CA3020243A1 - Exosomes carrying icp4-targeting mirna, pharmaceutical compositions and methods for treating hsv infection - Google Patents
Exosomes carrying icp4-targeting mirna, pharmaceutical compositions and methods for treating hsv infection Download PDFInfo
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Abstract
Disclosed is an exosome comprising an inhibitory amount of ICP4-targeting miRNA, wherein the ICP4-targeting miRNA has a seed sequence binding to mRNA of ICP-4; and an exo-motif operably linked to the seed sequence of the ICP4-targeting miRNA to enhance the packaging of the ICP4-targeting miRNA into the exosome. A pharmaceutical composition comprising the exosome and methods for treating HSV infection using the exosome are also disclosed.
Description
EXOSOMES CARRYING ICP4-TARGETING miRNA, PHARMACEUTICAL
COMPOSITIONS AND METHODS FOR TREATING HSV INFECTION
Technical Field The instant invention is related to a vehicle for delivery of miRNA to a cell, and in particular, to an exosome carrying ICP-4 targeting miRNA and a method for treating an HSV
infected cell. The instant invention is also related to a pharmaceutical composition comprising the ICP-4 targeting miRNA or the exosome carrying the ICP-4 targeting miRNA, and methods of using the pharmaceutical composition for treatment of HSV infection in a subject.
Background Virus infectious agents have been and continue to be significant pathogens in humans and animals including high density farming animals such as pigs and poultry. Herpes simplex viruses (HSV) infection-related diseases are a global health problem due to the high infection rates of the general population. Clinical manifestations include small, painful, vesicles affecting the skin, mouth, lips, eyes, or genitalia, and systemic symptoms such as fever and malaise. HSV
persists in sensory and autonomic neural ganglions for the life of the host and periodically reactivates. Clinical recurrences are triggered by several stimuli, such as stress, menstrual periods, fever or illness, sun exposure or sunburn.
Herpes viral infections include herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections.
HSV-2 is the cause of most genital herpes and is generally sexually transmitted. In contrast, HSV-1 is usually transmitted via nonsexual contacts. Preexisting HSV-1 antibodies can alleviate clinical manifestations of subsequently acquired HSV-2. Furthermore, HSV-1 has become an important cause of genital herpes in some developed countries. Additional manifestations of HSV viral infection may include encephalitis and keratitis.
Although proposals have been made for a cure for the above diseases, an unmet need continues to exist for methods of preventing or treating a viral infection of a host.
Summary In one aspect, an exosome carrying a miRNA targeting ICP4 is disclosed in which an exosome-packaging-associated motif (also referred to as "exo-motif' hereinafter) is operably linked to the miRNA targeting I0P4. In one embodiment, the exosome comprises an inhibitory amount of ICP4-targeting miRNA, wherein the ICP4-targeting miRNA has a seed sequence binding to mRNA of ICP-4; and an exo-motif operably linked to the seed sequence of the I0P4-targeting miRNA to enhance the packaging of the ICP4-targeting miRNA into the exosome. In some embodiments, the exo-motif is located downstream and covalently linked to the seed sequence of the CP4-targeting miRNA. In some embodiments, the exo-motif is obtained by mutation of one or more nucleic acids of the ICP-4 targeting miRNA except for the seed sequence. In some embodiments, the exo-motif is a two-fold motif generated through combination of two single exo-motifs. In some embodiments, the I0P4-targeting miRNA and the exo-motif, when operably linked, share at least one nucleotide.
Another aspect of the invention is related to a pharmaceutical composition comprising an exosome carrying a miRNA targeting CP4, and a pharmaceutically acceptable carrier. The exosome comprises an exosome-packaging-associated motif operably linked to the miRNA
targeting I0P4.
A further aspect of the invention is related to a cell comprising an exosome of the invention. The cell is preferably infected with HSV-1, HSV-2, or both, In one embodiment, the cell is a mammal cell.
A further aspect of the invention is related to a method for treating HSV
infection in a subject, the method comprising administering to the subject a pharmaceutically effective amount of the exosome or the pharmaceutical composition of the invention. In some embodiments, the HSV
infection includes diseases or conditions caused by HSV-1 or HSV-2 infection, including any of oral herpes, herpetic gingivo-stomatitis, cold sores, herpes pharyngitis, herpes keratitis, herpes
COMPOSITIONS AND METHODS FOR TREATING HSV INFECTION
Technical Field The instant invention is related to a vehicle for delivery of miRNA to a cell, and in particular, to an exosome carrying ICP-4 targeting miRNA and a method for treating an HSV
infected cell. The instant invention is also related to a pharmaceutical composition comprising the ICP-4 targeting miRNA or the exosome carrying the ICP-4 targeting miRNA, and methods of using the pharmaceutical composition for treatment of HSV infection in a subject.
Background Virus infectious agents have been and continue to be significant pathogens in humans and animals including high density farming animals such as pigs and poultry. Herpes simplex viruses (HSV) infection-related diseases are a global health problem due to the high infection rates of the general population. Clinical manifestations include small, painful, vesicles affecting the skin, mouth, lips, eyes, or genitalia, and systemic symptoms such as fever and malaise. HSV
persists in sensory and autonomic neural ganglions for the life of the host and periodically reactivates. Clinical recurrences are triggered by several stimuli, such as stress, menstrual periods, fever or illness, sun exposure or sunburn.
Herpes viral infections include herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections.
HSV-2 is the cause of most genital herpes and is generally sexually transmitted. In contrast, HSV-1 is usually transmitted via nonsexual contacts. Preexisting HSV-1 antibodies can alleviate clinical manifestations of subsequently acquired HSV-2. Furthermore, HSV-1 has become an important cause of genital herpes in some developed countries. Additional manifestations of HSV viral infection may include encephalitis and keratitis.
Although proposals have been made for a cure for the above diseases, an unmet need continues to exist for methods of preventing or treating a viral infection of a host.
Summary In one aspect, an exosome carrying a miRNA targeting ICP4 is disclosed in which an exosome-packaging-associated motif (also referred to as "exo-motif' hereinafter) is operably linked to the miRNA targeting I0P4. In one embodiment, the exosome comprises an inhibitory amount of ICP4-targeting miRNA, wherein the ICP4-targeting miRNA has a seed sequence binding to mRNA of ICP-4; and an exo-motif operably linked to the seed sequence of the I0P4-targeting miRNA to enhance the packaging of the ICP4-targeting miRNA into the exosome. In some embodiments, the exo-motif is located downstream and covalently linked to the seed sequence of the CP4-targeting miRNA. In some embodiments, the exo-motif is obtained by mutation of one or more nucleic acids of the ICP-4 targeting miRNA except for the seed sequence. In some embodiments, the exo-motif is a two-fold motif generated through combination of two single exo-motifs. In some embodiments, the I0P4-targeting miRNA and the exo-motif, when operably linked, share at least one nucleotide.
Another aspect of the invention is related to a pharmaceutical composition comprising an exosome carrying a miRNA targeting CP4, and a pharmaceutically acceptable carrier. The exosome comprises an exosome-packaging-associated motif operably linked to the miRNA
targeting I0P4.
A further aspect of the invention is related to a cell comprising an exosome of the invention. The cell is preferably infected with HSV-1, HSV-2, or both, In one embodiment, the cell is a mammal cell.
