and goatpox viruses, African horse sickness virus, Eastern equine encephalomyelitis virus, Western equine encephalomyelitis virus, Equine infectious anemia virus, Equine influenza virus, Equine herpesvirus 4, Equine arteritis virus, Venezuelan equine encephalomyelitis virus, Classical swine fever virus, Nipah virus, Porcine reproductive and respiratory syndrome virus, Swine vesicular disease virus, Transmissible gastroenteritis virus of swine, Avian infectious bronchitis virus, Infectious laryngotracheitis virus, Duck hepatitis virus, High and low pathogenic avian influenza viruses, Infectious bursal disease virus, Marek's disease virus, Newcastle disease virus, or Avian metapneumovirus. The virus can also be generally of the type papovaviruses, simian virus-40, adenoviruses, herpesviruses, pox viruses, picornaviruses, togaviruses, rabies viruses, influenza viruses, or reoviruses.
In performing the methods of the present invention, first, receptor screening is performed. A discovery platform is utilized (yeast two hybrid-based or biochemical interaction assays) for the identification of the cellular receptors that interact with one or more (or in any combination thereof) of the viral attachment and entry proteins/ligands such as CD2v (EP402R), I177L, p12 (061R), p30 (CP204L) receptor mediator, but not limited to these viral protein ligands.
There are multiple yeast two hybrid, mammalian two hybrid, and phage display approaches that can be used for this purpose. Luo, et al. (PMID: 9043710) describes a mammalian two-hybrid system. One protein of interest is expressed as a fusion to the Gal4 DNA-binding domain and another protein is expressed as a fusion to the activation domain of the VP16 protein of the herpes simplex virus. The vectors that express these fusion proteins are cotransfected with a reporter chloramphenicol acetyltransferase (CAT) vector into a mammalian cell line. The reporter plasmid contains a CAT gene under the control of five consensus Gal4 binding sites. If the two fusion proteins interact, there will be a significant increase in expression of the cat reporter gene. Fields, et al. (PMID: 2547163) describes a yeast two-hybrid system with a GAL4 DNA-binding domain fused to a protein X′ and a GAL4 activating region fused to a protein Y. If X and Y can form a protein-protein complex and reconstitute proximity of the GAL4 domains, transcription of a gene regulated by UASG occurs. Smith (Science. 1985 Jun. 14; 228(4705):1315-7) describes a phage two-hybrid system wherein foreign DNA fragments can be inserted into filamentous phage gene III to create a fusion protein with the foreign sequence in the middle. The fusion protein is incorporated into the virion, which retains infectivity and displays the foreign amino acids in immunologically accessible form. These “fusion phage” can be enriched more than 1000-fold over ordinary phage by affinity for antibody directed against the foreign sequence.
The receptor screening can be performed generally as follows. A library of swine/porcine genes is expressed in yeast or phage (phage can be used to screen far more). The expressed proteins then decorate the outside of the yeast cell/phage. An HPLC column can be made of the ASFV Capsid or of proteins or other potential ligands. The yeast cells or phage are incubated with the immobilized ASFV receptor ligand of choice. The cells or phage are washed, collected, and repeated to enrich. The sample is collected, and the receptor identified using typical biochemical/genetic methods defined by each hybrid/phage system.
The receptor and viral ligand interaction can be either competitive inhibition or noncompetitive inhibition. Competitive inhibition occurs when a chemical substance, small peptide, or antibody inhibits the effect of another by competing with it for binding, i.e., it resembles the normal substrate that binds to the receptor. Non-competitive inhibition occurs when the inhibitor reduces activity of the receptor and binds equally well to the receptor whether or not it has already bound the substrate.
A small molecule inhibition treatment can be derived upon the discovery of receptor. Once the interaction between viral ligand and cellular receptor is defined, small molecule disruptive screens (protein-protein interaction/disruption via two hybrid systems or others) is utilized to define small molecule candidates that can inhibit the interaction. Variations of the two hybrid systems can be used, for example a repressed transactivator (RTA) screen. In this screen, the small molecule library is added to yeast that only grows on selective media when the swine receptor peptide and the viral receptor/ligand peptide are locked in an interaction. By adding the small molecule library, one looks for those that disrupt the interaction. Once identified, which small molecule is the most robust, safe, and efficacious can be determined. Hirst, et al. (PMID: 11447261) describes a repressed transactivator (RTA) system employing the N-terminal repression domain of the yeast general repressor TUP1. TUP1-GAL80 fusion proteins, when co-expressed with GAL4, are shown to inhibit transcription of GAL4-dependent reporter genes. Joshi, et al. (PMID: 17515203) has used this system in screening for inhibitors of protein interactions from small molecule compound libraries. The libraries used for screening and testing for the present invention can come from the sea, rainforest, or be synthetic. Peptide and antibody libraries can also be used. Further screening and testing can be conducted to narrow the number of small molecules and test for the safety and efficacy in cell culture and animal models.
