Patent Application
20240240185
This application contains a Sequence Listing which has been submitted electronically in ST26 format and is hereby incorporated by reference in its entirety. Said ST26 file, created on Mar. 18, 2024, is named “800132US1.xml” and is 323,493 bytes in size.
Viruses are completely dependent upon the host for replication. Like all viruses, influenza virus exploits cellular processes to support its replication while simultaneously evading antiviral responses deployed by the cell in an attempt to block the infection. The balance between these pro- and anti-pathogen forces influences the outcome of an infection, the severity of disease, and even the potential to establish a pandemic outbreak.
Influenza virus is a serious public health threat causing annual epidemics and occasional pandemics with significant morbidity and mortality. Identifying cellular genes and proteins required by influenza virus is essential to understanding the viral life cycle and establishing a mechanistic foundation for the development of host-directed anti-viral therapeutics. Most genetic approaches to identify host factors regulating infection have relied upon loss-of-function screens, which only probe those genes already expressed in the system under study and are limited in their ability to detect contributions from genes essential for cell viability, genes with redundant functions, or gene products needed in limited quantities. Such studies leave a large amount of genetic space unexplored and raises the possibility that entirely new classes of viral co-factors have yet to be discovered.
Employing a screening approach for identifying host factors that impact influenza viral production after the initial infection, host factors that enhance influenza virus production were identified. Those factors are useful to study the regulation of the expression of viral genes and replication of the viral genome. Screening described herein include variations of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system, termed CRISPR activation (CRISPRa) and CRISPR inhibition (CRISPRi). In those methods, the sequence of a single guide RNA (“sgRNA”) directs Cas9 to a specific location, and the catalytically inactive Cas9 has been modified to recruit transcriptional activators or repressors to modify gene expression at that location.
An influenza virus was used to express the CRISPR sgRNA, in a technique referred to as transcriptional regulation by pathogen-programmed Cas9 (TRPPC). This way, the construct is inactive until after a virus infects a host cell and begins to be transcribed, and only the Cas9-expressing and influenza-infected cells are affected. To thoroughly blanket the genome, a library of 70,000 sequences (about 3 targeting sequences for each human gene) was prepared, which incorporated the sgRNA sequences into the influenza genome in between the two coding regions of the influenza NS gene segment and ensured proper cleavage via insertion of a microRNA sequence. In embodiments, the M gene segment may be employed. The library was used to perform a genetic selection by allowing all viruses to compete with each other through multiple rounds of replication in human lung cells. Viruses that activated pro-viral host factors gained a replicative advantage and came to quickly dominate the viral population, and those viruses and their host gene targets were easily determined by deep sequencing. This process can be adapted to any pathogen capable of delivering the targeting RNA.
In embodiments, a nucleic acid vector comprises a heterologous promoter operably linked to an open reading frame encoding a polypeptide having at least 80% amino acid sequence identity to one of SEQ ID Nos. 1-36 or 74-91 or a portion thereof with the activity of SEQ ID Nos. 1-36 or 74-91. In embodiments, the promoter is a viral promoter. In embodiments the promoter is a CMV promotes, retroviral LTRs (e g., HIV, MLV), or an adenovirus promoter like E1A. In embodiments, the polypeptide has at least 90% or 95% amino acid sequence identity to one of SEQ ID Nos. 1-36 or 74-91 or the portion thereof. In embodiments, the vector is a viral vector. In embodiments, the vector is a plasmid.
Further provided is a host cell having the vector or the genome of which is augmented with nucleic acid encoding a polypeptide having at least 80% amino acid sequence identity to one of SEQ ID Nos. 1-36 or 74-91 or a portion thereof with the activity of SEQ ID Nos. 1-36 or 74-91 or comprising a polypeptide having at least 80% amino acid sequence identity to one of SEQ ID Nos. 1-36 or 74-91 or a portion thereof with the activity of SEQ ID Nos. 1-36 or 74-91. In embodiments, the host cells can comprise eukaryotic cells. In embodiments, the host cells can comprise prokaryotic cells. The vector or nucleic acid can be maintained extrachromosomally or stably integrated into the genome of the host cell. In embodiments, the host cell can comprise an insect cell, a plant cell, or a mammalian cell. In embodiments, the host cell is a MDCK cell or derivatives thereof, MDBK, VERO, A549, 293T, CaLu3, MRC5, avian eggs such as chicken eggs. In embodiments, the host cell comprises transgenic eggs expressing a polypeptide having at least 80% amino acid sequence identity to one of SEQ ID Nos. 1-36 or 74-91 or a portion thereof with the activity of SEQ ID Nos. 1-36 or 74-91.
Also provided is method to increase influenza virus yield in cells, comprising: contacting influenza virus and cells comprising the vector comprising a nucleic acid encoding a polypeptide having at least 80% amino acid sequence identity to one of SEQ ID Nos 1-36 or 74-91 or a portion thereof with the activity of SEQ ID Nos. 1-36 or 74-91 or contacted with a polypeptide having at least 80% amino acid sequence identity to one of SEQ ID Nos. 1-36 or a portion thereof with the activity of SEQ ID Nos. 1-36 or 74-91; and collecting progeny influenza virus. The cells can be human, canine, or non-human primate cells. In embodiments, the cells are Vero cells, MDCK cells, 293T or PER C6® cells, or MvLu1 cells. The cells can be contacted with the vector or the polypeptide before contacting the cells with the influenza virus. In embodiments, the cell is contacted with the vector or the polypeptide after contacting the cells with the influenza virus. The yield of influenza virus in cells contacted with the vector or the polypeptide can be increased at least two-fold relative to the corresponding yield in host cells not contacted with the vector or the polypeptide.
In embodiments, a method to detect influenza virus in a sample is provided, comprising: contacting cells having the vector comprising a nucleic acid encoding a polypeptide having at least 80% amino acid sequence identity to one of SEQ ID Nos. 1-36 or 74-91 or a portion thereof with the activity of SEQ ID Nos. 1-36 or 74-91 and a biological sample; and determining whether the sample comprises influenza virus. In embodiments, the cells are human, canine or non-human primate cells. In embodiments, the cells are Vero cells, MDCK cells, 293T or PER.C6® cells, or MvLu1 cells. In embodiments, the sample is a physiological sample. In embodiments, the sample is a nasal sample. In embodiments, the sample is a physiological fluid sample. In embodiments, the method does not include employing nucleic acid amplification.