A further aspect of the invention is related to a method for treating HSV
infection in a subject, the method comprising administering to the subject a pharmaceutically effective amount of the exosome or the pharmaceutical composition of the invention. In some embodiments, the HSV
infection includes diseases or conditions caused by HSV-1 or HSV-2 infection, including any of oral herpes, herpetic gingivo-stomatitis, cold sores, herpes pharyngitis, herpes keratitis, herpes
2 whitlow, herpes gladiatorum, eczema herpeticum, neonatal herpes, genital herpes, herpetic proctitis, HSV encephalitis, HSV meningitis, and disseminated herpes simplex infection.
Other aspects of the invention will be readily available from reading the description below.
Brief Description of Drawings Figure 1. Derivation of stable cell lines expressing miRNAs targeting CP4.
Panel A. Schematic diagram of the plasmid encoding miRNAs targeting I0P4. The figure shows the nucleotide sequences of miR401, miR402, and miR403. The nucleotides highlighted in red indicate exosome-packaging-associated motifs (EXO-motifs). Panel B. Down-regulation of I0P4 by designed miRNA.
12-well plate of HEp-2 were co-transfected with 0.5 pg of plasmids expressing miR401, miR402, miR403 or non-target miRNA (NT) and 0.2 pg of plasmid encoding a His-tagged ICP4 (pICP4-his).
The cells were harvested after 48 h. Accumulations of I0P4, GFP and GAPDH were measured as described in Material and Methods. Panel C. Accumulation of GFP in stably transformed miR401 cells. The fluorescence associated with accumulation of GFP in HEp-2 cells stably transformed with miR401 (HEp-miR401) were captured with the aid of a Leica inverted fluorescence microscope by a computer-based imaging system as described in Material and Methods. Panel D.
Accumulation of miR401 in HEp-miR401 and parental HEp-2 cells. miR401 were quantified and normalized with respect to 18s rRNA.
Figure 2. Accumulation of viral proteins in stably transformed HEp-miR401 cells infected with HSV-1(F). 12-well-plate of HEp-miR401 or HEp-2 cells were mock infected or exposed to 0.1, 1, or 10 PFU of HSV-1(F) per cell. The cells were harvested at indicated hours post infection (hpi). The proteins were electrophoretically separated in 10% denaturing gels and reacted with antibodies against I0P27, CP8, VP16, GFP, or GAPDH. The protein bands were scanned with the aid of ImageJ scanner. The optical densities of the bands were normalized with respect to the optical density of corresponding bands generated from HEp-2 cells at 24 h (1.0, 10 PFU/cell) or 48 h (0.1 PFU/cell) after infection.
Other aspects of the invention will be readily available from reading the description below.
Brief Description of Drawings Figure 1. Derivation of stable cell lines expressing miRNAs targeting CP4.
Panel A. Schematic diagram of the plasmid encoding miRNAs targeting I0P4. The figure shows the nucleotide sequences of miR401, miR402, and miR403. The nucleotides highlighted in red indicate exosome-packaging-associated motifs (EXO-motifs). Panel B. Down-regulation of I0P4 by designed miRNA.
12-well plate of HEp-2 were co-transfected with 0.5 pg of plasmids expressing miR401, miR402, miR403 or non-target miRNA (NT) and 0.2 pg of plasmid encoding a His-tagged ICP4 (pICP4-his).
The cells were harvested after 48 h. Accumulations of I0P4, GFP and GAPDH were measured as described in Material and Methods. Panel C. Accumulation of GFP in stably transformed miR401 cells. The fluorescence associated with accumulation of GFP in HEp-2 cells stably transformed with miR401 (HEp-miR401) were captured with the aid of a Leica inverted fluorescence microscope by a computer-based imaging system as described in Material and Methods. Panel D.
Accumulation of miR401 in HEp-miR401 and parental HEp-2 cells. miR401 were quantified and normalized with respect to 18s rRNA.
Figure 2. Accumulation of viral proteins in stably transformed HEp-miR401 cells infected with HSV-1(F). 12-well-plate of HEp-miR401 or HEp-2 cells were mock infected or exposed to 0.1, 1, or 10 PFU of HSV-1(F) per cell. The cells were harvested at indicated hours post infection (hpi). The proteins were electrophoretically separated in 10% denaturing gels and reacted with antibodies against I0P27, CP8, VP16, GFP, or GAPDH. The protein bands were scanned with the aid of ImageJ scanner. The optical densities of the bands were normalized with respect to the optical density of corresponding bands generated from HEp-2 cells at 24 h (1.0, 10 PFU/cell) or 48 h (0.1 PFU/cell) after infection.
3 Figure 3. Virus yields recovered from infected HEp-miR401 cells. HEp-miR401 or parental HEp-2 cells were exposed to 0.01 or 0.1 PFU of HSV-1(F) per cell. After 2 h the inoculum was replaced with fresh medium. The virus progeny was harvested at times shown and titered in Vero cells.
Figure 4. Characterization of exosomes purified from extracellular medium harvested from HEp-2 or HEp-miR401 cell cultures. Panel A. Exosomes purified from HEp-miR401 or parental HEp-2 cells conform with known size of exosomal particles. Cultured HEp-2 or HEp-miR401 cells each containing 1x107cells were rinsed with PBS and incubated in serum-free medium.
After 18 h exosomes were isolated from collected cells by using Total Exosome Isolation ReagentTM (Thermo Fisher Cat. 4478359). Particle size distribution and number of isolated exosomes were determined by lzon's qNano technology as described in Materials and Methods. Panel B.
Characterization of purified exosomes with respect to the presence of exosome associated proteins.
Purified exosomes were lysed with RIPA lysis buffer, and 45 pg of exosome proteins were subjected to electrophoresis in denaturing gels and reacted with antibodies to exosome marker proteins CD9, Annexin V or Flotillin-1 respectively.
Figure 5. Analysis of miR401 from purified exosomes. Panel A and B.
Quantification of full length of C09 protein (28 kDa) from band intensities of a CD9 protein fragment (11 kDa). Panel A.
Immunoblot of standard dilution of CD9 protein fragment purchased from Sino Biological Inc. Band intensities were quantified using ImageJ. Panel B. Standard curve relating band intensities of the 11 kDa CD9 fragment in panel A to full size 28kDa CD9 protein. Panel C and Panel D.
Quantitative analysis of purified exosomes with respect to CD9 content.
Exosomes in 50 pl amounts purified from either extracellular medium of HEp-miR401 or HEp-2 cell cultures were lysed with RIPA lysis buffer and loaded in triplicate onto a 12% denaturing gel. The electrophoretically separated bands were reacted with CD9 antibody and band intensities were quantified using ImageJ (Panel C). Panel D shows the calculated amounts of CD9 protein in 50 pl of purified exosomes on the basis of data presented in Panel B. The amounts of recovered CD9 were 283 ng from HEp-miR401 vs 251 ng from HEp-2 cells. Panel E. Quantitative analysis of miR401 from purified exosomes. miR401 were extracted from exosomes containing 1 ng of equivalent of CD9.
Figure 4. Characterization of exosomes purified from extracellular medium harvested from HEp-2 or HEp-miR401 cell cultures. Panel A. Exosomes purified from HEp-miR401 or parental HEp-2 cells conform with known size of exosomal particles. Cultured HEp-2 or HEp-miR401 cells each containing 1x107cells were rinsed with PBS and incubated in serum-free medium.
After 18 h exosomes were isolated from collected cells by using Total Exosome Isolation ReagentTM (Thermo Fisher Cat. 4478359). Particle size distribution and number of isolated exosomes were determined by lzon's qNano technology as described in Materials and Methods. Panel B.
Characterization of purified exosomes with respect to the presence of exosome associated proteins.