A genetically modified cellular receptor can be used for prevention of the virus binding through dysfunction or other disruption of entry proteins. Once the cellular receptor is identified, specifically, the amino acids within the receptor that are critical for the recognition of the viral protein ligands, gene editing tools (such as, but not limited to, CRISPR, ZFNs, TALENs, further described below) can be used to alter (by substitution or deletion) the receptor encoding gene(s) with non-disruptive (functionally retainable protein) amino acid sequence(s) that block viral entry. The entry proteins are otherwise structural or functional membrane proteins. Their alteration can be at the genetic level affected by gene editing, but their natural function may need to be preserved so as to not disrupt or otherwise kill the target cells.
If glycosylation is needed for the receptor, swine macrophage cellular extracts can be added in the yeast/phage expressed libraries to force the glycosylation of the surface expressed peptide on the yeast/phage.
In an alternative to the above method, the viral protein can be isolated on a column as described above, then swine/porcine isolated macrophage/monocyte cells can be run over the column, incubated, then the cells can be enriched by elution (keeping the interaction intact). Once the isolated macrophage/monocyte is interacting with the viral receptor/ligand isolated, an antibody that recognizes the viral ligand can be added and then the synapse can be observed under a microscope. The single cell can be isolated and then the cellular receptor identified.
This gene editing approach can be conducted in swine embryonic lineages to create a genetically modified swine organism that is resistant to ASFV infection.
The gene editors used in the present invention can include any of the gene editors listed below. Any method of action can be used, including endonuclease cutting of DNA or RNA, guided by gRNAs. The nucleases work by cutting out or altering at the base pair level, the endogenous swine receptor sequences and replacing them using HDR with methods like HITI (non-dividing embryonic cells) or traditional HDR in dividing embryonic cells with one or more gRNAs. Gene editing can be used to create point mutations or multiple mutations that result in desired receptor. Cas/deaminase fusion proteins can be used to make point mutations.
Gene replacement can also be performed, which requires excision of a gene followed by replacement of the gene with a new gene that has an altered sequence that expresses a mutant (yet functional) receptor that blocks viral entry. Gene editing can be used to replace a wild type gene with an engineered gene that contains the mutant sequences allowing for the expression of the replacement receptor. Once the gene is excised, it can be replaced using gene replacement approaches (homology-directed recombination) in either dividing or non-dividing cells.
Zinc finger nuclease (ZFN) creates double-strand breaks at specific DNA locations. A ZFN has two functional domains, a DNA-binding domain that recognizes a 6 bp DNA sequence, and a DNA-cleaving domain of the nuclease Fok I.
TALENs (transcription activator-like effector nucleases) include a TAL effector DNA-binding domain fused to a DNA cleavage domain that creates double strand breaks in DNA.
Human WRN is a RecQ helicase encoded by the Werner syndrome gene. It is implicated in genome maintenance, including replication, recombination, excision repair and DNA damage response. These genetic processes and expression of WRN are concomitantly upregulated in many types of cancers. Therefore, it has been proposed that targeted destruction of this helicase could be useful for elimination of cancer cells. Reports have applied the external guide sequence (EGS) approach in directing an RNase P RNA to efficiently cleave the WRN mRNA in cultured human cell lines, thus abolishing translation and activity of this distinctive 3′-5′ DNA helicase-nuclease.
The Class 2 type VI-A CRISPR/Cas effector “C2c2” demonstrates an RNA-guided RNase function. C2c2 from the bacterium Leptotrichia shahii provides interference against RNA phage. In vitro biochemical analysis shows that C2c2 is guided by a single crRNA and can be programmed to cleave ssRNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific mRNAs. Cleavage is mediated by catalytic residues in the two conserved HEPN domains, mutations in which generate catalytically inactive RNA-binding proteins. The RNA-focused action of C2c2 complements the CRISPR-Cas9 system, which targets DNA, the genomic blueprint for cellular identity and function. The ability to target only RNA, which helps carry out the genomic instructions, offers the ability to specifically manipulate RNA in a high-throughput manner—and manipulate gene function more broadly. These results demonstrate the capability of C2c2 as a new RNA-targeting tool.
Another Class 2 type V-B CRISPR/Cas effector “C2c1” can also be used in the present invention for editing DNA. C2c1 contains RuvC-like endonuclease domains related distantly to Cpf1 (described below). C2c1 can target and cleave both strands of target DNA site-specifically. According to PMID: 27984729, a crystal structure confirms Alicyclobacillus acidoterrestris C2c1 (AacC2c1) binds to sgRNA as a binary complex and targets DNAs as ternary complexes, thereby capturing catalytically competent conformations of AacC2c1 with both target and non-target DNA strands independently positioned within a single RuvC catalytic pocket. PMID: 27984729 confirms that C2c1-mediated cleavage results in a staggered seven-nucleotide break of target DNA, crRNA adopts a pre-ordered five-nucleotide A-form seed sequence in the binary complex, with release of an inserted tryptophan, facilitating zippering up of 20-bp guide RNA:target DNA heteroduplex on ternary complex formation, and that the PAM-interacting cleft adopts a “locked” conformation on ternary complex formation.
C2c3 is a gene editor effector of type V-C that is distantly related to C2c1, and also contains RuvC-like nuclease domains. C2c3 is also similar to the CasY.1—CasY.6 group described below.