A method to decrease influenza virus replication in a mammal is provided, comprising: administering to the mammal a composition that inhibits or prevents expression of a polypeptide having at least 80% amino acid sequence identity to one of SEQ ID Nos 1-36 or 74-91 or a portion thereof with the activity of SEQ ID Nos. 1-36 or 74-91.
Further provided is a method to screen for compounds that alter the activity of a pathogen, comprising contacting cells expressing a polypeptide having at least 80% amino acid sequence identity to one of SEQ ID Nos. 1-36 or 74-91 or a portion thereof with the activity of SEQ ID Nos. 1-36 or 74-91 or an isolated polypeptide having at least 80% amino acid sequence identity to one of SEQ ID Nos. 1-36 or 74-91 or a portion thereof with the activity of SEQ ID Nos. 1-36 or 74-91 and a sample having a pathogen; and determining whether the polypeptide alters the activity of the pathogen. In embodiments, the pathogen is a virus. In embodiments, the cells are mammalian cells. For example, the cells can be canine, non-human primate, or human cells. In embodiments, the cells are MDCK cells. Any cell, e.g., any avian or mammalian cell, such as a human, e.g., 293T or PER.C6® cells, or canine, e.g., MDCK, bovine, equine, feline, swine, ovine, rodent, for instance mink, e.g., MvLu1 cells, or hamster, e.g., CHO cells, or non-human primate, e.g., Vero cells, including mutant cells, which supports efficient replication of influenza virus can be employed.
In embodiments, a method to inhibit expression of pro-viral genes in a mammal is provided, comprising administering to the mammal an effective amount a composition that specifically inhibits the expression of a polypeptide having at least 80% amino acid sequence identity to one of SEQ ID Nos. 1-36 or 74-91. In embodiments, the composition comprises RNA. In embodiments, the RNA comprises RNAi. In embodiments, the RNA comprises siRNA. In embodiments, the amount prevents or inhibits influenza virus replication.
In embodiments, a method to screen for inhibitory compounds is provided, comprising combining cells expressing a polypeptide having at least 80% amino acid sequence identity to one of SEQ ID Nos. 1-36 or 74-91 or a portion thereof with the activity of SEQ ID Nos. 1-36 or 74-91 or isolated nucleic acid that encodes a polypeptide having at least 80% amino acid sequence identity to one of SEQ ID Nos. 1-36 or 74-91 or a portion thereof with the activity of SEQ ID Nos. 1-36 or 74-91 and one or more test compounds; and determining whether the one or more test compounds inhibit expression of the polypeptide or inhibit transcription or translation of the isolated nucleic acid. Any cell, e.g., any avian or mammalian cell, such as a human, e.g., 293T or PER.C6® cells, or canine, e.g., MDCK, bovine, equine, feline, swine, ovine, rodent, for instance mink, e.g., MvLu1 cells, or hamster, e.g., CHO cells, or non-human primate, e.g., Vero cells, including mutant cells, which supports efficient replication of influenza virus can be employed.
In addition, disclosed herein are methods to prevent, inhibit, or treat influenza virus infection in an avian or a mammal is provided, comprising administering to the avian or mammal an effective amount of RNA that triggers RNA interference (RNAi), wherein the RNA encodes a polypeptide having at least 80% amino acid sequence identity to SEQ ID Nos. 1-36 or 74-91 or an antibody or antibody fragment thereof specific for one of SEQ ID Nos. 1-36 or 74-91. In embodiments, the mammal is a primate. In embodiments, the primate is a human. In embodiments, the RNA that triggers RNAi comprises small interfering RNAs (siRNA). In embodiments, the siRNA comprises microRNA (miRNA) or a binding site for miRNA. In embodiments, the miRNA binds to the S′UTR of RNA encoding one of SEQ ID Nos. 1-36 or 74-91. In embodiments, the RNAi binds to the 3′UTR of RNA encoding a polypeptide having at least 80% amino acid sequence identity to SEQ ID Nos. 1-36 or 74-91. In embodiments, the composition is locally administered, e.g., to the lungs. In embodiments, the composition is systemically administered or intranasally administered. The composition can comprise liposomes or nanoparticles comprising the siRNA. The antibody fragment can comprise Fab′, F(ab′)2, scFv or a single domain, e.g., of a heavy chain or light chain.
Described herein are methods to detect influenza virus in a sample, comprising: detecting in a biological sample the presence or amount of a polypeptide having at least 80% amino acid sequence identity to one of SEQ ID Nos. 1-36 or 74-91 or a portion thereof with the activity of SEQ ID Nos. 1-36 or 74-91.
(TRIPC) manipulates host gene expression to enable fitness-based screening.
Many approaches to identify host factors regulating infection have relied upon loss-of-function screens, which leaves a large amount of genetic space unexplored and raises the possibility that entirely new classes of viral co-factors have yet to be discovered. CRISPR activation (CRISPRa) and CRISPR inhibition (CRISPRi) may be used to exploit the programmable nature of Cas9 to recruit transcriptional activators or repressor to discrete genomic loci, respectively. CRISPRa and CRISPRi permit both gain- and loss-of-function screens, something not achievable in prior genome-wide surveys of viral host factors.
As disclosed herein, CRISPR-Cas9 technology was adapted to be programmed by the pathogen itself. The pathogen encodes and expresses the targeting RNA that places Cas9 as specific sites in the host genome, termed transcriptional regulation by pathogen-programmed Cas9 (TRPPC). Using the RNA virus influenza virus as an exemplar, TRPPC viruses were shown to modulate host gene expression. Thus, influenza virus can be engineered to specifically and potently modulate expression of discrete host genes. This process can be adapted to any pathogen capable of delivering the targeting RNA. Given that the pathogen expresses essential components of the TRPPC platform, the screen itself only begins during infection, and only in infected cells, which results in the identification of host regulators in the middle-to-late stages of replication.