Purified exosomes were lysed with RIPA lysis buffer, and 45 pg of exosome proteins were subjected to electrophoresis in denaturing gels and reacted with antibodies to exosome marker proteins CD9, Annexin V or Flotillin-1 respectively.
Figure 5. Analysis of miR401 from purified exosomes. Panel A and B.
Quantification of full length of C09 protein (28 kDa) from band intensities of a CD9 protein fragment (11 kDa). Panel A.
Immunoblot of standard dilution of CD9 protein fragment purchased from Sino Biological Inc. Band intensities were quantified using ImageJ. Panel B. Standard curve relating band intensities of the 11 kDa CD9 fragment in panel A to full size 28kDa CD9 protein. Panel C and Panel D.
Quantitative analysis of purified exosomes with respect to CD9 content.
Exosomes in 50 pl amounts purified from either extracellular medium of HEp-miR401 or HEp-2 cell cultures were lysed with RIPA lysis buffer and loaded in triplicate onto a 12% denaturing gel. The electrophoretically separated bands were reacted with CD9 antibody and band intensities were quantified using ImageJ (Panel C). Panel D shows the calculated amounts of CD9 protein in 50 pl of purified exosomes on the basis of data presented in Panel B. The amounts of recovered CD9 were 283 ng from HEp-miR401 vs 251 ng from HEp-2 cells. Panel E. Quantitative analysis of miR401 from purified exosomes. miR401 were extracted from exosomes containing 1 ng of equivalent of CD9.
4 Figure 6. Inhibition of viral gene expression and replication by exosome-mediated miR401. Panel A. Accumulation of selected viral proteins in cells exposed to exosomes containing miR401.
Cultures containing 2.5x105 HEp-2 cells were incubated with purified exosomes from HEp-miR401 cells. The miR401 copy numbers were determined as described in the legend of Figure 5.
Exosomes purified from HEp-2 parental cells served as controls. After 12 h of incubation the cells were exposed to 0.1 PFU of HSV-1(F) per cell. After 24 h the cells were harvested and the cells lysates were electrophoretically separated in a 10% denaturing gel, and reacted with indicated antibodies. The band density was normalized with respect to GAPDH and the yields obtained in mock-infected cells. Panel B. Virus yield from cells treated with exosomes containing miR401.
2.5x105HEp-2 cells were exposed to purified exosomes as described in panel A
and then infected with 0.01 PFU per cell. After 2 h, the inoculum was replaced with fresh culture medium. The infected cells were harvested at 48 h post infection (p.i.) and the virus yields were titered in Vero cells.
Detailed Description of the Invention Definitions It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "an exosome," is understood to represent one or more exosomes. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.
"Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, though preferably less than 25% identity, with one of the sequences of the present disclosure, A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 60 %, 65 %, 70 /0, 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 `)/0) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art.
As used herein, the terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of infection.
Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
By "subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on. The subject herein is preferably a human.
As used herein, phrases such as "to a patient in need of treatment" or "a subject in need of treatment" includes subjects, such as mammalian subjects, that would benefit from administration of a composition of the present disclosure used, e.g., for detection, for a diagnostic procedure and/or for treatment.
The present invention employs, among others, antisense oligomer and similar species for use in modulating the function or effect of nucleic acid molecules encoding I0P4. The hybridization of an oligomer of this invention with its target nucleic acid is generally referred to as "antisense".
Consequently, the preferred mechanism believed to be included in the practice of some preferred embodiments of the invention is referred to herein as "antisense inhibition."
Such antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded, or otherwise rendered inoperable. In this regard, it is presently preferred to target specific nucleic acid molecules and their functions for such antisense inhibition.
The functions of RNA to be interfered with can include functions such as translocation of the RNA
to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. One preferred result of such interference with target nucleic acid function is modulation of the expression of I0P4. In the context of the present invention, "modulation" and "modulation of expression" mean decrease (inhibition) in the amount or levels of a nucleic acid molecule encoding the gene, e.g., DNA or RNA. mRNA is often a preferred target nucleic acid.
In the context of this invention, "hybridization" means the pairing of complementary strands of oligomers. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.
An antisense oligomer is specifically hybridizable when binding of the oligomer to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense oligomer to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.
In the present invention the phrase "stringent hybridization conditions" or "stringent conditions"
refers to conditions under which a compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances and in the context of this invention, "stringent conditions"
under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomers and the assays in which they are being investigated.
"Complementary," as used herein, refers to the capacity for precise pairing between two nucleobases of an oligomeric compound. For example, if a nucleobase at a certain position of an oligonucleotide (an oligomeric compound), is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the oligonucleotide and a target nucleic acid, It is understood in the art that the sequence of an antisense oligomer need not be 100%
complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). It is preferred that the antisense compounds of the present invention comprise at least 70%, or at least 75%, or at least 80%, or at least 85% sequence complementarity to a target region within the target nucleic acid, more preferably that they comprise at least 90% sequence complementarity and even more preferably comprise at least 95% or at least 99% sequence complementarity to the target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleobases of the antisense oligomer are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense oligomer which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST
programs known in the art.
In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.
As used herein, the term "microRNA", "miRNA'', or "miR" refers to RNAs that function post-transcriptionally to regulate expression of genes, usually by binding to complementary sequences in the three prime (3') untranslated regions (3' UTRs) of target messenger RNA
(mRNA) transcripts, usually resulting in gene silencing. miRNAs are typically small regulatory RNA
molecules, for example, 21 or 22 nucleotides long. The terms "microRNA", "miRNA", and "miR" are used interchangeably.
As used herein, the term "infection" means the invasion by, multiplication and/or presence of a pathogen in a cell, tissue, or subject. In one embodiment, an infection is an "active' infection, i.e., one in which the pathogen is replicating in a cell, tissue, or subject. Such an infection may be characterized by the spread of the pathogen to other cells, tissues, organs, and/or subjects from the cells, tissues, organs, and/or subjects initially infected by the pathogen. An infection may also be a latent infection, i.e., one in which the pathogen is not replicating. In one embodiment, an infection refers to the pathological state resulting from the presence of the pathogen in a cell, tissue, or subject, or by the invasion of a cell, tissue, or subject by the pathogen.
The term "I0P4" as used herein refers to "infected cell polypeptide/protein 4"
which is one of the immediate-early proteins in HSV infection. Amino acid sequences of I0P4 are available from NCB!
through accession numbers YP_009137149 or YP_009137226. I0P4 has a Gene name of RS1 and NCB' sequence accession numbers NC_001806.2 or NC_001798.2. HSV-1 encodes more than 100 proteins, miRNAs and long non-coding RNAs. I0P4 is an essential regulatory protein expressed immediately after infection and controls its accumulation in infected cells. I0P4 binds as a dimer with high affinity to the consensus DNA sequence ATCGTCNNNNYCGRC (SEQ
ID NO.
39, wherein n is a, c, g, t or u, y is c, u or t, and r is a or g) and with lower affinities to a number of other sequences that are not represented by a unique consensus. DNA binding sites have been thoroughly mapped in the domains of several genes. Relevant to this invention is a site that conforms to the consensus and spans the transcription initiation site of the mRNA encoding ICP4.
