Correction of exon skipping in monocyte-derived cells improves immune responses

Abstract

The present disclosure generally relates to compositions and methods for inhibiting exon 3 skipping of the 6-pyruvoyltetrahydropterin synthase (PTS) gene in monocytes, monocyte-derived cells, such as macrophages and dendritic cells, or their precursor cells. Inhibition can be achieved by genome editing of the genomic sequence to remove certain splicing factor recognition sites, or by inhibiting the expression or activity of splicing factors that are responsible for cell-specific exon skipping. Monocytes and monocyte-derived cells with reduced exon skipping in the PTS gene can generate stronger immune responses and are therefore useful for the prevention and treatment of diseases such as infections and cancer.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 185,980, filed May 7, 2021, and U.S. Provisional Application No. 63 / 333,339, filed April 21, 2022, the entire contents of each of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing, which has been submitted in ASCII format via EFS-Web and is incorporated by reference in its entirety. The ASCII copy, created on May 3, 2022, is named 334203.txt and is 10310 bytes in size. [Background technology]

[0003] The immune system is a host defense system that protects the host from disease. It can detect a wide variety of agents known as pathogens, such as viruses, bacteria, and parasites, and distinguish them from the organism's own healthy tissue. Pathogens can evolve and adapt to avoid detection and neutralization by the immune system. However, jawed vertebrates, including humans, have developed sophisticated mechanisms to adapt to such changes.

[0004] Impairment of the immune system can lead to autoimmune diseases, inflammatory diseases, and cancer. Immunodeficiency occurs when the immune system becomes less active than normal, resulting in recurrent and life-threatening infections. In humans, immunodeficiency can be the result of either genetic diseases such as severe combined immunodeficiency, acquired conditions such as HIV / AIDS, or the use of immunosuppressant medications. Thus, stimulating the immune system can be useful in relieving immunodeficiencies and treating infectious diseases.

[0005] Cancer immunotherapy treats cancer by artificially stimulating the immune system to improve its natural ability to fight cancer. Cancer cells often carry tumor antigens that can be detected by antibodies capable of recognizing these antigens. Tumor antigens are often proteins or other macromolecules such as carbohydrates. Once detected, cancer cells can be killed by mechanisms such as antibody-dependent cellular cytotoxicity (ADCC). Summary of the Invention

[0006] In various embodiments, the present disclosure describes compositions and methods for inhibiting and / or correcting exon 3 skipping of the 6-pyruvoyltetrahydropterin synthase (PTS) gene in human monocytes, monocyte-derived cells (e.g., macrophages and dendritic cells), or their progenitors. Inhibition can be achieved by editing the genomic sequence of PTS to alter or remove certain splicing factor recognition sites, or by inhibiting the expression or activity of splicing factors that contribute to cell-type specific exon skipping. Human monocytes and monocyte-derived cells generally have no detectable normal protein product of the PTS gene (PTPS protein), as the majority of transcripts in these cells produce inactive proteins due to cell-type specific alternative splicing that skips exon 3. Thus, restoring PTPS protein activity in these cells may strengthen the immune system of human subjects and aid in the prevention and treatment of diseases, such as infectious diseases, cancer, cardiovascular disease, cerebrovascular disease, autoimmune disease, and liver damage.

[0007] One embodiment of the present disclosure provides a method of modifying the immune system in a human subject in need thereof, comprising editing a 6-pyruvoyltetrahydropterin synthase (PTS) genomic sequence or pre-mRNA of a monocyte, macrophage, dendritic cell, or a precursor thereof in the human subject, wherein the editing inhibits skipping of exon 3 of the PTS mRNA during splicing.

[0008] In some embodiments, the editing is to alter a recognition site for a splicing factor. In some embodiments, the recognition site is located within intron 2 or intron 3. In some embodiments, the recognition site is located within intron 2. In some embodiments, the splicing factor is selected from the group consisting of SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF8, SRSF9, SRSF10, SRSF11, and SRSF12. In some embodiments, the recognition site is selected from Table 3.

[0009] In some embodiments, the splicing factor is SRSF3. In some embodiments, the recognition site is CTCTTCC, which corresponds to nucleotides 2895-2901 of SEQ ID NO:1.

[0010] In some embodiments, the splicing factor is SRSF1. In some embodiments, the recognition site is selected from the group consisting of TGGTGGC (2833 to 2839 of SEQ ID NO: 1), TGATGCT (3541 to 3547 of SEQ ID NO: 1), TGGTGGA (3927 to 3933 of SEQ ID NO: 1), TGGTGCT (4105 to 4111 of SEQ ID NO: 1), and TGAAGGC (4143 to 4149 of SEQ ID NO: 1).

[0011] In some embodiments, the edit comprises an addition, deletion, and / or substitution of at least one, two, three, or four nucleotides of the recognition site, hi some embodiments, the edited recognition site cannot be bound by individual splicing factors.

[0012] In some embodiments, editing comprises introducing into the cell an editing system that targets the recognition site, hi some embodiments, the editing system is selected from a CRISPR / Cas (clustered regularly interspaced short palindromic repeats / CRISPR-associated proteins) system, a gene editor comprising a CRISPR / Cas system and cytosine deaminase, a meganuclease, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN) based gene editing system.

[0013] In some embodiments, the human subject is suffering from or at risk of developing an infection or condition, cancer, a cardiovascular disease, a cerebrovascular disease, an autoimmune disease, or a liver disorder.

[0014] Also provided in one embodiment is a method of modulating or modifying the immune system in a human subject in need thereof, comprising administering to the human subject human monocytes, macrophages, dendritic cells, or progenitor cells thereof, wherein a 6-pyruvoyltetrahydropterin synthase (PTS) genomic sequence in the cell has been edited to inhibit skipping of exon 3 of the PTS mRNA during splicing.

[0015] In some embodiments, the monocytes, macrophages, dendritic cells, or progenitor cells thereof are harvested from a human subject and edited in vitro or ex vivo, hi some embodiments, the monocytes or macrophages are dedifferentiated prior to administration.

[0016] In some embodiments, the editing alters a recognition site for a splicing factor. In some embodiments, the splicing factor is selected from the group consisting of SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF8, SRSF9, SRSF10, SRSF11, and SRSF12. In some embodiments, the recognition site is CTCTTCC, which corresponds to nucleotides 2895-2901 of SEQ ID NO:1.

[0017] Also provided in one embodiment is a method of modifying the immune system in a human subject in need thereof, comprising administering to the subject an agent that inhibits exon 3 skipping or promotes exon 3 recognition / inclusion in 6-pyruvoyltetrahydropterin synthase (PTS) pre-mRNA in monocytes, macrophages, dendritic cells, or precursor cells thereof, of the subject.

[0018] In some embodiments, the agent inhibits the biological activity of a splicing factor selected from the group consisting of SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF8, SRSF9, SRSF10, SRSF11, and SRSF12. In some embodiments, the splicing factor is SRSF3. In some embodiments, the agent is an antisense oligonucleotide or RNA, siRNA, or shRNA that inhibits expression of the splicing factor. In some embodiments, the agent is an antibody with specificity for the splicing factor.

[0019] In some embodiments, the agent is a small molecule agent selected from the group consisting of kinetin, epigallocatechin gallate (EGCG), genistein, daidzein, cardiac glycosides, and rectifiers of aberrant splicing (RECTAS). In some embodiments, the cardiac glycoside is selected from the group consisting of digoxin, digitonin, digitoxin, digoxigenin, digitoxigenin, acetyldigitoxin, bufalin, ouabagenin, and ouabain.

[0020] In some embodiments, the agent that promotes recognition or inclusion of exon 3 is U1 snRNA that recognizes exon 3 of the PTS pre-mRNA.

[0021] Also provided in one embodiment is a method of modifying the immune system in a mammalian subject in need thereof, comprising administering a nonsense suppressor to a tissue of the subject that produces or circulates monocytes, macrophages, dendritic cells, or their precursor cells.

[0022] In some embodiments, the nonsense suppressor is selected from the group consisting of stop codon readthrough drugs, suppressor tRNAs, stop codon pseudouridylation agents, and nonsense-mediated mRNA decay (NMD) inhibitors. In some embodiments, the nonsense suppressor is selected from the group consisting of ataluren, aminoglycosides, gentamicin, amikacin, negamycin, spiramycin, josamycin, tylosin, and amlexanox.