“CRISPR Cas9” as used herein refers to Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease Cas9. In bacteria the CRISPR/Cas loci encode RNA-guided adaptive immune systems against mobile genetic elements (viruses, transposable elements and conjugative plasmids). Three types (I-III) of CRISPR systems have been identified. CRISPR clusters contain spacers, the sequences complementary to antecedent mobile elements. CRISPR clusters are transcribed and processed into mature CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (crRNA). The CRISPR-associated endonuclease, Cas9, belongs to the type II CRISPR/Cas system and has strong endonuclease activity to cut target DNA. Cas9 is guided by a mature crRNA that contains about 20 base pairs (bp) of unique target sequence (called spacer) and a trans-activated small RNA (tracrRNA) that serves as a guide for ribonuclease III-aided processing of pre-crRNA. The crRNA:tracrRNA duplex directs Cas9 to target DNA via complementary base pairing between the spacer on the crRNA and the complementary sequence (called protospacer) on the target DNA. Cas9 recognizes a trinucleotide (NGG) protospacer adjacent motif (PAM) to specify the cut site (the 3rd nucleotide from PAM). The crRNA and tracrRNA can be expressed separately or engineered into an artificial fusion small guide RNA (sgRNA) via a synthetic stem loop (AGAAAU) to mimic the natural crRNA/tracrRNA duplex. Such sgRNA, like shRNA, can be synthesized or in vitro transcribed for direct RNA transfection or expressed from U6 or H1-promoted RNA expression vector, although cleavage efficiencies of the artificial sgRNA are lower than those for systems with the crRNA and tracrRNA expressed separately.
CRISPR/Cpf1 is a DNA-editing technology analogous to the CRISPR/Cas9 system, characterized in 2015 by Feng Zhang's group from the Broad Institute and MIT. Cpfl is an RNA-guided endonuclease of a class II CRISPR/Cas system. This acquired immune mechanism is found in Prevotella and Francisella bacteria. It prevents genetic damage from viruses. Cpf1 genes are associated with the CRISPR locus, coding for an endonuclease that use a guide RNA to find and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the CRISPR/Cas9 system limitations. CRISPR/Cpf1 could have multiple applications, including treatment of genetic illnesses and degenerative conditions.
A CRISPR/TevCas9 system can also be used. In some cases, it has been shown that once CRISPR/Cas9 cuts DNA in one spot, DNA repair systems in the cells of an organism will repair the site of the cut. The TevCas9 enzyme was developed to cut DNA at two sites of the target so that it is harder for the cells' DNA repair systems to repair the cuts (PMID: 27956611). The TevCas9 nuclease is a fusion of a I-Tevi nuclease domain to Cas9.
The Cas9 nuclease can have a nucleotide sequence identical to the wild type Streptococcus pyrogenes sequence. In some embodiments, the CRISPR-associated endonuclease can be a sequence from other species, for example other Streptococcus species, such as thermophilus; Pseudomona aeruginosa, Escherichia coli, or other sequenced bacteria genomes and archaea, or other prokaryotic microorganisms. Alternatively, the wild type Streptococcus pyrogenes Cas9 sequence can be modified. The nucleic acid sequence can be codon optimized for efficient expression in mammalian cells, i.e., “humanized.” A humanized Cas9 nuclease sequence can be for example, the Cas9 nuclease sequence encoded by any of the expression vectors listed in Genbank accession numbers KM099231.1 Gl:669193757; KM099232.1 Gl:669193761; or KM099233.1 Gl:669193765. Alternatively, the Cas9 nuclease sequence can be for example, the sequence contained within a commercially available vector such as PX330 or PX260 from Addgene (Cambridge, MA). In some embodiments, the Cas9 endonuclease can have an amino acid sequence that is a variant or a fragment of any of the Cas9 endonuclease sequences of Genbank accession numbers KM099231.1 Gl:669193757; KM099232.1 Gl:669193761; or KM099233.1 Gl:669193765 or Cas9 amino acid sequence of PX330 or PX260 (Addgene, Cambridge, MA). The Cas9 nucleotide sequence can be modified to encode biologically active variants of Cas9, and these variants can have or can include, for example, an amino acid sequence that differs from a wild type Cas9 by virtue of containing one or more mutations (e.g., an addition, deletion, or substitution mutation or a combination of such mutations). One or more of the substitution mutations can be a substitution (e.g., a conservative amino acid substitution). For example, a biologically active variant of a Cas9 polypeptide can have an amino acid sequence with at least or about 50% sequence identity (e.g., at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity) to a wild type Cas9 polypeptide. Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine. The amino acid residues in the Cas9 amino acid sequence can be non-naturally occurring amino acid residues. Naturally occurring amino acid residues include those naturally encoded by the genetic code as well as non-standard amino acids (e.g., amino acids having the D-configuration instead of the L-configuration). The present peptides can also include amino acid residues that are modified versions of standard residues (e.g., pyrrolysine can be used in place of lysine and selenocysteine can be used in place of cysteine). Non-naturally occurring amino acid residues are those that have not been found in nature, but that conform to the basic formula of an amino acid and can be incorporated into a peptide. These include D-alloisoleucine (2R,3S)-2-amino-3-methylpentanoic acid and L-cyclopentyl glycine (S)-2-amino-2-cyclopentyl acetic acid. For other examples, one can consult textbooks or the worldwide web (a site is currently maintained by the California Institute of Technology and displays structures of non-natural amino acids that have been successfully incorporated into functional proteins). The Cas-9 can also be shown in TABLE 3 below.