Binding of I0P4 to that site inhibits the transcription of its own mRNA. In essence ICP4 tightly regulates the synthesis of its own mRNA most likely to block the synthesis of excessive amounts of ICP4. In the absence of a surplus, the impact of decreased accumulation of ICP4 mRNA should be readily apparent.
miRNAs Targeting ICP4 The terms "miRNAs targeting ICP4", "a miRNA targeting ICP4", and "ICP4-targeting miRNA" which are used interchangeably herein, refer to a small non-coding RNA (microRNA or miRNA) designed to target or specifically bind to mRNA encoding protein ICP4 such that the transcription, translation and, in turn, expression of the I0P4 in a cell is impaired, reduced, or eliminated. As described above, miRNA is not necessarily bind to target mRNA by 100% specificity. It is known that miRNA
has a seed sequence (2-8 nucleotides from 5' end) which determines the specificity of biding to a target mRNA, while the remaining nucleotides are not necessarily exactly complementary to the target mRNA. Therefore, in one embodiment, the miRNA has a seed sequence of any of nucleotide sequences SEQ ID NO. 1 or SEQ ID NO: 2. In some embodiments, the miRNA
targeting ICP4 blocks the expression of I0P4 protein in a cell after delivered to the cell.
Exosomes Carrying miRNA Targeting ICP4 Exosomes are small, relatively uniform-sized vesicles derived from cellular membranes. For example, exosomes may have a diameter of about 30 to about 100 nm. They contain several key proteins (e.g. CD9, 0D63, CD81, 0D82, Annexin, Flotillin, etc) and in addition they package proteins, mRNAs, long non-coding RNAs and miRNAs. Exosomes transport the payload from cell to cell. On entry into recipient cells the exosome payload is released into cytoplasm.
In some embodiments, the miRNA targeting ICP4 is delivered to a cell via an exosome. Therefore, in one embodiment, an exosome carrying any of the I0P4-targeting miRNAs as described above is provided. The present invention uses a fragment of nucleotide sequence, referred to as "exo-motif' herein, to facilitate or enhance the packaging of a miRNA into an exosome. In one embodiment, the exo-motif is selected from any of the sequences identified in Table 1.
Table 1. Sequences of exo-motifs used with miRNAs of the invention Sequence ID Nucleotide Sequence Sequence ID Nucleotide Sequence SEQ ID NO. 5 5'-GGAG-3' SEQ ID NO. 19 5'-CGCG-3' SEQ ID NO. 6 5'-GGAC-3' SEQ ID NO. 20 5'-CGCC-3' SEQ ID NO. 7 5'-GGCG-3' SEQ ID NO. 21 5'-CGGG-3' SEQ ID NO. 8 5'-GGCC-3' SEQ ID NO. 22 5'-CGGC-3' SEQ ID NO. 9 5'-GGGG-3' SEQ ID NO. 23 5'-CCCU-3' SEQ ID NO. 10 5'-GGGC-3' SEQ ID NO. 24 5'-CCCG-3' Sequence ID Nucleotide Sequence Sequence ID Nucleotide Sequence SEQ ID NO. 11 5'-UGAG-3' SEQ ID NO. 25 5'-CCCA-3' SEQ ID NO, 12 5'-UGAC SEQ ID NO. 26 5'-UCCU-3' SEQ ID NO. 13 5'-UGCG SEQ ID NO. 27 5'-UCCG-3' SEQ ID NO. 14 5'-UGCC SEQ ID NO. 28 5'-UCCA-3' SEQ ID NO. 15 5'-UGGG SEQ ID NO. 29 5'-GCCU-3' SEQ ID NO. 16 5'-UGGC SEQ ID NO. 30 5'-GCCG-3' SEQ ID NO. 17 5'-CGAG SEQ ID NO. 31 5'-GCCA-3' SEQ ID NO. 18 5'-CGAC SEQ ID NO. 32 5'-UGACGAC-3' In some embodiments, the exo-motifs are used in combination. For example, two or more exo-motifs as identified in the Table are combined to form a two-fold exo-motif, The motifs can be combined linearly by linking the 5'-end of one exo-motif to the 3'-end of another exo-motif. In this context, when the first nucleotide of the 5'-end of one exo-motif is identical with the last nucleotide of the 3'-end of another exo-motif, one of the identical nucleotides can be designed to be omitted.
For example, "UGCG" (SEQ ID NO. 13) is combined with "GGAC" (SEQ ID NO. 6) to form a two-fold exo-motif "UGCGGAC" (SEQ ID NO. 32), The present invention also contemplates a three-fold or more exo-motif, i.e., an exo-motif consisted of three or more motifs of SEQ
ID NO. 5 to SEQ ID
NO. 31. Therefore, the term "exo-motif" used herein is meant to include nucleotide sequences that are able to enhance or facilitate packaging of miRNA to an exosome, including any of the single exo-motif of SEQ ID NO. 5 to SEQ ID NO. 31 and any two-fold (e.g. SEQ ID NO.
32), three-fold or more fold exo-motifs generated by the combinations of the single motifs.
In the present invention, the exo-motif is operably linked to the seed sequence of the miRNA. The term "operably linked" refers to functional linkage between a regulatory sequence (e.g. the exo-motif) and a nucleic acid sequence (e.g., the seed sequence of the miRNA) resulting in an enhance of, or facilitating the packaging of the miRNA into an exosome. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. Operably linked RNA sequences can be contiguous with each other.
In some embodiments, an exo-motif is located downstream the seed sequence of the miRNA. In some embodiments, an exo-motif is located upstream the seed sequence of the miRNA. In some embodiments, the seed sequence of the miRNA is flanked by exo-motifs. In one embodiment, an exo-motif is operably linked to the seed sequence of the miRNA. In one embodiment, an exo-motif is obtained by mutation of one or more of the nucleotide sequences of the miRNA except for the seed sequence. In one embodiment, the miRNA targeting I0P4 with exo-motif contains a nucleotide sequence of SEQ ID NO. 3 or SEQ ID NO. 4. In one embodiment, the miRNA targeting ICP4 with exo-motif is a nucleotide sequence of SEQ ID NO. 3 or SEQ ID NO. 4.
In some embodiments where an exo-motif is located downstream the seed sequence of the miRNA, the 3' end last nucleotide of the seed sequence and the 5' end first nucleotide of the exo-motif share a same nucleotide, for example, guanine nucleotide "G" or uracil nucleotide "U". For example, SEQ ID NO. 3 shows the sharing of the uracil nucleotide "U" between the exo-motif and the seed sequence. For example, SEQ ID NO. 4 shows the sharing of the guanine nucleotide "G"
between the exo-motif and the seed sequence.
In addition to the seed sequence and the exo-motif, the miRNA also includes additional nucleic acid sequence to facilitate binding to the target region of the mRNA. These additional nucleic acids are normally located downstream the exo-motif with a length of several nucleotides, e.g., 5 to 10 nucleotides. The additional nucleic acid sequences are preferably complementary to the corresponding segment of the target mRNA, but, as described above, not necessarily 100%
complementary.
Methods for transferring miRNAs into an exosome are available in the art, such as by co-transfecting a cell with a miRNA expression vector and a plasmid encoding ICP4, as described in the Example. Isolation, identification or characterization of an exosome is technically feasible in the art. Several proteins, e.g. CD9, 0D63, CD81, 0D82, Annexin, Flotillin, etc can be used as a marker of exosomes. Other methods for packaging miRNAs into exosomes may also be applicable with the present invention.
The exosome of the present invention contains an inhibitory amount of miRNA
targeting I0P4. An inhibitory amount is meant an amount sufficient for inhibiting the expression of the protein I0P4 once the miRNA in question was delivered into a cell infected with HSV virus.
The table below lists the nucleic acid sequences of miRNAs, seed sequences, and miRNA-motif used in the Example of the invention.