[0023] In some embodiments, the tissue is bone marrow.

[0024] In some embodiments, administration targets monocytes, macrophages, dendritic cells, or their precursors, hi some embodiments, the subject is suffering from or at risk of developing an infection or condition, or cancer.

[0025] In some embodiments, the subject has elevated immune activity when compared to a healthy individual. In some embodiments, the elevated immune activity is measured by a biomarker from a sample selected from the group consisting of serum, urine, cerebrospinal fluid, synovial fluid, saliva, peritoneal fluid, bile, pancreatic juice, and gastric juice. In some embodiments, the biomarker is selected from the group consisting of neopterin, C-reactive protein, interferon-gamma, interferon-alpha, interferon-beta, and procalcitonin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The following description describes exemplary embodiments of the present technology, but it should be understood that such description is not intended to limit the scope of the disclosure, but is provided as a description of exemplary embodiments.

[0027] Activation of PTPS-deficient monocytes PTPS (6-pyruvoyltetrahydropterin synthase, EC 4.2.3.12) is an enzyme that catalyzes the biosynthesis of 6-pyruvoyltetrahydropterin from GTP, which is used as a cofactor in the synthesis of aromatic amino acid monooxygenases and nitric oxide (NO) synthase. PTPS is a hexamer composed of identical subunits formed by the dimerization of trimers. A 12-stranded antiparallel β-barrel is formed by the dimerized trimers, forming a pore within PTPS. The enzymatic active site of PTPS is located where three monomers come together in each subunit of the hexamer. PTPS is an intermediate, or precursor, in the synthetic pathway of tetrahydrobiopterin (BH4), a cofactor for several enzyme systems, including aromatic amino acid monooxygenases (e.g., phenylalanine hydroxylase, tyrosine hydroxylase, tryptophan hydroxylase), nitric oxide synthases (e.g., inducible nitric oxide synthase, endothelial nitric oxide synthase, neuronal nitric oxide synthase), and ether lipid oxidase.

[0028] In humans, the PTPS protein is encoded by the PTS gene. Mutations in the PTS gene have been suggested to be responsible for inherited dystonia disorders. Four such mutations have been found, two of which are homozygous mutations, R25Q and I114V, and two of which are heterozygous mutations, R16C and K120stop.

[0029] Loss of PTPS activity is a common cause of tetrahydrobiopterin deficiency, which results in hyperphenylalaninemia and an inability to make neurotransmitters such as dopamine and serotonin. PTPS deficiency has also been shown to result in severe mental retardation, delayed motor development, and epileptic seizures. Low levels of tetrahydrobiopterin production, in contrast to near-total deficiency of tetrahydrobiopterin, can cause cycling symptoms.

[0030] The relationship between dysfunction of PTPS and immune deficiency or infectious diseases has not been established. However, by analyzing the biochemical role of PTPS in various cells and how PTPS evolved from animals to humans, the inventors determined that activating PTPS in human monocytes and monocyte-derived cells (e.g., macrophages and dendritic cells) can stimulate immune responses, which may be useful in the prevention and treatment of infectious diseases and cancer.

[0031] Unlike other tissues such as the liver and central nervous system, human monocytes and monocyte-derived cells have little detectable PTPS activity. The transcriptional levels of the PTS gene in monocytes and monocyte-derived cells are normal. Based on sequence analysis and mechanistic studies, the inventors assume that the reduced PTPS activity in human monocytes and monocyte-derived cells is due to exon 3 skipping rather than post-transcriptional mechanisms. Skipping of exon 3 results in truncated transcripts with premature stop codons. Similar differential expression patterns are also observed in higher primates such as chimpanzees. Such exon skipping does not occur, or at least is not universal, in many other human cell types or in monocytes and monocyte-derived cells of lower animals such as rats.

[0032] It is hypothesized that the reduced PTPS activity in human monocytes and monocyte-derived cells, although potentially evolutionarily advantageous, contributes to the weakening of the human immune system. Thus, it may be advantageous to reactivate PTPS in monocytes or monocyte-derived cells, at least in human subjects in which a stronger immune system is desirable, such as subjects suffering from or at risk of developing an infectious disease or cancer. Thus, in accordance with one embodiment of the present disclosure, compositions and methods are provided for modifying or increasing the activity of PTPS in human monocytes and monocyte-derived cells.

[0033] Gene editing As demonstrated in Example 1, intron 2 and intron 3 of the PTS gene harbor a number of potential binding sites for splicing factors such as serine / arginine-rich splicing factor 1 (SRSF1) and serine / arginine-rich splicing factor 3 (SRSF3) (previously known as SFRS1 and SFRS3, respectively). These splicing factor binding sites are postulated to be involved in exon 3 skipping leading to PTPS inactivation in human monocytes and monocyte-derived cells. Thus, modifying one or more of these binding sites to inhibit the binding of the respective splicing factors can reduce exon 3 skipping, thereby leading to the production of more active PTPS protein.

[0034] Thus, in some embodiments, the present disclosure provides a method of modifying or enhancing the immune system in a human subject in need thereof. The method can include editing a 6-pyruvoyltetrahydropterin synthase (PTS) genomic sequence or pre-mRNA in monocytes, macrophages, dendritic cells, or precursors thereof, of the human subject. In some embodiments, the editing inhibits skipping of exon 3 in the PTS mRNA during splicing. In some embodiments, the editing modifies a recognition site for a splicing factor. In some embodiments, the recognition site is located within intron 2 or intron 3 of the gene.

[0035] Splicing factors are proteins involved in removing introns from messenger RNA strands so that exons can join together. Splicing factors may be part of a complex known as the spliceosome. The splicing reaction is carried out by the spliceosome, which consists of five small nuclear ribonucleoprotein complexes, or snRNPs, and as many as 50-100 non-snRNP proteins. Assembly of the spliceosome also requires SR proteins, a family of sequence-specific splicing factors that have one or two RNA recognition motifs followed by an arginine / serine-rich domain. Non-limiting examples of splicing factors include serine / arginine-rich splicing factor 1 (SRSF1), SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF8, SRSF9, SRSF10, SRSF11, and SRSF12.

[0036] In one embodiment, at least one of the recognition sites of the PTS sequence is mutated. The mutation can occur in the genomic sequence of the host cell or can be introduced into the host cell as an extra copy. In some embodiments, the introduced sequence is a mutated genomic sequence. In some embodiments, the introduced sequence is a wild-type cDNA sequence or a biological equivalent.

[0037] The mutation can occur at the genome level or pre-mRNA level, without particular limitation. In some embodiments, the recognition site is recognized by any of SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF8, SRSF9, SRSF10, SRSF11, and / or SRSF12.

[0038] In some embodiments, the recognition site is recognized by SRSF3. In some embodiments, the recognition site has the sequence CTCTTCC (nucleotides 2895-2901 of SEQ ID NO: 1), which is located in intron 2 5' to exon 3. The variant can include one, two, three, four, five, six, or seven substitutions, deletions, additions, or combinations thereof. In some embodiments, one, two, three, four, five, six, or seven of the nucleotides are deleted. In some embodiments, one, two, three, four, five, six, or seven additional nucleotides are inserted. In some embodiments, one, two, three, four, five, six, or seven of the nucleotides are substituted, preferably with nucleotides that do not form the consensus sequence (CUCKUCY).

[0039] In some embodiments, the SRSF3 recognition site is selected from the group consisting of TCAAC (2505-2509 of SEQ ID NO:1), ACAAC (2569-2573 of SEQ ID NO:1), TCTTC (2896-2900 of SEQ ID NO:1), ACTAC (3102-3106 of SEQ ID NO:1), TCTAC (3508-3512 of SEQ ID NO:1), TCTAC (3508-3512 of SEQ ID NO:1), TCTAC (3514-3518 of SEQ ID NO:1), and TCTAC (4026-4030 of SEQ ID NO:1). The mutant can include one, two, three, four, or five substitutions, deletions, additions, or combinations thereof. In some embodiments, one, two, three, four, or five of the nucleotides are deleted. In some embodiments, one, two, three, four, five, or six additional nucleotides are inserted. In some embodiments, one, two, three, four, or five of the nucleotides are substituted, preferably with nucleotides that do not match the consensus sequence (WCWWC).