Although the RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform, some have reported that the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Accordingly, the six smaller Cas9 orthologues have been used and reports have shown that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. SaCas9 is 1053 bp, whereas SpCas9 is 1358 bp.
The Cas9 nuclease sequence, or any of the gene editor effector sequences described herein, can be a mutated sequence. For example, the Cas9 nuclease can be mutated in the conserved HNH and RuvC domains, which are involved in strand specific cleavage. For example, an aspartate-to-alanine (D10A) mutation in the RuvC catalytic domain allows the Cas9 nickase mutant (Cas9n) to nick rather than cleave DNA to yield single-stranded breaks, and the subsequent preferential repair through HDR can potentially decrease the frequency of unwanted indel mutations from off-target double-stranded breaks. In general, mutations of the gene editor effector sequence can minimize or prevent off-targeting.
The gene editor effector can be CasX or CasY or Cas Omega. CasX has a TTC PAM at the 5′ end (similar to Cpf1). The TTC PAM can have limitations in viral genomes that are GC rich, but not so much in those that are GC poor. The size of CasX (986 bp), smaller than other type V proteins, provides the potential for four gRNA plus one siRNA in a delivery plasmid. CasX can be derived from Deltaproteobacteria or Planctomycetes.
The gene editor effector can also be Archaea Cas9. The size of Archaea Cas9 is 950aa ARMAN 1 and 967aa ARMAN 4. The Archaea Cas9 can be derived from ARMAN-1 (Candidatus Micrarchaeum acidiphilum ARMAN-1) or ARMAN-4 (Candidatus Parvarchaeum acidiphilum ARMAN-4). The sequences for ARMAN 1 and ARMAN 4 are below.
In the present invention, when any of the compositions are contained within an expression vector, the CRISPR endonuclease can be encoded by the same nucleic acid or vector as the gRNA sequences. Alternatively, or in addition, the CRISPR endonuclease can be encoded in a physically separate nucleic acid from the gRNA sequences or in a separate vector.
Vectors containing nucleic acids such as those described herein are also provided. A “vector” is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Generally, a vector is capable of replication when associated with the proper control elements. Suitable vector backbones include, for example, those routinely used in the art such as plasmids, viruses, artificial chromosomes, BACs, YACs, or PACs. The term “vector” includes cloning and expression vectors, as well as viral vectors and integrating vectors. An “expression vector” is a vector that includes a regulatory region. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen/Life Technologies (Carlsbad, CA).
The vectors provided herein also can include, for example, origins of replication, scaffold attachment regions (SARs), and/or markers. A marker gene can confer a selectable phenotype on a host cell. For example, a marker can confer biocide resistance, such as resistance to an antibiotic (e.g., kanamycin, G418, bleomycin, or hygromycin). As noted above, an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or Flag TM tag (Kodak, New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus.
Additional expression vectors also can include, for example, segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col El, pCR1, pBR322, pMal-C2, pET, pGEX, pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e.g., the numerous derivatives of phage 1, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2p plasmid or derivatives thereof, vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences.
Yeast expression systems can also be used. For example, the non-fusion pYES2 vector (Xbal, Sphl, Shol, Notl, GstXl, EcoRl, BstXl, BamH1, Sacl, Kpn1, and Hindil cloning sites; Invitrogen) or the fusion pYESHisA, B, C (Xbal, Sphl, Shol, Notl, BstXl, EcoRl, BamH1, Sacl, Kpnl, and Hindil cloning sites, N-terminal peptide purified with ProBond resin and cleaved with enterokinase; Invitrogen), to mention just two, can be employed according to the invention. A yeast two-hybrid expression system can also be prepared in accordance with the invention.
The vector can also include a regulatory region. The term “regulatory region” refers to nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5′ and 3′ untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, nuclear localization signals, and introns.
As used herein, the term “operably linked” refers to positioning of a regulatory region and a sequence to be transcribed in a nucleic acid so as to influence transcription or translation of such a sequence. For example, to bring a coding sequence under the control of a promoter, the translation initiation site of the translational reading frame of the polypeptide is typically positioned between one and about fifty nucleotides downstream of the promoter. A promoter can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site or about 2,000 nucleotides upstream of the transcription start site. A promoter typically comprises at least a core (basal) promoter. A promoter also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR). The choice of promoters to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning promoters and other regulatory regions relative to the coding sequence.
Vectors include, for example, viral vectors (such as adenoviruses (“Ad”), adeno-associated viruses (AAV), and vesicular stomatitis virus (VSV) and retroviruses), liposomes and other lipid-containing complexes, and other macromolecular complexes capable of mediating delivery of a polynucleotide to a host cell. Vectors can also comprise other components or functionalities that further modulate gene delivery and/or gene expression, or that otherwise provide beneficial properties to the targeted cells. As described and illustrated in more detail below, such other components include, for example, components that influence binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector nucleic acid by the cell; components that influence localization of the polynucleotide within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the polynucleotide. Such components also might include markers, such as detectable and/or selectable markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector. Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities. Other vectors include those described in PMID: 12545555. A large variety of such vectors are known in the art and are generally available.