A pool of TRPPC influenza viruses was prepared targeting the entire genome and a genetic selection was performed allowing all viruses to compete with each other through multiple rounds of replication in human lung cells. Viruses within that population that activated pro-viral factors gained a replicative advantage and came to quickly dominate the viral population. Because the RNA programming Cas9 is encoded in the viral genome, the viruses with an advantage and their host gene targets are easily determined by deep sequencing. Moreover, as this is a fitness-based screen, TRPPC selections identify and inherently rank-order the most potent host regulators of viral replication. In short, the virus itself does the “heavy lifting” to pinpoint the cellular regulators of viral replication.
As an example, 36 host regulators of influenza virus replication whose expression enhances influenza virus replication, that were identified in a genome wide screen are disclosed herein. Several of these host regulators were individually tested for pro-viral properties for influenza virus. In embodiments, the host factor may increase viral yields ˜10-fold, e.g., in human lung cells. Importantly, over-expression of the host factors results in higher levels of virus replication. These are targets to generate cell lines to increase virus yields.
A “vector” or “delivery” vehicle refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide or polypeptide, and which can be used to mediate delivery of the polynucleotide or polypeptide to a cell or intercellular space, either in vitro or in vivo. Illustrative vectors include, for example, plasmids, viral vectors, liposomes, nanoparticles, or microparticles and other delivery vehicles. In embodiments, a polynucleotide to be delivered, sometimes referred to as a “target polynucleotide” or “transgene,” may comprise a coding sequence of interest in gene therapy (such as a gene encoding a protein of therapeutic interest), a coding sequence of interest and/or a selectable or detectable marker.
“Transduction,” “transfection,” “transformation” or “transducing” as used herein, are terms referring to a process for the introduction of an exogenous polynucleotide into a host cell leading to expression of the polynucleotide, e.g., the transgene in the cell, and includes the use of recombinant virus to introduce the exogenous polynucleotide to the host cell. Transduction, transfection or transformation of a polynucleotide in a cell may be determined by methods well known to the art including, but not limited to, protein expression (including steady state levels), e.g., by ELISA, flow cytometry and Western blot, measurement of DNA and RNA by hybridization assays, e.g., Northern blots, Southern blots and gel shift mobility assays. Methods used for the introduction of the exogenous polynucleotide include well-known techniques such as viral infection or transfection, lipofection, transformation and electroporation, as well as other non-viral gene delivery techniques. The introduced polynucleotide may be stably or transiently maintained in the host cell.
“Gene delivery” refers to the introduction of an exogenous polynucleotide into a cell for gene transfer, and may encompass targeting, binding, uptake, transport, localization, replicon integration and expression.
“Gene transfer” refers to the introduction of an exogenous polynucleotide into a cell which may encompass targeting, binding, uptake, transport, localization and replicon integration, but is distinct from and does not imply subsequent expression of the gene.
“Gene expression” or “expression” refers to the process of gene transcription, translation, and post-translational modification.
An “infectious” virus or viral particle is one that comprises a polynucleotide component which is capable of delivering into a cell for which the viral species is trophic. The term does not necessarily imply any replication capacity of the virus.
The term “polynucleotide” refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated or capped nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
A “transcriptional regulatory sequence” refers to a genomic region that controls the transcription of a gene or coding sequence to which it is operably linked. Transcriptional regulatory sequences of use generally include at least one transcriptional promoter and may also include one or more enhancers and/or terminators of transcription.
“Operably linked” refers to an arrangement of two or more components, wherein the components so described are in a relationship permitting them to function in a coordinated manner. By way of illustration, a transcriptional regulatory sequence or a promoter is operably linked to a coding sequence if the TRS or promoter promotes transcription of the coding sequence. An operably linked TRS is generally joined in cis with the coding sequence, but it is not necessarily directly adjacent to it.
“Heterologous” means derived from a genotypically distinct entity from the entity to which it is compared. For example, a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide). Similarly, a transcriptional regulatory element such as a promoter that is removed from its native coding sequence and operably linked to a different coding sequence is a heterologous transcriptional regulatory element.
A “terminator” refers to a polynucleotide sequence that tends to diminish or prevent read-through transcription (i.e., it diminishes or prevent transcription originating on one side of the terminator from continuing through to the other side of the terminator). The degree to which transcription is disrupted is typically a function of the base sequence and/or the length of the terminator sequence. In particular, as is well known in numerous molecular biological systems, particular DNA sequences, generally referred to as “transcriptional termination sequences” are specific sequences that tend to disrupt read-through transcription by RNA polymerase, presumably by causing the RNA polymerase molecule to stop and/or disengage from the DNA being transcribed. Typical example of such sequence-specific terminators include polyadenylation (“polyA”) sequences, e.g., SV40 polyA. In addition to or in place of such sequence-specific terminators, insertions of relatively long DNA sequences between a promoter and a coding region also tend to disrupt transcription of the coding region, generally in proportion to the length of the intervening sequence. This effect presumably arises because there is always some tendency for an RNA polymerase molecule to become disengaged from the DNA being transcribed, and increasing the length of the sequence to be traversed before reaching the coding region would generally increase the likelihood that disengagement would occur before transcription of the coding region was completed or possibly even initiated. Terminators may thus prevent transcription from only one direction (“uni-directional” terminators) or from both directions (“bi-directional” terminators), and may be comprised of sequence-specific termination sequences or sequence-non-specific terminators or both. A variety of such terminator sequences are known in the art, and illustrative uses of such sequences within the context of the present disclosure are provided below.
“Host cells,” “cell lines,” “cell cultures,” “packaging cell line” and other such terms denote higher eukaryotic cells, such as mammalian cells including human cells, useful in the present disclosure, e.g., to produce recombinant virus or recombinant polypeptide. These cells include the progeny of the original cell that was transduced. It is understood that the progeny of a single cell may not necessarily be completely identical (in morphology or in genomic complement) to the original parent cell.
“Recombinant,” as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction and/or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature. A recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.
A “control element” or “control sequence” is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3′ direction) from the promoter. Promoters include AAV promoters, e.g., P5, P19, P40 and AAV ITR promoters, as well as heterologous promoters.