[0040] In some embodiments, the splicing factor recognition site is recognized by SRSF1. In some embodiments, the splicing factor recognition site has a sequence selected from TGGTGGC (2833-2839 of SEQ ID NO:1), TGATGCT (3541-3547 of SEQ ID NO:1), TGGTGGA (3927-3933 of SEQ ID NO:1), TGGTGCT (4105-4111 of SEQ ID NO:1), or TGAAGGC (4143-4149 of SEQ ID NO:1). The variant can include one, two, three, four, five, six, or seven substitutions, deletions, additions, or combinations thereof. In some embodiments, one, two, three, four, five, six, or seven of the nucleotides are deleted. In some embodiments, one, two, three, four, five, or six additional nucleotides are inserted. In some embodiments, 1, 2, 3, 4, 5, 6, or 7 of the nucleotides are substituted, preferably with nucleotides that do not match the consensus sequence (UGRWGVH).

[0041] In some embodiments, the splicing factor recognition site is any one selected from Table 3. The variant can include 1, 2, 3, 4, 5, 6, or 7 substitutions, deletions, additions, or combinations thereof. In some embodiments, 1, 2, 3, 4, 5, 6, or 7 of the nucleotides are deleted. In some embodiments, 1, 2, 3, 4, 5, 6, or 6 additional nucleotides are inserted. In some embodiments, 1, 2, 3, 4, 5, 6, or 7 of the nucleotides are substituted, preferably with nucleotides that do not form a consensus sequence.

[0042] The mutation can be generated in situ by introducing a genome editing system into the target cell. Alternatively, the mutated sequence can be introduced into the target cell using a suitable vector, such as a viral vector. Methods for carrying out each step are known in the art.

[0043] Genome editing is a type of genetic engineering that uses engineered nucleases to insert, delete, or replace DNA in the genome of a living organism. Recently developed genome editing technology uses the CRISPR / Cas (clustered regularly interspaced short palindromic repeats / CRISPR-associated proteins) system. Directed by a guide RNA (gRNA), Cas nucleases can generate DNA double-strand breaks (DSBs) at targeted genomic sites in a variety of cells, both cell lines and cells derived from living organisms. These DSBs are then repaired by endogenous DNA repair systems. Endogenous DNA repair systems can be utilized to perform the desired genome edits.

[0044] In general, two major DNA repair pathways, non-homologous end joining (NHEJ) and homology-directed repair (HDR), can be activated by DSBs. NHEJ can introduce random insertions / deletions (indels) into the genomic DNA region surrounding the DSB, leading to open reading frame (ORF) shifts and ultimately gene inactivation. In contrast, when HDR is induced, the genomic DNA sequence at the target site can be replaced by the sequence of an exogenous donor DNA template through a homologous recombination mechanism, which can result in the correction of the gene mutation.

[0045] The later development of base editors (BEs) incorporating the APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) cytosine deaminase family into the CRISPR / Cas system has greatly improved the efficiency of CRISPR / Cas9-mediated gene correction. Through fusion with Cas9 nickase (nCas9), the cytosine (C) deamination activity of rat APOBEC1 (rA1) can be deliberately directed to target bases in the genome to catalyze the replacement of C with thymine (T) at these bases.

[0046] In some embodiments, Cas9 nickases are used in combination with a guide sequence(s), e.g., two guide sequences, each of which targets the sense and antisense strand of a DNA target. This combination allows both strands to be nicked and used to induce NHEJ. The ability of a candidate guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence can be assessed by any suitable assay. For example, sufficient components of a CRISPR system to form a CRISPR complex, including the guide sequence to be tested, can be provided to a host cell having the corresponding target sequence, such as by transfecting with a vector encoding the components of the CRISPR sequence, and then evaluating preferential cleavage within the target sequence. Similarly, cleavage of a target polynucleotide sequence can be evaluated in a test tube by providing the target sequence, the components of the CRISPR complex, including the guide sequence to be tested, and a control guide sequence that differs from the test guide sequence, and comparing the binding or cleavage rate at the target sequence between the test guide sequence reaction and the control guide sequence reaction. In some embodiments, the guide sequence is selected to reduce the degree of secondary structure within the guide sequence. The secondary structure can be determined by any suitable polynucleotide folding algorithm.

[0047] A few other classes of nucleases can also generate DSBs that are useful for genome editing, including meganucleases, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs).

[0048] Meganucleases are enzymes in the endonuclease family that are characterized by their ability to recognize and cleave large DNA sequences (14-40 base pairs). Meganucleases are found in microbial species, but it is difficult to find a natural meganuclease for a specific DNA sequence. However, huge banks containing tens of thousands of protein units have been created. These units can be combined to obtain chimeric meganucleases that recognize target sites.

[0049] Unlike meganucleases, ZFNs and TALENs utilize a non-specific DNA cleavage catalytic domain linked to a specific DNA sequence recognition peptide, such as zinc fingers and transcription activator-like effectors (TALEs). The first step was to find an endonuclease whose DNA recognition and cleavage sites are separate from each other, which is not the most common situation among restriction enzymes. Once this enzyme was found, the cleavage part of the enzyme can be isolated. This part is likely to be highly non-specific since it has no recognition ability. This part can then be attached to a sequence recognition peptide that can provide high specificity. The zinc finger motif occurs in several transcription factors. In transcription factors, it is most often located at the protein-DNA interaction site, stabilizing the motif. The C-terminal part of each finger is responsible for the specific recognition of the DNA sequence.

[0050] Transcription activator-like effector nucleases (TALENs) are specific DNA-binding proteins featuring an array of 33- or 34-amino acid repeats. TALENs are artificial restriction enzymes designed by fusing the DNA cleavage domain of a nuclease to a TALE domain, and can be tailored to specifically recognize unique DNA sequences. Such fusion proteins function as easily targetable "DNA scissors" for gene editing applications. The DNA-binding domain can be designed to bind any desired DNA sequence and is derived from TAL effectors, DNA-binding proteins excreted by plant pathogenic Xanthomonas species. TAL effectors consist of repeated domains, each of which contains a highly deliberate sequence of 34 amino acids to recognize a single DNA nucleotide within the target site. The nuclease can create a double-stranded break at the target site that can be repaired by error-prone non-homologous end joining (NHEJ), resulting in the introduction of small insertions or deletions to disrupt the gene. Such TALENs can be fused to the catalytic domain from the DNA nuclease FokI to generate transcription activator-like effector nucleases (TALENs). The resulting TALEN constructs combine specificity and activity, effectively generating engineered sequence-specific nucleases that bind and cleave DNA sequences only at preselected sites.

[0051] Genome editing can occur in vivo, for example, by administering a genome editing system (e.g., CRISPR / Cas and guide RNA) to a subject of interest. Administration can be systemic, but preferably targets monocytes or monocyte-derived cells, or bone marrow cells or other progenitor cells that can differentiate into monocytes or monocyte-derived cells. Alternatively, in some embodiments, monocytes, monocyte-derived cells, or progenitor cells thereof can be introduced into a subject after genome editing is completed in the cells in vitro or ex vivo.

[0052] Monocytes differentiate from hematopoietic stem cells, specifically granulocyte / macrophage progenitors in the bone marrow, and enter the periphery as circulating monocytes. Various microenvironmental cues can determine the fate of monocytes, leading to their differentiation into macrophages and dendritic cells. Monocytes are not just macrophage and dendritic cell precursors, but also immune effector cells. Under inflammatory conditions, circulating monocytes are recruited to sites of infection or injury, and once there, can differentiate.

[0053] Liposomes are a commonly used delivery system for monocyte / phagocyte targeted therapy, offering advantages such as low immunogenicity, biocompatibility, cell specificity, and drug protection. Parenterally administered liposomes are naturally cleared by the mononuclear phagocyte system (MPS). Liposomal drug delivery systems take advantage of the physiological role of these cells to provide specific targeting and improved delivery efficiency.

[0054] Targeting of liposomes to monocytes and monocyte-derived cells can be achieved by modifying the lipid composition to control physicochemical properties such as size and charge, as well as by including surface ligands, including proteins, peptides, antibodies, polysaccharides, glycolipids, glycoproteins, and lectins. Non-limiting examples of such ligands include anionic lipids, muramyl tripeptide (MTP), Arg-Gly-Asp (RGD), VACM-1 specific antibodies, CC52 specific antibodies, CC531 specific antibodies, CD11c / DEC-205 specific antibodies, lectins such as Mann-C4-Chol, maleylated bovine serum albumin (MBSA), O-steroly amylopectin (O-SAP), fibronectin, and galactosyl.