A “recombinant viral vector” refers to a viral vector comprising one or more heterologous gene products or sequences. Since many viral vectors exhibit size-constraints associated with packaging, the heterologous gene products or sequences are typically introduced by replacing one or more portions of the viral genome. Such viruses may become replication-defective, requiring the deleted function(s) to be provided in trans during viral replication and encapsidation (by using, e.g., a helper virus or a packaging cell line carrying gene products necessary for replication and/or encapsidation). Modified viral vectors in which a polynucleotide to be delivered is carried on the outside of the viral particle have also been described (PMID: 1681545).
Suitable nucleic acid delivery systems include recombinant viral vector, typically sequence from at least one of an adenovirus, adenovirus-associated virus (AAV), helper-dependent adenovirus, retrovirus, or hemagglutinating virus of Japan-liposome (HVJ) complex. In such cases, the viral vector comprises a strong eukaryotic promoter operably linked to the polynucleotide e.g., a cytomegalovirus (CMV) promoter. The recombinant viral vector can include one or more of the polynucleotides therein, preferably about one polynucleotide. In some embodiments, the viral vector used in the invention methods has a pfu (plague forming units) of from about 108 to about 5×1010 pfu. In embodiments in which the polynucleotide is to be administered with a non-viral vector, use of between from about 0.1 nanograms to about 4000 micrograms will often be useful e.g., about 1 nanogram to about 100 micrograms.
Additional vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include Moloney murine leukemia viruses and HIV-based viruses. One HIV-based viral vector comprises at least two vectors wherein the gag and pol genes are from an HIV genome and the env gene is from another virus. DNA viral vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector (PMID: 7830040, PMID: 8102799, PMID: 2153970,), Adenovirus Vectors (PMID: 8382374, PMID: 8387378, PMID: 7884845), and Adeno-associated Virus Vectors (PMID: 7842013).
Pox viral vectors introduce the gene into the cell's cytoplasm. Avipox virus vectors result in only a short-term expression of nucleic acid. Adenovirus vectors, adeno-associated virus vectors and herpes simplex virus (HSV) vectors may be an indication for some invention embodiments. The adenovirus vector results in a shorter-term expression (e.g., less than about a month) than adeno-associated virus, in some embodiments, may exhibit much longer expression. The particular vector chosen will depend upon the target cell and the condition being treated. The selection of appropriate promoters can readily be accomplished. An example of a suitable promoter is the 763-base-pair cytomegalovirus (CMV) promoter. Other suitable promoters which may be used for gene expression include, but are not limited to, the Rous sarcoma virus (RSV) (PMID: 8494924), the SV40 early promoter region, the herpes thymidine kinase promoter, the regulatory sequences of the metallothionein (MMT) gene, prokaryotic expression vectors such as the β-lactamase promoter, the tac promoter, promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter; and the animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells, insulin gene control region which is active in pancreatic beta cells, immunoglobulin gene control region which is active in lymphoid cells, mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells, albumin gene control region which is active in liver, alpha-fetoprotein gene control region which is active in liver, alpha 1-antitrypsin gene control region which is active in the liver, beta-globin gene control region which is active in myeloid cells, myelin basic protein gene control region which is active in oligodendrocyte cells in the brain, myosin light chain-2 gene control region which is active in skeletal muscle, and gonadotropic releasing hormone gene control region which is active in the hypothalamus. Certain proteins can express using their native promoter. Other elements that can enhance expression can also be included such as an enhancer or a system that results in high levels of expression such as a tat gene and tar element. This cassette can then be inserted into a vector, e.g., a plasmid vector such as, pUC19, pUC118, pBR322, or other known plasmid vectors, that includes, for example, an E. coli origin of replication. See Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory press, (1989). The plasmid vector may also include a selectable marker such as the 8-lactamase gene for ampicillin resistance, provided that the marker polypeptide does not adversely affect the metabolism of the organism being treated. The cassette can also be bound to a nucleic acid binding moiety in a synthetic delivery system, such as the system disclosed in WO 95/22618.
If desired, the polynucleotides of the invention can also be used with a microdelivery vehicle such as cationic liposomes and adenoviral vectors. For a review of the procedures for liposome preparation, targeting and delivery of contents, see PMID: 2856201. See also, Feigner and Holm, Bethesda Res. Lab. Focus, 11(2):21 (1989) and Maurer, R.A., Bethesda Res. Lab. Focus, 11(2):25 (1989).
Replication-defective recombinant adenoviral vectors can be produced in accordance with known techniques. See PMID: 1557362, PMID: 1644927, and PMID: 1370653.
Another delivery method is to use single stranded DNA producing vectors which can produce the expressed products intracellularly. See for example, PMID: 12545555), which is incorporated herein, by reference, in its entirety. Alternatively, RNA and/or protein therapeutic delivery can also be used.