Table 2. Nucleic acid sequences of miRNAs, seed sequences, and miRNAs linked with exo-motifs Sequence ID Identity Description Nucleic Acid Sequence SEQ ID NO. 1 miR401 Seed Sequence 5 ' -AGAGGAU- 3 ' SEQ ID NO. 2 miR402 Seed Sequence 5' -ACAGCAG- 3 ' SEQ ID NO. 3 miR401 miRNA + exo-motif 5' -AAGAGGAUGCGGACGACGAGG- 3 ' SEQ ID NO. 4 miR402 miRNA + exo-motif 5' -UACAGCAGCCGCGUGAUCAGG- 3 ' SEQ ID NO. 33 miR403 Seed Sequence 5'-UCAGCAG-3' SEQ ID NO. 34 miR403 miRNA + exo-motif 5' - GUCAGCAGGAAGCCCUUCUGC- 3 ' Methods and Therapies An aspect of the disclosure provides a method for prophylaxis or treatment of HSV viral infection in a subject comprising administering to the subject in need thereof a therapeutically effective amount of an exosome of the invention or a pharmaceutical composition comprising the exosome of the invention. Equally, the disclosure provides the exosome as described above for use in a method for treating or alleviating symptoms involved with viral infection. As described above, the exosome carries a miRNA targeting I0P4 to which an exo-motif is operably linked.
In certain embodiments, the exosome or the pharmaceutical composition is administered parenterally, e.g. intravenously, intramuscularly, percutaneously or intracutaneously.
In some embodiments, it may be desirable to combine an exosome of the invention with other agents effective in the treatment of viral infection. For example, the treatment of a viral infection may be implemented with an exosome of the invention and other anti-virus therapies, such as anti-virus agents.
The therapy may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments where the other agent and exosome are applied separately to the subject, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and exosome would still be able to exert an advantageously combined effect on the subject. In such instances, it is contemplated that one may administer the subject with both modalities within about 12 to 72 hrs of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.
In certain embodiments, the methods of treating a viral infection prevent the progression of the infection and/or the onset of disease caused by the viral infection. Thus, in some embodiments, a method for preventing the progression of a viral infection and/or the onset of disease caused by the viral infection, comprises administering an effective amount of an exosome of the invention or a composition comprising the exosome to a subject in need thereof. In certain embodiments, the methods of treating a viral infection prevent the onset, progression and/or recurrence of a symptom associated with a viral infection. Thus, in some embodiments, a method for preventing a symptom associated with a viral infection in a subject, comprises administering an effective amount of an exosome of the invention or a composition comprising the exosome to a subject in need thereof.
The viral infection that are treated by the present invention is caused by HSV-1 or HSV-2 virus.
Viral infections can cause diseases or conditions including oral herpes, herpetic gingivo-stomatitis, cold sores, herpes pharyngitis, herpes keratitis, herpes whitlow, herpes gladiatorum, eczema herpeticum, neonatal herpes, genital herpes, herpetic proctitis, HSV
encephalitis, HSV meningitis, disseminated herpes simplex infection and etc.
Oral herpes also known as cold sores can be the result of an HSV-1 or an HSV-2 infection.
Because of the association of HSV-2 with sexual transmission, infections in children are usually the result of HSV-1. The main diagnosis is herpetic gingivo-stomatitis, where the typical clear lesions first develop followed by ulcers that have a white appearance. The infection, often initially on the lips, spreads to all parts of the mouth and pharynx. Reactivation from the trigeminal ganglia can result in what are known as cold sores. Herpes pharyngitis is often associated with other viral infections of the upper respiratory tract. The disease is more severe in immunosuppressed people such as AIDS patients. Herpes keratitis is an infection of the eye and is primarily caused by HSV-1.
It can be recurrent and may lead to blindness. It is a leading cause of corneal blindness in the United States. Herpes whitlow affects people who come in manual contact with herpes-infected body secretions and can be caused by either type of HSV. HSV enters the body via small wounds on the hands or wrists. It can also be caused by transfer of HSV-2 from genitals to the hands.
Herpes gladiatorum is often found in wrestlers. It apparently spreads by direct contact from skin lesions on one wrestler to his/her opponent, and usually appears in the head and neck region (which are frequently sites of contact in wrestling holds). Oddly, the lesions are more often on the right side of the body (perhaps because most wrestlers are right handed). It is also seen in other contact sports such as rugby where it is known as scrum pox.
Eczema herpeticum is a pediatric condition found in children with active eczema or preexisting atopic dermatitis. HSV can spread over the skin at the site of eczema lesions.
The virus can spread to other organs such as liver. Neonatal herpes is a severe disease from HSV-2 and is often fatal, although such infection is rare. Infection is especially possible if the mother is shedding virus at the time of delivery. The virus can be contracted either in utero or during birth.
Because the neonate has an underdeveloped immune system, the virus can spread rapidly to many peripheral organs (e.g. lungs and liver) and can infect the central nervous system. Genital herpes and herpetic proctitis are usually the result of an HSV-2 infection with about 10% of cases being the result of HSV-1. Primary infection is often asymptomatic but many painful lesions can develop on the glans or shaft of the penis in men and on the vulva, vagina, cervix and perianal region of women where it may be accompanied by vaginal discharge. A variety of the infections also cause proctitis.
Secondary episodes of genital herpes, a result of reactivation of virus in the sacral ganglion, are frequently less severe (and last a shorter time) than the first episode.
Recurrent episodes seem usually to result from a primary HSV-2 infection. Whether there is an apparent active disease or not, an infected patient remains infectious without overt symptoms. HSV
encephalitis is the result of an HSV-1 infection and is the most common sporadic viral encephalitis. HSV
encephalitis is febrile and may result in damage to one of the temporal lobes, clinically marked by blood in the spinal fluid and seizures. The disease can be fatal and, in the US, fewer than 1000 cases per year are described. HSV meningitis is the result of an HSV-2 infection.
Disseminated herpes simplex infection is the spread of the infection throughout the body. This is a serious and life-threatening complication of HSV in patients with an impaired immune system.
Pharmaceutical Compositions An aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an exosome as described above and a pharmaceutically acceptable carrier. The pharmaceutical composition is useful for prophylaxis or treatment of HSV
infection in a subject. The exosome may be prepared in a suitable pharmaceutically acceptable carrier or excipient. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and 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 will be preferable to include isotonic agents, for example, sugars 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.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intratumoral and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 mL of isotonic NaCI
solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580).
Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The compositions disclosed herein may be formulated in a neutral or salt form.
Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
Examples In principle, controlled diminution of virus production in susceptible cells or accumulation of a specific viral protein can be done in only three ways, i.e. by the use of inhibitors of viral or cellular functions, by mutagenesis of the promoter regulating the expression of a key gene or by delivering to the susceptible cells a miRNA targeting the mRNA encoding the gene product.
In the studies described in this report the desired exosome payload was a miRNA, miRNAs are potent tools that in principle can be used to control the replication of infectious agents.
The objectives were to design miRNAs that can block the replication of herpes simplex virus 1 and which could be delivered to infected cells via exosomes. We designed 3 miRNAs targeting the mRNA encoding ICP4, an essential viral regulatory protein. Of the 3 miRNAs, one miRNA401 effectively blocked I0P4 accumulation and viral replication on transfection into susceptible cells. To facilitate packaging of the miRNA into exosomes we incorporated into the sequence of miRNA401 an exosome packaging motif. miRNA401 was shown to be packaged into exosomes and successfully delivered by exosomes to susceptible cells where it remained stable for at least 72 hrs. Finally, the results show that miRNA401 delivered to cells via exosomes effectively reduced virus yields in a miRNA401 dose dependent fashion. The protocol described in this report can be applied to study viral gene functions without actually deleting or mutagenizing the gene.