An “expression vector” is a vector comprising a region which encodes a gene product of interest, and is used for effecting the expression of the gene product in an intended target cell. An expression vector also comprises control elements operatively linked to the encoding region to facilitate expression of the protein in the target. The combination of control elements and a gene or genes to which they are operably linked for expression is sometimes referred to as an “expression cassette,” a large number of which are known and available in the art or can be readily constructed from components that are available in the art.
The terms “polypeptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, acetylation, phosphorylation, lipidation, or conjugation with a labeling component.
An “isolated” polynucleotide, e.g., plasmid, virus, polypeptide or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially prepared from. Thus, for example, an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Isolated nucleic acid, peptide or polypeptide is present in a form or setting that is different from that in which it is found in nature. For example, a given DNA sequence (e g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins. The isolated nucleic acid molecule may be present in single-stranded or double-stranded form. When an isolated nucleic acid molecule is to be utilized to express a protein, the molecule will contain at a minimum the sense or coding strand (i.e., the molecule may single-stranded), but may contain both the sense and anti-sense strands (i.e., the molecule may be double-stranded). Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. For example, a 2-fold enrichment, 10-fold enrichment, 100-fold enrichment, or a 1000-fold enrichment.
A “transcriptional regulatory sequence” refers to a genomic region that controls the transcription of a gene or coding sequence to which it is operably linked. Transcriptional regulatory sequences of use generally include at least one transcriptional promoter and may also include one or more enhancers and/or terminators of transcription.
“Operably linked” refers to an arrangement of two or more components, wherein the components so described are in a relationship permitting them to function in a coordinated manner. By way of illustration, a transcriptional regulatory sequence or a promoter is operably linked to a coding sequence if the TRS or promoter promotes transcription of the coding sequence. An operably linked TRS is generally joined in cis with the coding sequence, but it is not necessarily directly adjacent to it.
“Conservative” amino acid substitutions are, for example, aspartic-glutamic as polar acidic amino acids; lysine/arginine/histidine as polar basic amino acids; leucine/isoleucine/methionine/valine/alanine/glycine/proline as non-polar or hydrophobic amino acids; serine/threonine as polar or uncharged hydrophilic amino acids. Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting polypeptide. Whether an amino acid change results in a functional polypeptide can readily be determined by assaying the specific activity of the polypeptide. Naturally occurring residues are divided into groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues that influence chain orientation: gly, pro, and (6) aromatic; trp, tyr, phe.
The disclosure also envisions polypeptides with non-conservative substitutions. Non-conservative substitutions entail exchanging a member of one of the classes described above for another.
As used herein, “individual” (as in the subject of the treatment) means a mammal. Mammals include, for example, humans; non-human primates, e.g., apes and monkeys; and non-primates, e.g., dogs, cats, rats, mice, cattle, horses, sheep, and goats. Non-mammals include, for example, fish and birds.
“Substantially” as the term is used herein means completely or almost completely; for example, a composition that is “substantially free” of a component either has none of the component or contains such a trace amount that any relevant functional property of the composition is unaffected by the presence of the trace amount, or a compound is “substantially pure” is there are only negligible traces of impurities present.
“Treating” or “treatment” within the meaning herein refers to an alleviation of symptoms associated with a disorder or disease, “inhibiting” means inhibition of further progression or worsening of the symptoms associated with the disorder or disease, and “preventing” refers to prevention of the symptoms associated with the disorder or disease.
As used herein, an “effective amount” or a “therapeutically effective amount” of an agent, refers to an amount of the agent that alleviates, in whole or in part, symptoms associated with the disorder or condition, or halts or slows further progression or worsening of those symptoms, or prevents or provides prophylaxis for the disorder or condition, e.g., an amount that is effective to prevent, inhibit or treat in the individual one or more symptoms.
In particular, a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result A therapeutically effective amount is also one in which any toxic or detrimental effects of the agent(s)are outweighed by the therapeutically beneficial effects.
The term “sequence” refers to a nucleotide sequence of any length, which can be DNA or RNA; can be linear, circular or branched and can be either single-stranded or double stranded. The term “donor sequence” refers to a nucleotide sequence that is inserted into a genome. A donor sequence can be of any length, for example between 2 and 10,000 nucleotides in length (or any integer value therebetween or thereabove), e.g., between about 100 and 1,000 nucleotides in length (or any integer therebetween), e.g., between about 200 and 500 nucleotides in length. For example, an exogenous nucleic acid can comprise an infecting viral genome, a plasmid or episome introduced into a cell, or a chromosome that is not normally present in the cell. Methods for the introduction of exogenous molecules into cells are known to those of skill in the art and include, but are not limited to, lipid-mediated transfer (e.g., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer. An exogenous molecule can also be the same type of molecule as an endogenous molecule but derived from a different species than the cell is derived from. For example, a human nucleic acid sequence may be introduced into a cell line originally derived from a mouse or hamster.
The term “exogenous,” when used in relation to a protein, gene, nucleic acid, or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid, or polynucleotide which has been introduced into the cell or organism by artificial or natural means. An exogenous nucleic acid may be from a different organism or cell, or it may be one or more additional copies of a nucleic acid which occurs naturally within the organism or cell. By way of a non-limiting example, an exogenous nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature, e.g., an expression cassette which links a promoter from one gene to an open reading frame for a gene product from a different gene.
“Transformed” or “transgenic” is used herein to include any host cell or cell line, which has been altered or augmented by the presence of at least one recombinant DNA sequence. The host cells are typically produced by transfection with a DNA sequence in a plasmid expression vector, as an isolated linear DNA sequence, or infection with a recombinant viral vector.
The term “sequence homology” means the proportion of base matches between two nucleic acid sequences or the proportion amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the proportion of matches over the length of a selected sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, or 6 bases or less or 2 bases or less. When using oligonucleotides as probes or treatments, the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally not less than 17 target base matches out of 20 possible oligonucleotide base pair matches (85%); not less than 9 matches out of 10 possible base pair matches (90%), or not less than 19 matches out of 20 possible base pair matches (95%).
Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less or 2 or less. Alternatively, two protein sequences (or polypeptide sequences derived from them of at least 30 amino acids in length) are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater. The two sequences or parts thereof are more homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program.