As described above, the compositions of the present invention can be prepared in a variety of ways known to one of ordinary skill in the art. Regardless of their original source or the manner in which they are obtained, the compositions of the invention can be formulated in accordance with their use. For example, the nucleic acids and vectors described above can be formulated within compositions for application to cells in tissue culture or for administration to a patient or subject. Any of the pharmaceutical compositions of the invention can be formulated for use in the preparation of a medicament, and particular uses are indicated below in the context of treatment. When employed as pharmaceuticals, any of the nucleic acids and vectors can be administered in the form of pharmaceutical compositions. These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular, oral or parenteral. Methods for ocular delivery can include topical administration (eye drops), subconjunctival, periocular or intravitreal injection or introduction by balloon catheter or ophthalmic inserts surgically placed in the conjunctival sac. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular administration. Parenteral administration can be in the form of a single bolus dose, or maybe, for example, by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, and the like. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
This invention also includes pharmaceutical compositions which contain, as the active ingredient, nucleic acids and vectors described herein in combination with one or more pharmaceutically acceptable carriers. We use the terms “pharmaceutically acceptable” (or “pharmacologically acceptable”) to refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal or a human, as appropriate. The term “pharmaceutically acceptable carrier,” as used herein, includes any and all solvents, dispersion media, coatings, antibacterial, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants and the like, that may be used as media for a pharmaceutically acceptable substance. In making the compositions of the invention, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, tablet, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semisolid, or liquid material (e.g., normal saline), which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), lotions, creams, ointments, gels, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. As is known in the art, the type of diluent can vary depending upon the intended route of administration. The resulting compositions can include additional agents, such as preservatives. In some embodiments, the carrier can be, or can include a lipid-based or polymer-based colloid. In some embodiments, the carrier material can be a colloid formulated as a liposome, a hydrogel, a microparticle, a nanoparticle, or a block copolymer micelle. As noted, the carrier material can form a capsule, and that material may be a polymer-based colloid.
The nucleic acid sequences of the invention can be delivered to an appropriate cell of a subject. This can be achieved by, for example, the use of a polymeric, biodegradable microparticle or microcapsule delivery vehicle, sized to optimize phagocytosis by phagocytic cells such as macrophages. For example, PLGA (poly-lacto-co-glycolide) microparticles approximately 1-10 μm in diameter can be used. The polynucleotide is encapsulated in these microparticles, which are taken up by macrophages and gradually biodegraded within the cell, thereby releasing the polynucleotide. Once released, the DNA is expressed within the cell. A second type of microparticle is intended not to be taken up directly by cells, but rather to serve primarily as a slow-release reservoir of nucleic acid that is taken up by cells only upon release from the micro-particle through biodegradation. These polymeric particles should therefore be large enough to preclude phagocytosis (i.e., larger than 5 μm and preferably larger than 20 μm). Another way to achieve uptake of the nucleic acid is using liposomes, prepared by standard methods. The nucleic acids can be incorporated alone into these delivery vehicles or co-incorporated with tissue-specific antibodies, for example antibodies that target cell types that are commonly latently infected reservoirs of viral infection, for example, brain macrophages, microglia, astrocytes, and gut-associated lymphoid cells. Alternatively, one can prepare a molecular complex composed of a plasmid or other vector attached to poly-L-lysine by electrostatic or covalent forces. Poly-L-lysine binds to a ligand that can bind to a receptor on target cells. Delivery of “naked DNA” (i.e., without a delivery vehicle) to an intramuscular, intradermal, or subcutaneous site, is another means to achieve in vivo expression. In the relevant polynucleotides (e.g., expression vectors) the nucleic acid sequence encoding an isolated nucleic acid sequence comprising a sequence encoding a CRISPR-associated endonuclease and a guide RNA is operatively linked to a promoter or enhancer-promoter combination. Promoters and enhancers are described above.
In some embodiments, the compositions of the invention can be formulated as a nanoparticle, for example, nanoparticles comprised of a core of high molecular weight linear polyethylenimine (LPEI) complexed with DNA and surrounded by a shell of polyethyleneglycol-modified (PEGylated) low molecular weight LPEI.
The nucleic acids and vectors may also be applied to a surface of a device (e.g., a catheter) or contained within a pump, patch, or other drug delivery device. The nucleic acids and vectors of the invention can be administered alone, or in a mixture, in the presence of a pharmaceutically acceptable excipient or carrier (e.g., physiological saline). The excipient or carrier is selected on the basis of the mode and route of administration. Suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences (E. W. Martin), a well-known reference text in this field, and in the USP/NF (United States Pharmacopeia and the National Formulary).
The methods of the invention can be expressed in terms of the preparation of a medicament. Accordingly, the invention encompasses the use of the agents and compositions described herein in the preparation of a medicament. The compounds described herein are useful in therapeutic compositions and regimens or for the manufacture of a medicament for use in treatment of diseases or conditions as described herein.
Any composition described herein can be administered to any part of the host's body for subsequent delivery to a target cell. A composition can be delivered to, without limitation, the brain, the cerebrospinal fluid, joints, nasal mucosa, blood, lungs, intestines, muscle tissues, skin, or the peritoneal cavity of a mammal. In terms of routes of delivery, a composition can be administered by intravenous, intracranial, intraperitoneal, intramuscular, subcutaneous, intramuscular, intrarectal, intravaginal, intrathecal, intratracheal, intradermal, or transdermal injection, by oral or nasal administration, or by gradual perfusion over time. In a further example, an aerosol preparation of a composition can be given to a host by inhalation.