[0055] Monocytes, macrophages, dendritic cells, or their precursors can also be edited outside the subject's body and then introduced into the subject. Such monocytes, macrophages, dendritic cells, or their precursors can be obtained from a donor subject or from the target subject itself. In some embodiments, the isolated cells can be enriched for monocytes, macrophages, dendritic cells, or their precursors before editing. In some embodiments, the cell population comprises more than 80%, even more preferably more than 90%, monocytes, macrophages, dendritic cells, or their precursors.

[0056] In some embodiments, the isolated progenitor cells can be differentiated into monocytes or monocyte-derived cells as needed for the human subject, hi some embodiments, the isolated cells (e.g., monocytes) can be dedifferentiated into progenitor cells that can differentiate into monocytes.

[0057] Monocytes are a hematopoietic cell lineage derived from bone marrow progenitor cells. Committed bone marrow progenitor cells differentiate to form blood monocytes, which circulate in the blood, invade tissues, and become resident macrophages. Thus, progenitor cells can be bone marrow precursor cells or other types of progenitor cells in the bone marrow.

[0058] In some embodiments, the cells can be activated, for example with interferon gamma (INF-γ), to enhance cytotoxicity. Monocyte maturation to macrophages and activation with interferon gamma can be achieved by contact with INF-γ. Alternatively, activation can be achieved by transformation with recombinant DNA expressing INF-γ.

[0059] The edited monocytes, monocyte-derived cells, or precursors thereof are then introduced into a subject. In some embodiments, the cells are delivered systemically, including, but not limited to, by injection into the blood, bone marrow, or target tissue. Moreover, the cells can be used alone or in combination with other pharmaceutical compositions.

[0060] Exon skipping inhibitors Inhibition of exon 3 skipping in the PTS gene in human cells (or skipping of the corresponding exon in other mammalian cells, particularly primate cells) can also be achieved or assisted by using an agent that inhibits the biological activity of any of the individual splicing factors, for example, SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF8, SRSF9, SRSF10, SRSF11, and SRSF12. Thus, in one embodiment, a method of modifying or enhancing the immune system in a mammalian subject in need thereof is provided. In some embodiments, the method involves administering to the subject an agent that inhibits exon 3 skipping in 6-pyruvoyltetrahydropterin synthase (PTS) pre-mRNA.

[0061] It is contemplated that each of the splicing factors may play a role in exon 3 skipping. In a preferred embodiment, the agent targets SRSF3. SRSF3 has a recognition site in intron 2 of the PTS gene, as shown in Example 1. In some embodiments, the agent targets SRSF1 or SRSF9.

[0062] Inhibiting the biological activity of a splicing factor, i.e. reducing the biological activity, can be achieved by decreasing the expression of the splicing factor and / or interfering with the function of the splicing factor protein.

[0063] Methods for reducing protein expression include, without limitation, the use of antisense or RNAi technology. "RNA interference" (RNAi) refers to the sequence-specific or gene-specific suppression of gene expression (protein synthesis) mediated by small interfering RNA (siRNA).

[0064] "Small interfering RNA" (siRNA) generally refers to a double-stranded RNA molecule, about 10 to about 30 nucleotides in length, capable of mediating RNA interference (RNAi). The term siRNA includes small hairpin RNA (shRNA). shRNAs include single-stranded RNA that form a stem-loop structure, the stem being composed of complementary sense and antisense strands that make up the double-stranded siRNA, and the loop being a linker of various sizes. The stem structure of shRNAs is generally about 10 to about 30 nucleotides in length.

[0065] In some embodiments, the agent that inhibits the biological activity of a splicing factor is an antibody. As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen-binding fragment or single chain thereof. That is, the term "antibody" includes any protein or peptide containing molecule that includes at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen.

[0066] Antibodies, or antigen-binding polypeptides, variants, or derivatives thereof of the present disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, such as Fab, Fab', and F(ab')2, Fd, Fv, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (sdFv), fragments comprising either the VK or VH domains, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to the LIGHT antibodies disclosed herein). Immunoglobulin or antibody molecules of the present disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule.

[0067] The antibodies disclosed herein can be of any animal origin, including birds and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or avian antibodies. The antibodies can also be of animal origin and subsequently humanized.

[0068] In some embodiments, the antibody has specificity for splicing factors such as SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF8, SRSF9, SRSF10, SRSF11, or SRSF12. The antibody cannot be bispecific or multispecific, targeting more than one of the splicing factors. In one embodiment, the antibody is a monoclonal antibody with specificity for SRSF3.

[0069] Small molecule inhibitors of splicing factors can also be used. The agent can also be a molecule that generally reduces aberrant splicing or promotes normal splicing by any other means. Non-limiting examples of agents include kinetin, epigallocatechin gallate (EGCG), genistein, daidzein, cardiac glycosides (e.g., digoxin), and rectifiers of aberrant splicing (RECTAS).

[0070] Kinetin (6-furfurylaminopurine) belongs to a family of N6-substituted adenine derivatives known as cytokinins, i.e., plant growth factors, which also include zeatin, benzyladenine, and 2iP. Kinetin has been shown to be able to rescue human mRNA splicing defects and increase production of normal mRNA (see, e.g., Slaugenhaupt et al., Human Mol. Genet. 2004, 13(4):429-36), likely through inhibition of splicing factor activity.

[0071] Genistein (5,7-dihydroxy-3-(4-hydroxyphenyl)chromen-4-one) is an isoflavone that has been used as an angiogenesis inhibitor and phytoestrogen. Daidzein (7-hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one) is a structurally distinct naturally occurring isoflavone found in soybeans and other legumes. Genistein and daidzein, as well as other isoflavones, are produced in plants through the phenylpropanoid pathway of secondary metabolism and are used as a defense response against single carrier and pathogenic attack. In humans, recent studies have shown that daidzein may be useful in mitigating menopause, osteoporosis, blood cholesterol, and reducing the risk of some hormone-related cancers, and heart disease.

[0072] Epigallocatechin gallate (EGCG), also called epigallocatechin-3-gallate, is an ester of epigallocatechin and gallic acid and is a type of catechin. EGCG, a polyphenol with antioxidant activity, is the most abundant catechin in tea.

[0073] Genistein and daidzein, optionally in combination with EGCG, have been shown to have potential activity in rescuing defective splicing (see, e.g., Anderson, et al., Mol Nutr Food Res. 2012 56(4):570-579).

[0074] Cardiac glycosides are a class of organic compounds that are common secondary metabolites of several plants, such as the foxglove plant, and increase the cardiac output and rate of contraction by acting on the cellular sodium potassium ATPase pump. The general structure of a cardiac glycoside consists of a steroid molecule with a sugar (glycoside) and an R group attached. The steroid nucleus consists of five fused rings to which other functional groups, such as methyl, hydroxyl, and aldehyde groups, can be attached to affect the biological activity of the entire molecule. Non-limiting examples include digoxin, digitonin, digitoxin, digoxigenin, digitoxigenin, acetyldigitoxin, bufalin, ouabagenin, and ouabain.

[0075] Researchers have shown that cardiac glycosides, including digoxin, digitonin, digitoxin, digoxigenin, digitoxigenin, acetyldigitoxin, bufalin, ouabagenin, and ouabain, were able to block the production of exon-skipped transcripts by inhibiting SRSF3 (Liu et al., FEBSJ. 2013 280(15):3632-46).

[0076] The name "aberrant splicing rectifier" or "RECTAS" refers to compounds identified from drug screens that can promote exon recognition and therefore reduce aberrant splicing (see Yoshida et al., Proc Natl Acad Sci USA. 2015;112(9):2764-2769). RECTAS has the chemical name 2-chloro-N-(furan-2-ylmethyl)-7H-purin-6-amine.

[0077] Additional agents can be identified by screening, for example, using cultured human monocytes as a test model. Correction of exon skipping can be tested, for example, using antibodies that target wild-type PTPS protein.

[0078] Antisense technology, including the use of antisense RNA or antisense oligonucleotides, can be readily used against cis-regulatory element sequences to inhibit their effect on exon skipping. Such cis-regulatory element sequences can be, for example, exon or intron splicing silencers.