The term “corresponds to” is used herein to mean that a polynucleotide sequence is structurally related to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is structurally related to all or a portion of a reference polypeptide sequence, e.g., they have at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97% or more, e.g., 99% or 100%, sequence identity. In contradistinction, the term “complementary to” is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For illustration, the nucleotide sequence “TATAC” corresponds to a reference sequence “TATAC” and is complementary to a reference sequence “GTATA”.
The term “sequence identity” means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term “percentage of sequence identity” means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A. T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms “substantial identity” as used herein denote a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, e.g., at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 20-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
As used herein, “substantially pure” or “purified” means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), for instance, a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, or more than about 85%, about 90%, about 95%, and about 99%. The object species may be purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
To prepare expression cassettes encoding one of SEQ ID Nos. 1-36 or 74-91 or truncated forms thereof (a “portion”), a peptide thereof, or a fusion thereof, for transformation, the recombinant DNA sequence or segment may be circular or linear, double-stranded or single-stranded. A DNA sequence which encodes an RNA sequence that is substantially complementary to a mRNA sequence encoding a gene product of interest is typically a “sense” DNA sequence cloned into a cassette in the opposite orientation (i.e., 3′ to 5′ rather than 5′ to 3′). Generally, the DNA sequence or segment is in the form of chimeric DNA, such as plasmid DNA, that can also contain coding regions flanked by control sequences which promote the expression of the DNA in a cell. As used herein, “chimeric” means that a vector comprises DNA from at least two different species, or comprises DNA from the same species, which is linked or associated in a manner which does not occur in the “native” or wild-type of the species.
Aside from DNA sequences that serve as transcription units, or portions thereof, a portion of the DNA may be untranscribed, serving a regulatory or a structural function. For example, the DNA may itself comprise a promoter that is active in eukaryotic cells, e.g., mammalian cells, or in certain cell types, or may utilize a promoter already present in the genome that is the transformation target of the lymphotrophic virus. Such promoters include the CMV promoter, as well as the SV40 late promoter and retroviral LTRs (long terminal repeat elements), although many other promoter elements well known to the art may be employed, e.g., the MMTV, RSV, MLV or HIV LTR. In embodiments, expression is inducible. In embodiments, a tissue-specific promoter (or enhancer) is employed.
Other elements functional in the host cells, such as introns, enhancers, polyadenylation sequences and the like, may also be a part of the recombinant DNA. Such elements may or may not be necessary for the function of the DNA but may provide improved expression of the DNA by affecting transcription, stability of the mRNA, or the like. Such elements may be included in the DNA as desired to obtain the optimal performance of the transforming DNA in the cell. The recombinant DNA to be introduced into the cells may contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of transformed cells from the population of cells sought to be transformed. Alternatively, the selectable marker may be carried on a separate piece of DNA and used in a co-transformation procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are well known in the art and include, for example, antibiotic and herbicide-resistance genes, such as neo, hpt, dhfr, bar, aroA, puro, hyg, dapA and the like. See also, the genes listed on Table 1 of Lundquist et. al. (U.S. Pat. No. 5,848,956).
Reporter genes are used for identifying potentially transformed cells and for evaluating the functionality of regulatory sequences. Reporter genes which encode for easily assayable proteins are well known in the art. In general, a reporter gene is a gene which is not present in or expressed by the recipient organism or tissue and which encodes a protein whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Example reporter genes include the chloramphenicol acetyl transferase gene (cat) from Tn9 of E. coli, the beta-glucuronidase gene (gus) of the uidA locus of E. coli, the green, red, or blue fluorescent protein gene, and the luciferase gene. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
The general methods for constructing recombinant DNA which can transform target cells are well known to those skilled in the art, and the same compositions and methods of construction may be utilized to produce the DNA useful herein.
The recombinant DNA can be readily introduced into the host cells, e.g., mammalian, bacterial, yeast or insect cells, or prokaryotic cells, by transfection with an expression vector comprising the recombinant DNA by any procedure useful for the introduction into a particular cell, e.g., physical or biological methods, to yield a transformed (transgenic) cell having the recombinant DNA so that the DNA sequence of interest is expressed by the host cell. In embodiments, the recombinant DNA is stably integrated into the genome of the cell.
Physical methods to introduce a recombinant DNA into a host cell include calcium-mediated methods, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods to introduce the DNA of interest into a host cell include the use of DNA and RNA viral vectors. Viral vectors, e.g., retroviral or lentiviral vectors, have become a widely used method for inserting genes into eukaryotic cells, such as mammalian, e.g., human cells. Other viral vectors can be derived from poxviruses, e.g., vaccinia viruses, herpes viruses, adenoviruses, adeno-associated viruses, baculoviruses, and the like.
To confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; biochemical assays, such as detecting the presence or absence of a particular gene product, e.g., by immunological means (ELISAs and Western blots) or by other molecular assays.
To detect and quantitate RNA produced from introduced recombinant DNA segments, RT-PCR may be employed. In this application of PCR, it is first necessary to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then through the use of conventional PCR techniques amplify the DNA. In most instances PCR techniques, while useful, will not demonstrate integrity of the RNA product. Further information about the nature of the RNA product may be obtained by Northern blotting. This technique demonstrates the presence of an RNA species and gives information about the integrity of that RNA. The presence or absence of an RNA species can also be determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northern blotting and only demonstrate the presence or absence of an RNA species.
While Southern blotting and PCR may be used to detect the recombinant DNA segment in question, they do not provide information as to whether the recombinant DNA segment is being expressed. Expression may be evaluated by specifically identifying the peptide products of the introduced DNA sequences or evaluating the phenotypic changes brought about by the expression of the introduced DNA segment in the host cell.
Delivery vectors or vehicles include, for example, viral vectors, microparticles, nanoparticles, liposomes and other lipid-containing complexes, and other macromolecular complexes capable of mediating delivery of a gene or a protein to a host cell, e.g., a gene to provide for recombinant expression of a polypeptide encoded by the gene. Vectors or vehicles can also comprise other components or functionalities that further modulate gene delivery and/or gene expression, or that otherwise provide beneficial properties. 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 by the cell; components that influence localization of the transferred gene within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the gene. 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 or have taken up protein delivered by a vehicle. 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. Selectable markers can be positive, negative or bifunctional. Positive selectable markers allow selection for cells carrying the marker, whereas negative selectable markers allow cells carrying the marker to be selectively eliminated. A variety of such marker genes have been described, including bifunctional (i.e., positive/negative) markers (see, e.g., WO 92/08796; and WO 94/28143). Such marker genes can provide an added measure of control that can be advantageous in gene therapy contexts. A large variety of such vectors are known in the art and are generally available.