The dosage required will depend on the route of administration, the nature of the formulation, the nature of the animal's illness, the animal's size, weight, surface area, age, and sex, other drugs being administered, and the judgment of the attending clinicians. Wide variations in the needed dosage are to be expected in view of the variety of cellular targets and the differing efficiencies of various routes of administration. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art. Administrations can be single or multiple (e.g., 2- or 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold). Encapsulation of the compounds in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery. Dosage can be given to provide total viral load elimination. Dosage can also be given to reduce viral load within the animal to allow for the immune destruction of the remainder of the viral load.
The duration of treatment with any composition provided herein can be any length of time from as short as one day to as long as the life span of the host (e.g., many years). For example, a compound can be administered once a week (for, for example, 4 weeks to many months or years); once a month (for, for example, three to twelve months or for many years); or once a year for a period of 5 years, ten years, or longer. It is also noted that the frequency of treatment can be variable. For example, the present compounds can be administered once (or twice, three times, etc.) daily, weekly, monthly, or yearly.
An effective amount of any composition provided herein can be administered to an individual in need of treatment. The term “effective” as used herein refers to any amount that induces a desired response while not inducing significant toxicity in the patient. Such an amount can be determined by assessing a patient's response after administration of a known amount of a particular composition. In addition, the level of toxicity, if any, can be determined by assessing an individual's clinical symptoms before and after administering a known amount of a particular composition. It is noted that the effective amount of a particular composition administered to a patient can be adjusted according to a desired outcome as well as the individual's response and level of toxicity. Significant toxicity can vary for each particular individual and depends on multiple factors including, without limitation, the individual's disease state, age, and tolerance to side effects.
Any method known to those in the art can be used to determine if a particular response is induced. Clinical methods that can assess the degree of a particular disease state can be used to determine if a response is induced. The particular methods used to evaluate a response will depend upon the nature of the individual's disorder, the individual's age, and sex, other drugs being administered, and the judgment of the attending clinician. The viral load in the individual can be monitored, for example, with a blood test that measures viral RNA per milliliter of blood. Examples of such tests include quantitative branched DNA (bDNA), reverse transcriptase-polymerase chain reaction (RT-PCR), and qualitative transcription-mediated amplification.
The present invention also provides for specific methods of treating ASFV. It is hypothesized that ASFV is both lytic and lysogenic (
Antibody and antigen-based vaccines have not worked, and it is likely because the strategies for their development have not taken into account both types of replication cycle—lysogenic and lytic. For example, antibodies targeting the capsid protein (antibodies that are either directly injected as a therapeutic, or antibodies that are stimulated in vivo from an immune response to a viral peptide) may not neutralize the ASFV virion because the capsid is protected by an outer membrane (i.e., it is inaccessible). As the viral replication cycle shifts to a lytic cycle, the antibodies may indeed interact with their respective capsid epitopes, but at this stage, the in vivo viral titre is likely too high to have effective and lasting neutralizing responses. This, combined with rapid viral expansion (correlated with a 24- to 72-hour 100% mortality rate associated with the virus) contributes to the body being overwhelmed by virus. Antigen stimulation as a preventative has not worked in the past as the antigens are almost always capsid-based. Therefore, the B-cell response producing the IgG does not recognize early ASFV that is surrounded by an outer membrane (shown in
Therefore, the present invention provides for a method of treating a viral infection in an individual with a virus that is both lysogenic and lytic, by administering a viral antigen (to stimulate a B-cell response) and/or antibody therapeutic that targets protein on an outer membrane of a lysogenic phase of the virus, administering a viral antigen (to stimulate a B-cell response) and/or antibody therapeutic that targets protein on a capsid of a lytic phase of the virus, and treating the viral infection (
To overcome these challenges a new two-fold strategy is necessary.