[0079] Improved exon recognition The process of exon recognition may require canonical sequences, such as splice sites, polypyrimidine tracts, and branch sites, as well as exon and intron cis-acting regulatory element sequences. Furthermore, whether an exon is recognized also depends on the cellular environment that provides splicing factors. In cells that do not provide the appropriate cellular environment, agents can be added to improve / rescue exon recognition.

[0080] Exon-specific U1 (ExSpeU1) has been used to promote the recognition of defective or normal exons in non-cooperative environments. ExSpeU1 is a U1 snRNP-like particle that binds by complementarity to intronic regions downstream of the 5' splice site of skipped exons. ExSpeU1 promotes exon recognition by recruiting spliceosomal elements. ExSpeU1 can be used to correct exon skipping at consensus 5' splice sites, polypyrimidine tracts, or exonic regulatory element sequences. ExSpeU1 has been successful in restoring splicing in genes of various genetic disorders, including hemophilia B, cystic fibrosis, Netherton syndrome, and spinal muscular atrophy. ExSpeU1 can be designed to bind at non-conserved intronic sequences, so ExSpeU1 rarely results in off-target events.

[0081] ExSpeU1 can comprise a single stranded U1 snRNA molecule having a portion capable of hybridizing to a target nucleotide sequence on the primary transcript of a target gene of therapeutic interest. The target nucleotide sequence of the U1 snRNA molecule can be located in a region of the pre-mRNA comprised between 2 base pairs and 50 base pairs downstream of an exon / intron junction site.

[0082] The snRNA can be introduced into the target cell by a polynucleotide encoding the U1snRNA molecule. Preferably, the polynucleotide comprises a promoter sequence and a polyadenylation signal sequence. The promoter is preferably the endogenous promoter of the gene encoding the human U1snRNA.

[0083] Nonsense suppressors and methods In some embodiments, normal expression / activity of PTS in cells in which exon 3 (or the corresponding exon in mammalian or primate cells) is skipped can be restored by nonsense suppression therapy. Nonsense suppression therapy aims to suppress translation arrest at an in-frame premature stop codon (PTC, also known as a nonsense mutation) to restore defective protein function. Exon skipping in PTS leads to the generation of a stop / stop codon, resulting in a truncated inactive transcript. The assumption of nonsense suppression therapy is that it can restore most of the amino acid residues and functional domains of the PTPS protein.

[0084] Non-limiting examples of nonsense suppression therapies include read-through drugs, suppressor tRNA, PTC pseudouridylation, and inhibition of nonsense-mediated mRNA decay.

[0085] Aminoglycosides are a class of antibiotics that have been shown to bind to eukaryotic ribosomes and lead to the misincorporation of near-cognate aminoacyl-tRNA at PTCs. Gentamicin, the most commonly used aminoglycoside in nonsense suppression studies, has been shown to restore functional protein in short-term studies of DMD, CF, nephrogenic diabetes insipidus, hemophilia, retinal degeneration, APC-mediated colon cancer, and mucopolysaccharidosis type I-Hurler.

[0086] Another example of an aminoglycoside is amikacin. In particular, intravenous administration of unilamellar, low-clearance liposomes containing amikacin (MiKasome™) has shown good efficacy with reduced toxicity.

[0087] Some non-aminoglycoside antibiotics can also suppress PTC in mammalian cells. Negamycin, a peptide antibiotic that binds to the eukaryotic small ribosomal subunit, suppresses nonsense mutations and restores protein function in the APC gene associated with colon cancer, the laminin alpha-2 gene associated with congenital muscular dystrophy, and the dystrophin gene associated with DMD.

[0088] Additionally, several macrolide antibiotics, such as spiramycin, josamycin, and tylosin, can suppress APC nonsense mutations and restore APC protein function, and these macrolide antibiotics can reduce the number and size of tumors and intestinal polyps in mice carrying nonsense mutations in the APC gene.

[0089] Ataluren, formerly known as PTC124, is an oxadiazole compound that inhibits termination at PTC without affecting translation termination at the natural stop codon in mammalian cells. Comprehensive preclinical studies have shown that PTC124 is safe, has minimal off-target side effects, has no antibacterial activity, and is orally available.

[0090] High-throughput drug screening has identified other compounds that suppress nonsense mutations in the ATM gene that cause ataxia-telangiectasia (Du, et al. 2009. J. Exp. Med. 206:2285-97; Du, et al. 2013. Mol. Ther. 21:1653-60). Such drugs include RT13, RT14, GJ071, and GJ072, and their derivatives. These drugs can restore expression of full-length, functional ATM protein.

[0091] Compounds that inhibit nonsense-mediated mRNA decay (NMD) are also available. NMD is a pathway that allows mRNAs containing PTCs to be degraded. Amlexanox is an NMD inhibitor that also inhibits PTCs.

[0092] A "nonsense suppressor tRNA" is a tRNA derivative whose anticodon has been altered to recognize a stop codon, thus allowing the incorporation of an amino acid at the stop codon and bypassing translation termination. A nonsense suppressor tRNA can be constructed, for example, by modifying the anticodon of tRNALy to recognize the UAG nonsense codon instead of the normal AAA (lysine) codon.

[0093] "Pseudouridation" is the isomerization of the ribonucleoside uridine to the 5'-ribosyl isomer pseudouridine (Ψ). H / ACA guide RNAs can be used to provide the possibility of site-specific pseudouridation via sequence homology with any RNA sequence. Pseudouridation of tRNA molecules results in alternative codon recognition through changes in the anticodon loop structure. As all three nonsense codons have a uridine (U) in the first position (UAA, UAG, UGA), pseudoouridation of the uridine of the stop codon can modify the efficiency of PTC recognition.

[0094] Patient selection for exon skipping correction As presented, restoration of PTPS protein activity may strengthen the immune system and aid in the prevention and treatment of diseases such as infections and cancer. Such restoration may occur at any stage in an individual, particularly in an individual suspected of developing an infection or cancer. Likewise, this may be useful at any time point when improved immune activity is required for any individual.

[0095] In some embodiments, amelioration can be achieved in subjects at risk of or infected with an infectious disease, for example, through administration of an agent as disclosed herein. In some embodiments, amelioration can be achieved in subjects at risk of developing or with cancer. In some embodiments, amelioration can be achieved in subjects with a certain degree of oxidative stress. In some embodiments, amelioration can be achieved in subjects with increased immune activity.

[0096] The immune activity of an individual can be measured by suitable biomarkers. Non-limiting examples of biomarkers include neopterin, C-reactive protein, and procalcitonin. Other examples include interferon gamma, interferon-alpha, and interferon-beta. For example, elevated levels of neopterin and / or interferon gamma, or decreased levels of tetrahydrobiopterin and / or nitric oxide indicate that the subject may benefit from the treatment of the present disclosure.

[0097] Samples that can be used to measure biomarkers include, without limitation, serum, urine, cerebrospinal fluid, synovial fluid, saliva, peritoneal fluid, bile, pancreatic juice, and gastric juice.

[0098] The baseline levels of these biomarkers are also known. For example, more than 97% of healthy individuals (both children and adults) have serum neopterin concentrations less than 10 nmol / L. Thus, if an individual has a serum neopterin concentration greater than 10 nmol / L, the individual can be selected for restoration of PTPS protein activity using the techniques of the present disclosure. In some embodiments, subjects are selected for treatment if their serum neopterin concentration is greater than 10 nmol / L, 20 nmol / L, 30 nmol / L, 50 nmol / L, 100 nmol / L, or 200 nmol / L.

[0099] Similarly, urinary neopterin levels, expressed as mol neopterin / mol creatinine, greater than 350 μmol neopterin / mol creatinine in subjects under 7 years of age or greater than 250 μmol neopterin / mol creatinine in older patients indicate that the subject may benefit from restoration of PTPS protein activity. Similarly, salivary neopterin concentrations greater than 3.4 nmol / L, synovial fluid neopterin concentrations greater than 9 nmol / L, peritoneal neopterin concentrations greater than 26.3 nmol / L, or bronchoalveolar lavage fluid neopterin concentrations greater than 85.6 nmol / L indicate increased immune activity.