Vectors or vehicles within the scope of the disclosure include, but are not limited to, isolated nucleic acid, e.g., plasmid-based vectors which may be extrachromosomally maintained, and viral vectors, e.g., recombinant adenovirus, retrovirus, lentivirus, herpesvirus, poxvirus, papilloma virus, or adeno-associated virus, including viral and non-viral vectors, or proteins which are present in liposomes, e.g., neutral or cationic liposomes, such as DOSPA/DOPE, DOGS/DOPE or DMRIE/DOPE liposomes, and/or associated with other molecules such as DNA-anti-DNA antibody-cationic lipid (DOTMA/DOPE) complexes. Vectors or vehicles may be administered via any route including, but not limited to, intramuscular, buccal, rectal, intravenous or intracoronary administration, and transfer to cells may be enhanced using electroporation and/or iontophoresis. In embodiments, vectors are locally administered.
In embodiments, an isolated polynucleotide or vector having that polynucleotide, encoding a polypeptide or fusion protein that has substantial identity, e.g., at least 80% or more, e.g., 85%, 87%, 90%, 92%, 95%, 97%, 98%, 99% and up to 100%, amino acid sequence identity to one of SEQ ID NOs. 1-36 or 74-91, or a portion thereof, is envisioned.
Retroviral vectors exhibit several distinctive features including their ability to stably and precisely integrate into the host genome providing long-term transgene expression. These vectors can be manipulated ex vivo to eliminate infectious gene particles to minimize the risk of systemic infection and patient-to-patient transmission. Pseudotyped retroviral vectors can alter host cell tropism.
Lentiviruses are derived from a family of retroviruses that include human immunodeficiency virus and feline immunodeficiency virus. However, unlike retroviruses that only infect dividing cells, lentiviruses can infect both dividing and nondividing cells. Although lentiviruses have specific tropisms, pseudotyping the viral envelope with vesicular stomatitis virus yields virus with a broader range (Schnepp et al., Meth. Mol. Med., 69:427 (2002)).
Adenoviral vectors may be rendered replication-incompetent by deleting the early (E1A and E1B) genes responsible for viral gene expression from the genome and are stably maintained into the host cells in an extrachromosomal form. These vectors have the ability to transfect both replicating and nonreplicating cells and, in particular, these vectors have been shown to efficiently infect cardiac myocytes in vivo, e.g., after direction injection or perfusion. Adenoviral vectors have been shown to result in transient expression of therapeutic genes in vivo, peaking at 7 days and lasting approximately 4 weeks. The duration of transgene expression may be improved in systems utilizing neural specific promoters. In addition, adenoviral vectors can be produced at very high titers, allowing efficient gene transfer with small volumes of virus.
Recombinant adeno-associated viruses (rAAV) are derived from nonpathogenic parvoviruses, evoke essentially no cellular immune response, and produce transgene expression lasting months in most systems. Moreover, like adenovirus, adeno-associated virus vectors also have the capability to infect replicating and nonreplicating cells and are believed to be nonpathogenic to humans.
AAV vectors include but are not limited to AAV1, AAV2, AAV5, AAV7, AAV8, AAV9 or AAVrh. 10.
Plasmid DNA is often referred to as “naked DNA” to indicate the absence of a more elaborate packaging system. Direct injection of plasmid DNA to myocardial cells in vivo has been accomplished. Plasmid-based vectors are relatively nonimmunogenic and nonpathogenic, with the potential to stably integrate in the cellular genome, resulting in long-term gene expression in postmitotic cells in vivo. Plasmid DNA may be delivered to cells as part of a macromolecular complex, e.g., a liposome or DNA-protein complex, and delivery may be enhanced using techniques including electroporation.
The peptide, polypeptide or fusion proteins can be synthesized in vitro, e.g., by the solid phase peptide synthetic method or by recombinant DNA approaches (see above). The solid phase peptide synthetic method is an established and widely used method. These polypeptides can be further purified by fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on an anion-exchange resin such as DEAE; chromatofocusing, SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; or ligand affinity chromatography.
Once isolated and characterized, chemically modified derivatives of a given peptide, polypeptide or fusion thereof, can be readily prepared. For example, amides of the peptide, polypeptide or fusion thereof may also be prepared by techniques well known in the art for converting a carboxylic acid group or precursor, to an amide. One method for amide formation at the C-terminal carboxyl group is to cleave the peptide, polypeptide or fusion thereof from a solid support with an appropriate amine, or to cleave in the presence of an alcohol, yielding an ester, followed by aminolysis with the desired amine.
Salts of carboxyl groups of a peptide, polypeptide or fusion thereof may be prepared in the usual manner by contacting the peptide, polypeptide, or fusion thereof with one or more equivalents of a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide; a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate; or an amine base such as, for example, triethylamine, triethanolamine, and the like.
N-acyl derivatives of an amino group of the peptide, polypeptide or fusion thereof may be prepared by utilizing an N-acyl protected amino acid for the final condensation, or by acylating a protected or unprotected peptide, polypeptide, or fusion thereof. O-acyl derivatives may be prepared, for example, by acylation of a free hydroxy polypeptide or polypeptide resin. Either acylation may be carried out using standard acylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both N- and O-acylation may be carried out together, if desired.
Formyl-methionine, pyroglutamine and trimethyl-alanine may be substituted at the N-terminal residue of the polypeptide. Other amino-terminal modifications include aminooxypentane modifications.
In embodiments, an isolated peptide, polypeptide or fusion protein has substantial identity, e.g., at least 80% or more, e.g., 85%, 87%, 90%, 92%, 95%, 97%, 98%, 99% and up to 100%, amino acid sequence identity to one of SEQ ID NOs. 1-36 or 74-91 or portion thereof, is envisioned.