Incorporation of RNAs into exosomes is sequence dependent and facilitated by hnRNPA2B1, a component of exosomes. hnRNPA2B1 sorts into exosomes RNAs containing one of two known exosome-packaging motifs (EXO-motifs). A key function of hnRNPA2B1 is to regulate mRNA
trafficking to axons in neural cells that is mediated by binding a 21-nt RNA
sequence called RNA
trafficking sequence (RTS). This sequence contains both of the EXO-motifs.
The Examples show that HSV-1 replication can be reduced in a dose dependent manner by delivering to the cells via exosomes a miRNA designed to target mRNA encoding I0P4, a major regulatory protein of HSV-1.
Materials and Methods Exosome Isolation and Quantification. Cells seeded in 1150 flask for 24 h were extensively rinsed with PBS and then incubated n serum free medium. After 18 h the cell-free extracellular medium was centrifuged at 2000g for 30 min, The supernatant fluid was harvested mixed with recommended dose of Total Exosome Isolation kit reagent (Thermo Fisher Cat No. 4478359), stored overnight at 4 C and then centrifuged for 1 h. The pelleted exosomes were then resuspended in 200 pl of PBS or were lysed in RIPA buffer and then quantified by a BCA assay using the Enhanced BCA
Protein Assay Kit (Beyotime Biotechnology, China) according to manufacturer's instructions.
Exosome protein content was determined by calibration against standard curve, which was prepared by plotting the absorbance at 562 nm versus bovine serum albumin standard concentration.
Exosome Size Analysis. Exosome size distribution analysis was done using the qNano system (Izon, Christchurch, New Zealand). lzon's qNano technology (www.izon.com) was employed to detect extracellular vesicles passing through a nanopore by way of a single-molecule electrophoresis. In practice it enables accurate particle-by-particle characterization of vesicles from 75 to 150 nm in size of exosomes, without averaging the particle sizes. Purified exosomes were diluted to 1:10 in PBS with 0.05% Tween-20, vigorously shaken, and measured by using an NP150 (A45540) nanopore aperture according to the manufacturer's instructions. Data processing and analysis were carried out on the Izon Control Suite software v3.3 (Izon Science).
Quantitative RT-PCR for miRNA. Total RNAs from cells and liquid exosomes were isolated using TRIzol reagent (Thermo Fisher Scientific) and TRIzol LS reagent (Thermo Fisher Scientific) according to the respective manufacturer's instructions. The procedure was performed as described. The miRNA
tested were reverse-transcribed from 50 ng total RNA in duplicate by specific stem-loop primer as described in the TaqMan miRNA reverse transcription kit (Applied Biosystems, Inc.). The expression of miRNA was determined by real-time PCR using TaqMan Universal Master Mix II kit purchased from Applied Biosystems, Inc. miRNA copy number was normalized by comparison with cellular 18s rRNA.
The primers of miR401 were designed according to Chen et al. and synthesized by Ige Biotechnology.
The sequences are as follows: miR401 stem loop primer, 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCTCGT-3' (SEQ ID NO. 35);
forward primer, 5'-TGCTGGCGAAGAGGATGC-3' (SEQ ID NO, 36); reverse primer, 5'-CCAGTGCAGGGTCCGAGGTA-3' (SEQ ID NO. 37); probe, 5'-(6-FAM) CTGGATACGA000TCGTC(MGB)-3 (SEQ ID NO. 38).
Results Design and construction of a miRNA capable of suppressing the replication of HSV-1.
The objective of the first series of experiments was to design a miRNA
targeting I0P4, the major regulatory protein of HSV-1. To this end we have constructed 3 miRNAs designated miR401, mi402 and miR403. The sequence of each of the miRNAs shown in Figure 1 Panel A
contains downstream of miRNA seed sequence additional sequences embodying exosome-packaging-associated motifs (exo-motifs). As illustrated in Panel A, the miRNAs were cloned downstream of an open reading frame encoding EGFP into a miRNA expression vector named "pcDNA6,2-GW/EmGFP-miR-neg control plasmid" as described in Materials and Methods.
To test the miRNAs HEp-2 cells were co-transfected with the miRNA expression vectors (pmiR401, pmiR402, pmiR403) described above and a plasmid encoding ICP4 tagged at the C
terminus with His (pICP4-His). As shown in Figure 1 panel B miR401 was the most effective of the 3 constructs in suppressing the accumulation of CP4. The results show that the accumulation of ICP4 is repressed by miR401 at higher efficiency. miR402 showed moderate effect, whereas the non-targeting (NT) and miR403 plasmids had no effect on accumulation of ICP4 (Figure 1B). Figure 1 panel B shows the expression of EGFP in cells transfected with the construct containing miR401 suggesting that that the plasmid is efficiently transfected into HEp-2 cells.
Therefore, miR401 was selected for further studies.
-The first step in this process was the construction of a stable cell line by transfection of the plasmid into HEp-2 cell under selection of antibiotic of Blasticidin. Single cell clone from EGPF positive cells was selected, amplified and designated HEp-miR401 (Figure 1C), Figure 1D shows expression of mature miR401 by qPCR analysis. As expected the parental HEp-2 cells failed to express detectable expression of EGFP and miR401 (Figure 10 and 1D, respectively).
Accumulation of viral proteins is reduced in HEp-miR401 cells.
In this series of experiments replicate cultures of HEp-2 or HEp-miR401 cells were exposed to 0.1, 1, or 10 PFU of HSV-1(F) per cell. The cultures were harvested at indicated times after infection and then solubilized, subjected to electrophoresis in denaturing gels, reacted with antibodies to ICP27, ICP8 or VP16 representing different kinetic classes of virus replication. GFP is positive indicator of HEp-miR401 stable cell line and GAPDH served as a loading control (Figure 2). The protein bands were scanned with the aid of ImageJ scanner. The optical density of the bands was normalized with respect to the optical density of corresponding bands generated from HEp-2 cells at 24 h (1.0, 10 PFU/cell) 0r48 h (0.1 PFU/cell) after infection. The results show that in cells exposed to 0.1 PFU or 1.0 PFU/cell the amounts of the viral proteins decreased to undetected level. Accumulation of viral proteins in cells exposed to 10 PFU/cell was significantly lower than those detected in the HEp-2 cells (Figure 2).
miR401 inhibits the replication of HSV-1(F) virus.
In this series of experiments replicate cultures of HEp-2 or HEp-miR401 cells were exposed for 0.1 or 0.01 PFU of HSV-1(F) per cell. The cells harvested at the times shown in Figure 3 and viral progeny were titrated on Vero cells. The results shown indicate that the accumulations of virus in HEp-miR401 cells were significantly lower than those obtained from infected HEp-2 cells.
Characterization of miR401 packaged in exosomes.
Several experiments were carried out to test the hypothesis that miR401 produced in HEp-miR401 cells is packaged into exosomes. First, exosomes produced in HEp-2 or HEp-miR401 were purified as described in Materials and Methods. Next, 200 pl of purified exosomes obtained from cultures grown in T150 cells were then measured with respect to size by nanoparticle tracking analysis using lzon's qNano technology. The results (Figure 4A) show that the exosomes purified from HEp-2 cells or HEp-miR401 cells over lapped in size and formed a single band ranging from 75 and 150 nm in size.