[0100] A baseline level of nitric oxide can also be identified for each individual. In some embodiments, a nitric oxide level (e.g., in serum or urine) of greater than 10 μmol / L can be considered as not requiring treatment. Thus, in some embodiments, if an individual has a nitric oxide concentration of less than 10 μmol / L, the individual can be selected for restoration of PTPS protein activity using the techniques of the present disclosure. In some embodiments, a subject is selected for treatment if the nitric oxide concentration is less than 0.1 μmol / L, 0.2 μmol / L, 0.5 μmol / L, 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 10 μmol / L, 15 μmol / L, 20 μmol / L, 30 μmol / L, 40 μmol / L, 50 μmol / L, 60 μmol / L, 70 μmol / L, 80 μmol / L, 90 μmol / L, 100 μmol / L, 150 μmol / L, 200 μmol / L, 300 μmol / L, or 500 μmol / L.

[0101] Such biomarkers can also be used to measure the effectiveness of correcting / inhibiting exon skipping of the PTS gene. For example, a decrease in neopterin levels, a decrease in interferon gamma levels, or an increase in either BH4, tetrahydrobiopterin, or nitric oxide levels can be indicative of effective correction of exon skipping. In some embodiments, efficacy can also be measured directly by detecting mature PTS mRNA with the skipped exon present in the target cell.

[0102] Therapeutic Compositions and Methods The genome editing system, the in vitro or ex vivo treated cells, the antisense or RNAi molecules, the antibodies, or the small molecule agents can be prepared as pharmaceutical compositions and administered to mammalian, particularly primate and human subjects in need thereof. The pharmaceutical composition can further comprise a pharma- ceutical acceptable carrier.

[0103] The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or state government or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, and especially humans. Moreover, a "pharmaceutically acceptable carrier" will generally be any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary.

[0104] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle used in administering a therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is a suitable carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, and the like. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates, or phosphates, if desired. Antibacterial agents such as benzyl alcohol or methyl parabens, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and agents for adjusting tonicity such as sodium chloride or glucose are also contemplated. Such compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The compositions can be formulated as suppositories, using traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference.

[0105] In one embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition can further include a solubilizing agent and a local anesthetic, such as lidocaine, to alleviate pain at the injection site. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example as a dry lyophilized powder or water-free concentrate in an airtight sealed container, for example an ampoule or sachette labeled with the quantity of active agent. If the composition is to be administered by infusion, the composition can be dispensed into an infusion bottle containing pharmaceutical grade sterile water or sterile saline. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0106] In some embodiments, the pharmaceutical composition is formulated for targeted delivery to target cells. For example, the agent can be packaged in liposomes. Liposomes are a commonly used delivery system for monocyte / phagocyte targeted therapy, offering advantages such as low immunogenicity, biocompatibility, cell specificity, and drug protection. Parenterally administered liposomes are naturally cleared by the mononuclear phagocyte system (MPS). The liposomal drug delivery system takes advantage of the physiological role of these cells to provide specific targeting and improved delivery efficiency.

[0107] Targeting of liposomes to monocytes and monocyte-derived cells can be achieved by modifying the lipid composition to control physicochemical properties such as size and charge, as well as by including surface ligands, including proteins, peptides, antibodies, polysaccharides, glycolipids, glycoproteins, and lectins. Non-limiting examples of such ligands include anionic lipids, muramyl tripeptide (MTP), Arg-Gly-Asp (RGD), VACM-1-specific antibodies, CC52-specific antibodies, CC531-specific antibodies, CD11c / DEC-205-specific antibodies, lectins such as Mann-C4-Chol, maleylated bovine serum albumin (MBSA), O-steroliamylopectin (O-SAP), fibronectin, and galactosyl.

[0108] The molecules, cells, systems, and compositions of the present disclosure may be useful for enhancing the immune system in a human subject in need thereof and for treating a disease or condition in the subject. The human subject may be one suffering from a disease or condition, the treatment of which may benefit from an improved immune system. Human subjects at risk of developing such a disease or condition may also benefit from the treatment.

[0109] As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, where the subject is to prevent or slow (reduce) the progression of an undesirable physiological change or disorder, such as an infection or cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or not, reduction in the extent of disease, stabilization of the disease state (i.e., no progression), delay or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or total). "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to the condition or disorder or those in whom the condition or disorder is to be prevented.

[0110] infectious disease In some embodiments, the subject is suffering from or at risk of infection with an infectious disease. Infectious disease is when disease-causing agents invade the body tissues of an organism, where they multiply, and the host tissues react against them, producing toxins. Infectious diseases can be caused by infectious agents, such as viruses, viroids, prions, bacteria, nematodes, such as parasitic roundworms and pinworms, arthropods, such as ticks, mites, fleas, and lice, fungi, such as ringworm, and other macroparasites, such as tapeworms and other worms. In one embodiment, the infectious agent is a bacterium, such as a gram-negative bacterium. In one embodiment, the infectious agent is a virus, such as a DNA virus, an RNA virus, and a reverse transcription virus.

[0111] Non-limiting examples of viruses include herpesviruses, coronaviruses, influenza viruses, filoviruses, flaviviruses, arenaviruses, togaviruses, bunyaviridae, poxviruses, picornaviruses, retroviruses, paramyxoviruses, hantaviruses, measles viruses, mumps viruses, rubella viruses, viral hepatitis (all types) viruses, viral meningitis viruses, human T-lymphotropic viruses, Crimean-Congo hemorrhagic fever viruses, and other viral families.

[0112] Non-limiting examples of diseases associated with bacterial infections include sepsis / septic shock, Pseudomonas infections, Brucella infections, Streptococcus infections, Mycobacterium infections, Salmonella infections, Borrelia infections, Listeria infections, Shigella infections, and bacterial meningitis.

[0113] Non-limiting examples of diseases associated with parasitic infections include Plasmodium infections, Leishmaniasis, Schistosoma infections, Trypanosoma infections, and Giardia infections.

[0114] cancer In some embodiments, the subject has cancer or is at risk of developing cancer. The method can generally enhance the immune system of a human subject, and therefore may be useful in treating a number of cancer types. In some embodiments, the cancer is selected from bladder cancer, non-small cell lung cancer, renal cancer, breast cancer, urethral cancer, colorectal cancer, head and neck cancer, squamous cell carcinoma, Merkel cell carcinoma, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, and small cell lung cancer.

[0115] In some embodiments, the subject is afflicted with a disease selected from leukemia (including acute leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, including sarcomas and cell types such as, for example, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangio ... These include, but are not limited to, lymphangioendotheliosarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, prostate cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic lung carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0116] In some embodiments, the method further comprises administration of a chemotherapeutic agent. Chemotherapeutic agents that can be administered with the compositions of the present disclosure include antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, and vincristine). sulfates); hormones (e.g., medroxyprogesterone, estramustine sodium phosphate, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone); nitrogen mustard derivatives (e.g., melphalan, chlorambucil, mechlorethamine (nitrogen mustard), and thiotepa); steroids and combination drugs (e.g., betamethasone sodium phosphate); and others (e.g., dacarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[0117] In some embodiments, the compositions of the present disclosure are administered in combination with cytokines, including, but not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

[0118] In further embodiments, the compositions of the present disclosure are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.

[0119] Cardiovascular diseases and disorders In some embodiments, the subject has cardiovascular disease or disorder. Non-limiting examples of cardiovascular disease and disorder include coronary heart disease, cerebrovascular disease, peripheral artery disease, rheumatic heart disease, congenital heart disease, and deep vein thrombosis and pulmonary embolism. Other examples include heart attack and stroke. Improving the subject's immune function may help reduce the risk of these diseases or treat these diseases.

[0120] In some embodiments, the subject has a cerebrovascular disease, e.g., stroke (e.g., ischemic stroke, hemorrhagic stroke, transient ischemic attack (TIA)), carotid artery stenosis, cerebral aneurysm, vascular malformation, moyamoya disease, venous hemangioma, and venous malformation of Galen (VGM).

[0121] Further examples of cardiovascular diseases and conditions include, without limitation, dilated cardiomyopathy, chronic myocarditis, acute rheumatic fever, aortic regurgitation, mitral regurgitation, atherosclerosis, arteriosclerosis, acute coronary syndromes, chronic coronary syndromes, coronary artery disease, acute myocardial infarction, unstable angina, non-Q wave myocardial infarction, congestive heart failure, left ventricular dysfunction, peripheral arterial disease, critical limb ischemia, acute stroke, acute cerebral ischemia, ischemic stroke, non-ischemic heart failure, pulmonary arterial hypertension, and chronic thromboembolic pulmonary hypertension (CTEPH).