Substitutions may include substitutions which utilize the D rather than L form, as well as other well known amino acid analogs, e.g., unnatural amino acids such as a, a-disubstituted amino acids, N-alkyl amino acids, lactic acid, and the like. These analogs include phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, citruline, α-methyl-alanine, para-benzoyl-phenylalanine, phenylglycine, propargylglycine, sarcosine, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, w-N-methylarginine, and other similar amino acids and imino acids and tert-butylglycine.
Conservative amino acid substitutions may be employed—that is, for example, aspartic-glutamic as acidic amino acids; lysine/arginine/histidine as polar basic amino acids; leucine/isoleucine/methionine/valine/alanine/proline/glycine non-polar or hydrophobic amino acids; serine/threonine as polar or hydrophilic amino acids Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting peptide, polypeptide or fusion polypeptide. Whether an amino acid change results in a functional peptide, polypeptide or fusion polypeptide can readily be determined by assaying the specific activity of the peptide, polypeptide or fusion polypeptide.
Amino acid substitutions are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:
The disclosure also envisions a peptide, polypeptide or fusion polypeptide with non-conservative substitutions. Non-conservative substitutions entail exchanging a member of one of the classes described above for another.
Acid addition salts of the peptide, polypeptide or fusion polypeptide or of amino residues of the peptide, polypeptide or fusion polypeptide may be prepared by contacting the polypeptide or amine with one or more equivalents of the desired inorganic or organic acid, such as, for example, hydrochloric acid. Esters of carboxyl groups of the polypeptides may also be prepared by any of the usual methods known in the art.
The polypeptides or fusions thereof, or nucleic acid encoding the polypeptide or fusion or the complement thereof, e.g., RNAi, can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, e.g., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.
In embodiments, the polypeptides or fusions thereof, or nucleic acid encoding the polypeptide or fusion, or the complement thereof, may be administered by infusion or injection. Solutions of the polypeptides or fusions thereof, or nucleic acid encoding the polypeptide or fusion or the complement thereof, or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it may be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active agent in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
Useful solid carriers may include finely divided solids such as tale, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as antimicrobial agents can be added to optimize the properties for a given use. Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
Useful dosages of the polypeptides or fusions thereof, or nucleic acid encoding the polypeptide or fusion, can be determined by comparing their in vitro activity and in vivo activity in animal models thereof. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.
Generally, the concentration of the polypeptides or fusions thereof, or nucleic acid encoding the polypeptide or fusion, or the complement thereof, in a liquid composition, may be from about 0.1-25 wt-%, e.g., from about 0.5-10 wt-%. The concentration in a semi-solid or solid composition such as a gel or a powder may be about 0.1-5 wt-%, e.g., about 0.5-2.5 wt-%.
The amount of the polypeptides or fusions thereof, or nucleic acid encoding the polypeptide or fusion required for use alone or with other agents will vary with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.
The polypeptides or fusions thereof, or nucleic acid encoding the polypeptide or fusion, or the complement thereof, may be conveniently administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, or conveniently 50 to 500 mg of active ingredient per unit dosage form.
In general, however, a suitable dose may be in the range of from about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, for example in the range of 6 to 90 mg/kg/day, e.g., in the range of 15 to 60 mg/kg/day.
In embodiments, the pro-viral factor comprises a sodium/hydrogen exchanger 10 isoform 1 (SLC9C1) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a treslin isoform 1 (TICRR) [Homo sapiens]
a different isoform of the protein, a polypeptide having the sequence in NP_689472.3, which is incorporated by reference herein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises an olfactory receptor 4C6 (OR4C6) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a C-type lectin domain family 4 member C isoform 1 (CLEC4C) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 7(NDUFA7) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises an olfactory receptor 51A7 (OR51A7) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a chloride channel protein CIC-Kb isoform 1 (CLCNKB) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-5 (GNG5) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a S-adenosyl-L-methionine-dependent tRNA 4-demethylwyosine synthase (TYW1) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a ras-related protein Rab-42 isoform 1 (RAB42) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 3 (HCN3) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a rasGAP-activating-like protein 1 isoform 1 (RASAL1) [Homo sapiens]
a different isoform of the protein, a polypeptide having the sequence in NP_001288131.1, which is incorporated by reference herein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a UL16-binding protein 1 isoform 1 precursor (ULBP1) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a macrophage immunometabolism regulator (C5orf30) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a protein mono-ADP-ribosyltransferase PARP15 isoform 1 [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a neuroligin-4, X-linked (NLGN4X) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a CD59 glycoprotein preproprotein (CD59) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a cofilin-2 isoform 1 (CFL2) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a gasdermin-B isoform 1 (GSDMB) [Homo sapiens]
a different isoform of the protein, a polypeptide having the sequence in NP_001159430.1, which is incorporated by reference herein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a bromodomain-containing protein 4 isoform long (BRD4) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises an interferon-induced protein with tetratricopeptide repeats 3 isoform a (IFIT3) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises an opioid growth factor receptor (OGFR) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises an epimerase family protein SDR39U1 isoform 1 (SDR39U1) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a regulating synaptic membrane exocytosis protein 2 isoform a (RIMS2) [Homo sapiens]
a different isoform of the protein, or a polypeptide having the sequence in NP_001335413.1, which is incorporated by reference herein or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a sia-alpha-2,3-Gal-beta-1,4-G1cNAc-R:alpha 2,8-sialyltransferase (ST8SIA3) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a cyclin-dependent kinase inhibitor 3 isoform 1 (CDKN3) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a T-cell immunoglobulin and mucin domain-containing protein 4 isoform 1 precursor (TIMD4) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a protein SYS1 homolog isoform a (SYS1) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises an ubiquitin D (UBD) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a mediator of RNA polymerase II transcription subunit 17 (MED17) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a peroxisome biogenesis factor 13 (PEX13) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises an ubiquitin carboxyl-terminal hydrolase 17-like protein 13 (USP17L13) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a mirror-image polydactyly gene 1 protein isoform 1 (MIPOL1) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a ribokinase isoform 1 (RBKS) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises an ubiquitin carboxyl-terminal hydrolase 17 (USP17L2) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the pro-viral factor comprises a dystrophin isoform Dp427m (DMD) [Homo sapiens]
a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In embodiments, the disclosure provides for nucleic acid sequences useful to inhibit transcription or translation of mRNA in a host organism, e.g., useful to inhibit RNA, or to enhance viral production. The nucleic acid sequences encode a polypeptides having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity to one of SEQ ID Nos. 1-36 or 74-91.