Next, the purified exosomes were tested for the presence of proteins associated with exosomes that is 009, Annexin V, and Flotillin-1. In brief, purified exosomes were lysed by RIPA lysis buffer, 45 pg of solubilized exosome protein subjected to electrophoresis in denaturing gels, and reacted with antibodies to 009, AnnexinV, or Flotillin-1 (Figure 4B). The results indicate that the exosome proteins were present in purified exosomes derived from HEp-2 or HEp-m1R401 cell line, To quantify the amounts of miRNA packaged in exosomes we selected CD9 as an indicator of exosome concentration. To quantify CD9 protein, we used an 11 kDa 009 fragment purchased from Sino Biological Inc as a substitute for the full size CD9 protein. In brief, serial dilutions of 009 protein fragment were subjected to electrophoresis in denaturing gels, reacted with antibodies to CD9 (Figure 5A), the bands intensities were quantified using ImageJ to construct a standard curve.
Equivalence of full length of CD9 protein (28 KDa) was calculated accordingly (Figure 5B), Next, 200 pl of exosomes preparations purified from either T150 of HEp-miR401 or T150 of parental HEp-2 cells in three independent experiments. Fifty microliters of preparation from each was subjected to electrophoresis in denaturing gels, reacted with antibodies to 009 (Figure 50) and the bands intensities were quantified using ImageJ. The results showed that on average, 50 pl of purified exosomes equaled 283 ng of 0D9 obtained from HEp-miR401 and 251 ng of 009 obtained from HEp-2 parental cells (Figure 50). We measured the amounts of miR401 packaged in exosomes produced in the two cell lines. The results were that in exosomes produced in HEp-miR401 cell 1 ng of 009 corresponded to 1X106 miR401. miR401 was not detected in exosomes produced in HEp-2 cells (Figure 5E).
Exosome-delivered miR401 inhibits HSV-1 replication in a dose dependent manner.
In this series of experiments, we examined whether miR401 delivered via exosome is effective in diminishing the replication of HSV-1. We report 2 series of experiments.
In the first replicate cultures each containing 2.5x105 HEp-2 cells were exposed for 12 h to purified exosomes produced in HEp-miR401 cells or HEp-2 cells. The amounts of miR401 contained in the exosomes ranged from 360 to 10,000 copies per cell. The culture exposed to exosomes produced in HEp-2 cell was equivalent in amount to the highest concentration of exosomes containing miRNA401. After 12 h of incubation the cells were rinsed, exposed to 0.1 PFU
of HSV-1 (F) per cell and harvested at 24 h. The cell lysate was subjected to an electrophoresis in denaturing gels and reacted with antibodies to I0P4, I0P27, I0P8 or US11. Figure 6A shows that the accumulations of viral proteins ICP27, I0P8 and US11 decreased in HEp-2 exposed to exosomes containing miR401 in a dose dependent manner.
The second series of experiments was a replica of the first except that the infected cells were harvested at 48 h after infection and the virus produced in the infected cells was titered in Vero cells. The results shown in Figure 6B indicate that the accumulation of virus in cells exposed to exosome containing miR401 decreased significantly in a dose-dependent manner as predicted by the effects of miR401 on the accumulation of viral proteins.
The results show that miRNA401 is packaged in exosomes and is readily delivered to susceptible cells. We have also shown that the miRNA delivered via exosomes persists in recipient cells for at least 72 h. We have quantified the packaged miRNA in terms of miRNA copies per ng of CD9, a key protein component of the exosomes. Lastly, we have shown that miRNA401 delivered to cells in exosomes reduces viral yields in a dose dependent manner. The results presented herein also show that miRNAs can be used to define the targeted gene function as well as block viral replication if the targeted gene plays an essential role in viral replication.
In many instances it may obviate the cumbersome process of deleting the targeted gene to define its function.
It should be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the disclosures embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure.
All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.
The disclosures illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising," "including," containing," etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed.
Appendix A lists the sequences as described herein.
APPENDIX A
<110> IMMVIRA CO., LIMITED
<120> EXOSOMES CARRYING ICP4-TARGETING miRNA, PHARMACEUTICAL
COMPOSITIONS AND METHODS FOR TREATING HSV INFECTION
<130> PAT 104740-1 <160> 39 <170> PatentIn version 3.5 <210> 1 <211> 7 <212> RNA
<213> Artificial Sequence <220>
<223> miR401 seed sequence <400> 1 agaggau 7 <210> 2 <211> 7 <212> RNA
<213> Artificial Sequence <220>
<223> miR402 seed sequence <400> 2 acagcag 7 <210> 3 <211> 21 <212> RNA
<213> Artificial Sequence <220>
<223> miRNA401 + exo-motif <400> 3 aagaggaugc ggacgacgag g 21 <210> 4 <211> 21 <212> RNA
<213> Artificial Sequence <220>
<223> miRNA402 + exo-motif <400> 4 uacagcagcc gcgugaucag g 21 <210> 5 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 5 ggag 4 <210> 6 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 6 ggac 4 <210> 7 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 7 ggcg 4 <210> 8 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 8 ggcc 4 <210> 9 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 9 gggg 4 <210> 10 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 10 gggc 4 <210> 11 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 11 ugag 4 <210> 12 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 12 ugac 4 <210> 13 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 13 ugcg 4 <210> 14 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 14 ugcc 4
Cultures containing 2.5x105 HEp-2 cells were incubated with purified exosomes from HEp-miR401 cells. The miR401 copy numbers were determined as described in the legend of Figure 5.
Exosomes purified from HEp-2 parental cells served as controls. After 12 h of incubation the cells were exposed to 0.1 PFU of HSV-1(F) per cell. After 24 h the cells were harvested and the cells lysates were electrophoretically separated in a 10% denaturing gel, and reacted with indicated antibodies. The band density was normalized with respect to GAPDH and the yields obtained in mock-infected cells. Panel B. Virus yield from cells treated with exosomes containing miR401.
2.5x105HEp-2 cells were exposed to purified exosomes as described in panel A
and then infected with 0.01 PFU per cell. After 2 h, the inoculum was replaced with fresh culture medium. The infected cells were harvested at 48 h post infection (p.i.) and the virus yields were titered in Vero cells.
Detailed Description of the Invention Definitions It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "an exosome," is understood to represent one or more exosomes. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.
"Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, though preferably less than 25% identity, with one of the sequences of the present disclosure, A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 60 %, 65 %, 70 /0, 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 `)/0) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art.
As used herein, the terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of infection.
Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
By "subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on. The subject herein is preferably a human.
As used herein, phrases such as "to a patient in need of treatment" or "a subject in need of treatment" includes subjects, such as mammalian subjects, that would benefit from administration of a composition of the present disclosure used, e.g., for detection, for a diagnostic procedure and/or for treatment.
The present invention employs, among others, antisense oligomer and similar species for use in modulating the function or effect of nucleic acid molecules encoding I0P4. The hybridization of an oligomer of this invention with its target nucleic acid is generally referred to as "antisense".
Consequently, the preferred mechanism believed to be included in the practice of some preferred embodiments of the invention is referred to herein as "antisense inhibition."
Such antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded, or otherwise rendered inoperable. In this regard, it is presently preferred to target specific nucleic acid molecules and their functions for such antisense inhibition.
The functions of RNA to be interfered with can include functions such as translocation of the RNA
to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. One preferred result of such interference with target nucleic acid function is modulation of the expression of I0P4. In the context of the present invention, "modulation" and "modulation of expression" mean decrease (inhibition) in the amount or levels of a nucleic acid molecule encoding the gene, e.g., DNA or RNA. mRNA is often a preferred target nucleic acid.