[0122] Autoimmune Diseases and Disorders In some embodiments, the subject has an autoimmune disease or disorder. In some embodiments, the autoimmune disease or condition to be treated includes Aicardi-Goutieres syndrome, alopecia areata, ankylosing spondylitis, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus (type 1), celiac disease, autoimmune juvenile idiopathic arthritis, Crohn's disease, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, hemophagocytic lymphohistiocytosis, idiopathic thrombocytopenic purpura, myasthenia gravis, autoimmune myocarditis, Examples of the conditions include one or more of multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, peripheral neuropathy, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis including juvenile rheumatoid arthritis, sarcoidosis, scleroderma / systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus, autoimmune thyroiditis, Hashimoto's thyroiditis, autoimmune uveitis, ulcerative colitis, vitiligo, and granulomatosis with polyangiitis (Wegener's granulomatosis).

[0123] Rheumatoid arthritis (RA) is a long-term autoimmune disorder that primarily affects the joints. Rheumatoid arthritis typically results in hot, swollen, painful joints. The pain and stiffness often worsen after rest. Most commonly, the wrists and hands are affected, with the same joints on both sides of the body typically affected. The disease may also affect other parts of the body. The cause of rheumatoid arthritis is unknown, but is thought to involve a combination of genetic and environmental factors. The underlying mechanism involves the body's immune system attacking the joints. This leads to inflammation and thickening of the joint capsule. The goal of treatment is to decrease pain, reduce inflammation, and improve the individual's overall function. Symptomatic analgesics, steroids, and NSAIDs are frequently used. A group of drugs called disease-modifying antirheumatic drugs (DMARDs), such as hydroxychloroquine and methotrexate, may be used in an attempt to slow the progression of the disease.

[0124] Osteoarthritis (OA) is a type of joint disease resulting from the destruction of articular cartilage and the underlying bone. The most common symptoms are joint pain and stiffness. Initially, symptoms may occur only after exercise, but over time, they may become chronic. Other symptoms may include joint swelling, reduced range of motion, and weakness or numbness in the arms and legs if the back is affected. Causes include previous joint injury, abnormal development of joints or limbs, and genetic factors. People who are overweight, have different lengths of legs, or have a job that causes high levels of joint stress are at higher risk. Osteoarthritis is thought to be caused by mechanical stress on the joints and a low-grade inflammatory process. Treatments include exercise, efforts to reduce joint stress, supports, and painkillers.

[0125] Multiple sclerosis (MS) is a demyelinating disease that damages the insulating covering of nerve cells in the brain and spinal cord. This damage disrupts the ability of parts of the nervous system to communicate, resulting in a variety of signs and symptoms, including physical, mental, and in some cases, psychiatric problems. Specific symptoms may include double vision, blindness in one eye, muscle weakness, problems with sensation, or problems with coordination. Although the cause is unknown, the underlying mechanism is thought to be either destruction by the immune system or a failure of myelin-producing cells. There is no known cure for multiple sclerosis. Treatment attempts to improve function after an attack and prevent new attacks.

[0126] Asthma is a common long-term inflammatory disease of the airways of the lungs. It is characterized by variable and recurrent symptoms, reversible airflow obstruction, and bronchospasm. Symptoms include episodes of wheezing, coughing, chest tightness, and shortness of breath. Asthma is thought to be caused by a combination of genetic and environmental factors. Environmental factors include exposure to air pollution and allergens. Asthma is classified according to the frequency of symptoms, forced expiratory volume in 1 second (FEV1), and peak expiratory flow rate. Asthma can also be classified as atopic or non-atopic, with atopic referring to a tendency to develop type 1 hypersensitivity reactions. There is no cure for asthma. Symptoms can be prevented by avoiding trigger substances, such as allergens and irritants, and by using inhaled corticosteroids. If asthma symptoms remain uncontrolled, long-acting beta agonists (LABAs) or anti-leukotriene agents can be used in addition to inhaled corticosteroids. Treatment for rapidly worsening symptoms usually involves inhaled short-acting beta-2 agonists, such as salbutamol, and corticosteroids taken monthly. In very severe cases, intravenous corticosteroids, magnesium sulfate, and hospitalization may be required.

[0127] Chronic obstructive pulmonary disease (COPD) is a type of obstructive lung disease characterized by poor airflow over a long period of time. COPD can have two major conditions: emphysema and chronic bronchitis. In emphysema, the walls between the numerous air sacs are damaged. As a result, the air sacs lose their shape and become flat. This damage can also destroy the walls of the air sacs, leading to fewer large air sacs instead of many small ones. When this happens, the amount of gas exchanged in the lungs decreases. In chronic bronchitis, the lining of the airways remains irritated and inflamed on a regular basis, which causes the lining to swell. Numerous thickened mucous membranes form in the airways, making it difficult to breathe. There is no known cure for COPD, but the symptoms can be treated and its progression can be slowed.

[0128] Neurodegenerative Diseases and Disorders In some embodiments, the subject being treated has a neurodegenerative disease or disorder, examples of which include, but are not limited to, multiple sclerosis, Alzheimer's disease, Parkinson's disease, vascular dementia, Huntington's disease, Creutzfeldt-Jakob disease, Down's syndrome, depression, autism spectrum disorder, ADHD / ADD, amyotrophic lateral sclerosis, and neuromyelitis optica spectrum disorder.

[0129] Liver, kidney, and other diseases and conditions In some embodiments, the subject has liver disease or disorder.The non-limiting examples of liver disease or disorder include hepatitis, liver cirrhosis, liver cancer, liver failure, ascites, gallstones, hemochromatosis, primary sclerosing cholangitis and primary biliary cirrhosis.The examples also include non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).

[0130] In some embodiments, the subject has a renal disease or disorder, such as, but not limited to, diabetic nephropathy, nephrotic syndrome, glomerulonephritis, chronic renal disease, and end-stage renal disease.

[0131] In some embodiments, the subject has a condition associated with transplantation. In one example, the subject has kidney, heart, liver, or pancreas transplant rejection. In another example, the subject has graft-versus-host disease (GVHD). In another example, the subject has transfusion-associated rejection.

[0132] Further diseases and conditions that may be suitably treated with the present technology include, without limitation, sarcoidosis, periodontitis, acute pancreatitis, intrauterine infection, burns, aging, macrophage activation syndrome, hip fracture, pneumoconiosis, acute exacerbation of chronic liver failure, systemic inflammatory response syndrome, obesity, and diabetes mellitus. EXAMPLES

[0133] The following examples are included to demonstrate specific embodiments of the present disclosure. Those skilled in the art will recognize that the techniques disclosed in the examples are in accordance with the techniques of the present invention and work well in implementing the present disclosure, and therefore can be considered to constitute specific modes for its implementation. However, those skilled in the art will recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the present disclosure and still obtain the same or similar results.

[0134] Example 1: Identification of cis-regulatory elements that affect exon skipping This example examines the genomic sequence of human 6-pyruvoyltetrahydropterin synthase (PTS) for cis-regulatory elements involved in the regulation of exon 3 skipping.

[0135] The human genomic sequence of the PTS can be found in GenBank ID: 5805 (NG_008743.1: nucleotides 5001 to 12609), which is reproduced in Table 1 below.

[0136] TIFF2024516738000001.tif228170TIFF2024516738000002.tif232170TIFF2024516738000003.tif114170

[0137] The sequences were inspected to identify cis-regulatory sequence element motifs targeted by protein factors involved in splicing, in particular splicing factors, arginine / serine-rich proteins, e.g., SRSF1-SRSF12 (see Table 2).

[0138] TIFF2024516738000004.tif64170

[0139] The search was focused within two introns surrounding exon 3 that is skipped in human monocytes and macrophages. The most significant findings are summarized in Table 3.

[0140] TIFF2024516738000005.tif93170

[0141] TIFF2024516738000006.tif92170

[0142] It is assumed that each of these identified cis-regulatory sequence elements may play a role in regulating exon 3 skipping in PTS genes. In particular, the only SRSF3 binding site in intron 2, CTCTTCC (2895-2901 in SEQ ID NO: 1), may play an important role in SRSF3-mediated exon 3 skipping.