In embodiments, the nucleic acid inhibits expression of a Homo sapiens solute carrier family 9 member C1 (SLC9C1), transcript variant 1, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of an Homo sapiens olfactory receptor family 4 subfamily C member 6 (OR4C6), mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of a Homo sapiens C-type lectin domain family 4 member C (CLEC4C), transcript variant 1, mRNA NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of an Homo sapiens NADH:ubiquinone oxidoreductase subunit A7 (NDUFA7),
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of a Homo sapiens olfactory receptor family 51 subfamily A member 7 (OR51A7), mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of an Homo sapiens chloride voltage-gated channel Kb (CLCNKB), transcript variant 1, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of an Homo sapiens G protein subunit gamma 5 (GNG5), mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of an Homo sapiens tRNA-yW synthesizing protein 1 homolog (TYW1), transcript variant 1, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of an Homo sapiens RAB42, member RAS oncogene family (RAB42), transcript variant 1, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of an Homo sapiens hyperpolarization activated cyclic nucleotide gated potassium channel 3 (HCN3), transcript variant 1, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of an Homo sapiens RAS protein activator like 1 (RASAL1), transcript variant 1, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of an Homo sapiens UL16 binding protein 1 (ULBP1), transcript variant 1, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of an Homo sapiens macrophage immunometabolism regulator (MACIR), transcript variant 1, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of an Homo sapiens poly(ADP-ribose) polymerase family member 15 (PARP15), transcript variant 1, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of an Homo sapiens neuroligin 4 X-linked (NLGN4X), transcript variant 1, mRNA having NCBI Reference Sequence.
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of an Homo sapiens CD59 molecule (CD59 blood group) (CD59), transcript variant 1, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of an Homo sapiens cofilin 2 (CFL2), transcript variant 1, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88% 90%. 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of treslin isoform 1 [Homo sapiens] mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of Homo sapiens macrophage immunometabolism regulator (MACIR), transcript variant 1, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of Homo sapiens gasdermin B (GSDMB), transcript variant 1, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of Homo sapiens bromodomain containing 4 (BRD4), transcript variant long, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of Homo sapiens interferon induced protein with tetratricopeptide repeats 3 (IFIT3), transcript variant 1, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of Homo sapiens opioid growth factor receptor (OGFR), mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of Homo sapiens short chain dehydrogenase/reductase family 39U member 1 (SDR39U1), transcript variant 1, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of Homo sapiens regulating synaptic membrane exocytosis 2 (RIMS2), transcript variant 1, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of Homo sapiens ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 3 (ST8SIA3), mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of Homo sapiens cyclin dependent kinase inhibitor 3 (CDKN3), transcript variant 1, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of Homo sapiens T cell immunoglobulin and mucin domain containing 4 (TIMD4), transcript variant 1, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of Homo sapiens SYS1 golgi trafficking protein (SYS1), transcript variant 1, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%. 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of Homo sapiens ubiquitin D (UBD), mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of Homo sapiens mediator complex subunit 17 (MED17), mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of Homo sapiens peroxisomal biogenesis factor 13 (PEX13), mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of Homo sapiens ubiquitin specific peptidase 17 like family member 13 (USP17L13), mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of Homo sapiens mirror-image polydactyly 1 (MIPOL1), transcript variant 1, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of Homo sapiens ribokinase (RBKS), transcript variant 1, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of Homo sapiens ubiquitin specific peptidase 17 like family member 2 (USP17L2), mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In embodiments, the nucleic acid inhibits expression of Homo sapiens dystrophin (DMD), transcript variant Dp427m, mRNA having NCBI Reference Sequence:
a different transcript variant of the gene, or a sequence with at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
The amino acid sequences for the polypeptides of SEQ ID NOS: 74-91 and the corresponding nucleic acid sequences of SEQ ID NOs: 92-109 that encode the polypeptides of SEQ ID NOS: 74-91 are provided in
The invention will be described by the following non-limiting example.
Most genetic approaches to identify host factors regulating infection have relied upon loss-of-function screens. Knock-out screens are limited in genes they can query, as genes essential for cell survival cannot be investigated. Moreover, existing screens often rely on proxy phenotypes instead of directly measuring viral replication. This leaves a large amount of genetic space unexplored and raises the possibility that entirely new classes of viral co-factors have yet to be discovered. TRPPC overcomes this in at least 3 ways: 1) it is a fitness-based screen dependent on viral replication; 2) TRPPC inherently rank orders host factors, as the abundance of any particular virus reflects the importance of the modulated host gene; and 3) TRPPC can be used for both loss- and gain-of-function screening, exploring new genetic space including essential genes. Furthermore, this system is entirely portable, functioning with any pathogen that can deliver a targeting RNA, amenable to various iterations changing the selective pressure or modes of replication to focus on different aspects of infection, and in principle can also be performed in vivo in transgenic animals expressing the CRISPRa/i machinery.
TRPPC can be used to identify host factors regulating pathogen replication. The top hits identified by the inventors increase replication of influenza virus, and this information can be used to increase virus yield in commercial settings, and even a modest gain in viral yield would have large impacts on production. Similarly, adenovirus-based vaccines like the adenovirus based COVID19 vaccine are produced in cell culture, and engineering host gene expression to increase yields would have a major impact on this process.
Rank-ordered top hits from influenza virus TRPPC screen in human lung cells:
Additional hits are provided in amino acid sequences of SEQ ID NOs: 74-91 shown in
All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification, this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details herein may be varied considerably without departing from the basic principles of the invention.
This application is a continuation of U.S. Provisional Application Ser. No. 63/384,541, filed Nov. 21, 2022, the contents of which are specifically incorporated herein by reference in its entirety.
This invention was made with government support under AI125897 awarded by the National Institutes of Health awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63384541 | Nov 2022 | US |