1. The swine needs to be stimulated with a viral antigen derived from a viral protein that exists on the outer membrane of the ASFV to neutralize virions that bud (lysogenic) in early infection. These viral antigens include EP402R (CD2v), EP153R, p21 (063L), and I177L (but not limited to these outer membrane proteins, per strategy) which are viral outer membrane proteins. Further, E183L (p54) is an integral viral inner membrane protein that plays a critical role in virions trafficking to viral factories in the ER during the early lysogenic stages of viral replication, as mentioned in detail above (
2. The swine also needs to be treated with a viral antigen derived from a viral protein that exists on the capsid to neutralize virions that do not have an outer membrane (lytic) late infection. These viral antigens can include at least one type of capsid protein such as, p30 (CP204L), p72 (B646L), p49 (B438L), and other capsid proteins that are accessible to antibody neutralization and elicit a strong immune response (
Recently, two viral outer membrane proteins were identified that is responsible for the extracellular viral docking with erythrocytes (a likely mechanism to rapidly distribute the virus through the circulating blood), T-cell suppression and MCH Class I blocking. These proteins are called EP402R (CD2v) and EP153R. EP402R has also been shown to be responsible for immunosuppressive activity by inhibiting lymphocyte proliferation. EP153R has been shown to be responsible for blocking the MCH Class I complexes on infected macrophages. Therefore, by targeting the EP402R (CD2v) and EP153R protein for vaccine or therapeutic purposes, the extracellular virus will be greatly inhibited to spread throughout the organism as well as prevent lymphocyte inhibition (shown in
Other key proteins for viral structure that compose the capsid include p14.5 (E120R), p72 (B646L), p30 (CP204L), and p49 (B438L). These three proteins can be targeted for vaccine and/or therapeutic approaches, in order to neutralize the extracellular virions that lack an outer membrane (as result of the lytic cycle) (shown in
The swine can therefore be treated with either whole proteins, or a peptide (surface exposed), or a mixture of peptides derived from: 1) the proteins involved in the early stage lysogenic cycle of ASFV replication such as i) the outer membrane proteins EP402R (CD2v) and EP153R and/or ii) the integral viral inner membrane protein E183L (p54), and in combination with 2) the proteins involved in the lytic cycle of ASFV replication such as i)p14.5 (E120R), p72 (B646L), p49 (B438L) and/or ii) the inner and outer membrane protein p12 (016R. The treatment produces a B-cell response (immediate and memory) in the swine as a prophylactic measure against ASFV lysogenic and lytic replication cycles. Peptide segments of any of these proteins (and not whole protein) can be used to create an immune stimulating response. This strategy is shown in
The present invention also provides for a composition for treating a viral infection in an individual with a virus that is both lysogenic and lytic including a viral antigen (that stimulates a B-cell response) and/or antibody therapeutic that targets protein on an outer membrane of a lysogenic phase of the virus and a viral antigen that targets protein on a capsid of a lytic phase of the virus (
The most optimal antibodies and epitope sequences can be found for each of the proteins that define the lysogenic and lytic stages of the virus using screening or Al based approaches. Once the antibodies are defined, they can be manufactured and injected into healthy individuals (i.e., swine) to protect them from ASFV infection. Alternatively, once the antibody epitopes are defined, they can be used to engineer new antibodies, such as 1) a bispecific antibody that recognizes two viral epitopes and therefore neutralizes multiple points of the virus. The two epitopes (if bispecific they can also be used to target a protein of the lysogenic cycle and a protein of the lytic cycle simultaneously, and/or 2) the addition of CD47/mCD47 to the Fc region of the antibody (
The treatment can include an antigen stimulation approach using at least one of:
Each of these strategies are not limited to CD2v (EP402R), EP153R, p54 (E183L), I177L pE120R, p72 (B646L), p30 (CP204L), p49 (B438L), or p12 (061R) additional outer membrane and capsid proteins can be exploited for the same purpose/outcome.
CD2v (EP402R), EP153R, p54 (E183L), I177L and/or pE120R, p72 (B646L), p30 (CP204L), p49 (B438L), p12 (061R) whole proteins or any combination of peptide(s) thereof, can be used as antigens to discover antibodies using any type of antibody discovery platform. Some of these platforms include gene editing-driven antibody over-expression systems in B-cells, phage libraries, yeast expression systems, nano well GFP-labeling systems, to name a few. Once the antibodies are discovered, they can be tested for affinity, avidity, specificity, selectivity, stability, precision, robustness, and the best candidates (derived from a platform screen) can be used as a therapeutic treatment to neutralize viral CD2v (EP402R), EP153R, p54 (E183L), I177L and/or pE120R, p72 (B646L), p30 (CP204L), p49 (B438L), p12 (061R) (or other outer membrane and/or capsid proteins) after the swine have been infected.
The therapeutic treatment can include at least one of: two separate injections, one each of an antibody (or several neutralizing antibodies) raised against CD2v (EP402R), EP153R, p54 (E183L), I177L and/or pE120R, p72 (B646L), p30 (CP204L), p49 (B438L), p12 (061R); or one injection containing a pool of antibodies raised against CD2v (EP402R), EP153R, p54 (E183L), I177L and/or pE120R, p72 (B646L), p30 (CP204L), p49 (B438L), p12 (061R). This strategy can also be used in treating humans if the virus jumps species.
Therefore, the present invention provides for a method of finding antibodies for treating a viral infection in an individual with a virus that is both lysogenic and lytic, by using whole proteins or peptides of target protein on an outer membrane of a lysogenic phase of the virus and target protein on a capsid of a lytic phase of the virus as antigens to discover antibodies with an antibody discovery platform, testing discovered antibodies for affinity, avidity, specificity, selectivity, stability, precision, and robustness, and selecting a best candidate antibody as a therapeutic treatment for the viral infection. The present invention also provides for the antibodies found by this method.
The present invention also provides for a vaccine for preventing viral infection, including whole and/or partial domains of proteins of both a lysogenic and lytic phase of a virus. The domains can include at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity similarity. The proteins can be expressed using an mRNA deliverable to stimulate B-cells in the individual to produce the proteins and corresponding neutralizing antibodies.
Throughout this application, various publications, including United States patents, are referenced by author and year and patents by number. Full citations for the publications are listed below. The disclosures of these publications and patents in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation.
Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims, the invention can be practiced otherwise than as specifically described.
| Number | Date | Country | |
|---|---|---|---|
| 62900816 | Sep 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17535545 | Nov 2021 | US |
| Child | 18072783 | US | |
| Parent | PCT/US20/50939 | Sep 2020 | US |
| Child | 17535545 | US |