[0143] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0144] The invention described herein can suitably be practiced in the absence of any element(s), limitation(s), not specifically disclosed herein. That is, for example, terms such as "comprising," "including," "containing," etc., should be read broadly and without particular limitation. Furthermore, the terms and expressions employed herein are used as terms of description, not of limitation, and it is not intended that such terms and expressions be used as the exclusion of any equivalents of the features shown and described or portions thereof, but rather that various modifications are possible within the scope of the invention as claimed.

[0145] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated herein by reference in their entirety to the same extent as if each was individually incorporated herein by reference. In case of conflict, the present specification, including definitions, will control.

Claims

1. An ex vivo method for inhibiting or correcting exon 3 skipping of 6 - pyruvoyltetrahydropterin synthase (PTS) mRNA or promoting recognition of said exon 3 in cells, comprising editing the genomic sequence of said PTS of the pre - mRNA or contacting said cells with an agent that inhibits exon skipping or promotes exon recognition or inclusion.

2. A composition for regulating the immune system in a mammalian subject in need thereof, comprising an editing system that edits the genomic sequence or pre - mRNA of 6 - pyruvoyltetrahydropterin synthase (PTS) in monocytes, macrophages, dendritic cells, or their progenitor cells in said mammalian subject, wherein said editing system inhibits exon 3 skipping of said PTS mRNA during splicing.

3. The composition according to claim 2, wherein said editing is to modify the recognition site of a splicing factor.

4. The composition according to claim 3, wherein said recognition site is located within intron 2 or intron 3.

5. The composition according to claim 3, wherein said splicing factor is selected from the group consisting of SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF8, SRSF9, SRSF10, SRSF11, and SRSF12.

6. The composition according to claim 5, wherein said recognition site is selected from Table 3.

7. The composition according to claim 5, wherein said splicing factor is SRSF3.

8. The composition according to claim 7, wherein said recognition site is CTCTTCC, corresponding to nucleotides 2895 - 2901 of SEQ ID NO:

1.

9. The composition according to claim 5, wherein the splicing factor is SRSF1.

10. The composition according to claim 9, wherein the recognition site is selected from the group consisting of TGGGTGG C (2833-2839 of SEQ ID NO: 1), TGATGCT (3541-3547 of SEQ ID NO: 1), TGGGGA (3927-3933 of SEQ ID NO: 1), TGGGTGC (4105-4111 of SEQ ID NO: 1), and TGA AGGC (4143-4149 of SEQ ID NO: 1).

11. The composition according to claim 3, wherein the editing of the editing system includes addition, deletion, and / or substitution of at least one, two, three, or four of the nucleotides at the recognition site.

12. The composition according to claim 11, wherein the edited recognition site cannot be bound by an individual splicing factor.

13. The composition according to claim 3, wherein the editing system is introduced into the cell and the editing system targets a recognition site.

14. The composition according to claim 13, wherein the editing system is selected from a CRISPR / Cas (clustered regularly interspaced short palindromic repeat / CRISPR-associated protein) system, a gene editor comprising a CRISPR / Cas system and a cytosine deaminase, a meganuclease, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN)-based gene editing system.

15. The composition according to claim 2, for treating or preventing the risk of developing an infectious disease or a pathological condition, or cancer, or an infectious disease or a pathological condition, cancer, a cardiovascular disease, a cerebrovascular disease, an autoimmune disease, a kidney disease, a transplantation-related pathological condition, or a liver disorder.

16. A composition for regulating the immune system in a mammalian subject in need thereof, the composition comprising mammalian monocytes, macrophages, dendritic cells, or progenitor cells thereof, wherein the 6-pyruvoyltetrahydropterin synthase (PTS) genomic sequence is edited to inhibit exon 3 skipping of PTS mRNA during splicing or to promote exon 3 recognition. **Claim 17** The composition according to claim 16, wherein the monocytes, macrophages, dendritic cells, or progenitor cells thereof are recovered from the mammalian subject and are edited in vitro or ex vivo. **Claim 18** The composition according to claim 16, wherein the monocytes or macrophages are dedifferentiated prior to administration. **Claim 19** The composition according to claim 16, wherein the editing modifies the recognition site of a splicing factor. **Claim 20** The composition according to claim 19, wherein the splicing factor is selected from the group consisting of SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF8, SRSF9, SRSF10, SRSF11, and SRSF12. **Claim 21** The composition according to claim 20, wherein the recognition site is CTCTTCC and corresponds to nucleotides 2895-2901 of SEQ ID NO:

1. **Claim 22** A composition for regulating the immune system in a mammalian subject in need thereof, the composition comprising an agent that inhibits exon 3 skipping or promotes exon 3 recognition in the 6-pyruvoyltetrahydropterin synthase (PTS) pre-mRNA of monocytes, macrophages, dendritic cells, or progenitor cells of the subject. **Claim 23** The composition according to claim 22, wherein the agent inhibits the biological activity of a splicing factor selected from the group consisting of SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF8, SRSF9, SRSF10, SRSF11, and SRSF12.

24. The composition according to claim 23, wherein the splicing factor is SRSF3.

25. The composition according to claim 23, wherein the agent is an antisense oligonucleotide or RNA, siRNA, or shRNA that inhibits the expression of the splicing factor.

26. The composition according to claim 23, wherein the agent is an antibody that has specificity for the splicing factor.

27. The composition according to claim 22, wherein the agent is a small molecule agent selected from the group consisting of kinetin, epigallocatechin gallate (EGCG), genistein, daidzein, cardiac glycosides, and abnormal splicing rectifiers (RECTAS).

28. The composition according to claim 27, wherein the cardiac glycoside is selected from the group consisting of digoxin, digitonin, digoxigenin, digitoxigenin, acetyl digitoxin, bufalin, ouabagenin, and ouabain.

29. The composition according to claim 22, wherein the agent is U1snRNA that recognizes exon 3 in the PTS pre-mRNA.

30. A composition for regulating the immune system in a mammalian subject in need thereof, comprising a nonsense suppressor, wherein the nonsense suppressor is administered to a tissue of the subject that produces or circulates monocytes, macrophages, dendritic cells, or progenitor cells thereof.

31. The nonsense suppressor of the composition according to claim 30 is selected from the group consisting of a stop codon readthrough agent, a suppressor tRNA, a stop codon pseudouridylation agent, and a nonsense-mediated mRNA decay (NMD) inhibitor.

32. The nonsense suppressor of the composition according to claim 30 is selected from the group consisting of ataluren, aminoglycoside, gentamicin, amikacin, negamycin, spiramycin, josamycin, tylosin, and anlequinox.

33. The tissue of the composition according to claim 30 is bone marrow.

34. The administration of the composition according to claim 22 targets the monocytes, macrophages, dendritic cells, or their progenitor cells.

35. The composition according to claim 22 for treating or preventing an infectious disease or condition, or cancer, or the risk of developing an infectious disease or condition, or cancer.

36. The subject of the composition according to claim 2 has increased immune activity compared to a healthy individual.

37. The increased immune activity of the composition according to claim 36 is measured by a biomarker from a sample selected from the group consisting of serum, urine, cerebrospinal fluid, synovial fluid, saliva, ascites, bile, pancreatic juice, and gastric juice.

38. The biomarker of the composition according to claim 36 is selected from the group consisting of neopterin, nitric oxide, C-reactive protein, interferon-gamma, interferon-alpha, interferon-beta, and procalcitonin.

39. The biomarker includes neopterin, and the subject of the composition according to claim 38 has a serum neopterin concentration greater than 10 nmol / L, 20 nmol / L, 30 nmol / L, 50 nmol / L, 100 nmol / L, or 200 nmol / L.

40. The biomarker contains nitric oxide, and in the subject, the serum nitric oxide concentration is less than 0.1 μmol / L, 0.2 μmol / L, 0.5 μmol / L, 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 10 μmol / L, 15 μmol / L, 20 μmol / L, 30 μmol / L, 40 μmol / L, 50 μmol / L, 60 μmol / L, 70 μmol / L, 80 μmol / L, 90 μmol / L, or 100 μmol / L. The composition according to claim 38.

41. The composition according to claim 2, wherein the biomarker is selected from the group consisting of neopterin, nitric oxide, C-reactive protein, interferon-gamma, interferon-alpha, interferon-beta, and procalcitonin, and the regulation is monitored.