In the context of this invention, "hybridization" means the pairing of complementary strands of oligomers. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.
An antisense oligomer is specifically hybridizable when binding of the oligomer to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense oligomer to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.
In the present invention the phrase "stringent hybridization conditions" or "stringent conditions"
refers to conditions under which a compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances and in the context of this invention, "stringent conditions"
under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomers and the assays in which they are being investigated.
"Complementary," as used herein, refers to the capacity for precise pairing between two nucleobases of an oligomeric compound. For example, if a nucleobase at a certain position of an oligonucleotide (an oligomeric compound), is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the oligonucleotide and a target nucleic acid, It is understood in the art that the sequence of an antisense oligomer need not be 100%
complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). It is preferred that the antisense compounds of the present invention comprise at least 70%, or at least 75%, or at least 80%, or at least 85% sequence complementarity to a target region within the target nucleic acid, more preferably that they comprise at least 90% sequence complementarity and even more preferably comprise at least 95% or at least 99% sequence complementarity to the target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleobases of the antisense oligomer are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense oligomer which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST
programs known in the art.
In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.
As used herein, the term "microRNA", "miRNA'', or "miR" refers to RNAs that function post-transcriptionally to regulate expression of genes, usually by binding to complementary sequences in the three prime (3') untranslated regions (3' UTRs) of target messenger RNA
(mRNA) transcripts, usually resulting in gene silencing. miRNAs are typically small regulatory RNA
molecules, for example, 21 or 22 nucleotides long. The terms "microRNA", "miRNA", and "miR" are used interchangeably.
As used herein, the term "infection" means the invasion by, multiplication and/or presence of a pathogen in a cell, tissue, or subject. In one embodiment, an infection is an "active' infection, i.e., one in which the pathogen is replicating in a cell, tissue, or subject. Such an infection may be characterized by the spread of the pathogen to other cells, tissues, organs, and/or subjects from the cells, tissues, organs, and/or subjects initially infected by the pathogen. An infection may also be a latent infection, i.e., one in which the pathogen is not replicating. In one embodiment, an infection refers to the pathological state resulting from the presence of the pathogen in a cell, tissue, or subject, or by the invasion of a cell, tissue, or subject by the pathogen.
The term "I0P4" as used herein refers to "infected cell polypeptide/protein 4"
which is one of the immediate-early proteins in HSV infection. Amino acid sequences of I0P4 are available from NCB!
through accession numbers YP_009137149 or YP_009137226. I0P4 has a Gene name of RS1 and NCB' sequence accession numbers NC_001806.2 or NC_001798.2. HSV-1 encodes more than 100 proteins, miRNAs and long non-coding RNAs. I0P4 is an essential regulatory protein expressed immediately after infection and controls its accumulation in infected cells. I0P4 binds as a dimer with high affinity to the consensus DNA sequence ATCGTCNNNNYCGRC (SEQ
ID NO.
39, wherein n is a, c, g, t or u, y is c, u or t, and r is a or g) and with lower affinities to a number of other sequences that are not represented by a unique consensus. DNA binding sites have been thoroughly mapped in the domains of several genes. Relevant to this invention is a site that conforms to the consensus and spans the transcription initiation site of the mRNA encoding ICP4.
Binding of I0P4 to that site inhibits the transcription of its own mRNA. In essence ICP4 tightly regulates the synthesis of its own mRNA most likely to block the synthesis of excessive amounts of ICP4. In the absence of a surplus, the impact of decreased accumulation of ICP4 mRNA should be readily apparent.
miRNAs Targeting ICP4 The terms "miRNAs targeting ICP4", "a miRNA targeting ICP4", and "ICP4-targeting miRNA" which are used interchangeably herein, refer to a small non-coding RNA (microRNA or miRNA) designed to target or specifically bind to mRNA encoding protein ICP4 such that the transcription, translation and, in turn, expression of the I0P4 in a cell is impaired, reduced, or eliminated. As described above, miRNA is not necessarily bind to target mRNA by 100% specificity. It is known that miRNA
has a seed sequence (2-8 nucleotides from 5' end) which determines the specificity of biding to a target mRNA, while the remaining nucleotides are not necessarily exactly complementary to the target mRNA. Therefore, in one embodiment, the miRNA has a seed sequence of any of nucleotide sequences SEQ ID NO. 1 or SEQ ID NO: 2. In some embodiments, the miRNA
targeting ICP4 blocks the expression of I0P4 protein in a cell after delivered to the cell.
Exosomes Carrying miRNA Targeting ICP4 Exosomes are small, relatively uniform-sized vesicles derived from cellular membranes. For example, exosomes may have a diameter of about 30 to about 100 nm. They contain several key proteins (e.g. CD9, 0D63, CD81, 0D82, Annexin, Flotillin, etc) and in addition they package proteins, mRNAs, long non-coding RNAs and miRNAs. Exosomes transport the payload from cell to cell. On entry into recipient cells the exosome payload is released into cytoplasm.
In some embodiments, the miRNA targeting ICP4 is delivered to a cell via an exosome. Therefore, in one embodiment, an exosome carrying any of the I0P4-targeting miRNAs as described above is provided. The present invention uses a fragment of nucleotide sequence, referred to as "exo-motif' herein, to facilitate or enhance the packaging of a miRNA into an exosome. In one embodiment, the exo-motif is selected from any of the sequences identified in Table 1.
Table 1. Sequences of exo-motifs used with miRNAs of the invention Sequence ID Nucleotide Sequence Sequence ID Nucleotide Sequence SEQ ID NO. 5 5'-GGAG-3' SEQ ID NO. 19 5'-CGCG-3' SEQ ID NO. 6 5'-GGAC-3' SEQ ID NO. 20 5'-CGCC-3' SEQ ID NO. 7 5'-GGCG-3' SEQ ID NO. 21 5'-CGGG-3' SEQ ID NO. 8 5'-GGCC-3' SEQ ID NO. 22 5'-CGGC-3' SEQ ID NO. 9 5'-GGGG-3' SEQ ID NO. 23 5'-CCCU-3' SEQ ID NO. 10 5'-GGGC-3' SEQ ID NO. 24 5'-CCCG-3' Sequence ID Nucleotide Sequence Sequence ID Nucleotide Sequence SEQ ID NO. 11 5'-UGAG-3' SEQ ID NO. 25 5'-CCCA-3' SEQ ID NO, 12 5'-UGAC SEQ ID NO. 26 5'-UCCU-3' SEQ ID NO. 13 5'-UGCG SEQ ID NO. 27 5'-UCCG-3' SEQ ID NO. 14 5'-UGCC SEQ ID NO. 28 5'-UCCA-3' SEQ ID NO. 15 5'-UGGG SEQ ID NO. 29 5'-GCCU-3' SEQ ID NO. 16 5'-UGGC SEQ ID NO. 30 5'-GCCG-3' SEQ ID NO. 17 5'-CGAG SEQ ID NO. 31 5'-GCCA-3' SEQ ID NO. 18 5'-CGAC SEQ ID NO. 32 5'-UGACGAC-3' In some embodiments, the exo-motifs are used in combination. For example, two or more exo-motifs as identified in the Table are combined to form a two-fold exo-motif, The motifs can be combined linearly by linking the 5'-end of one exo-motif to the 3'-end of another exo-motif. In this context, when the first nucleotide of the 5'-end of one exo-motif is identical with the last nucleotide of the 3'-end of another exo-motif, one of the identical nucleotides can be designed to be omitted.
For example, "UGCG" (SEQ ID NO. 13) is combined with "GGAC" (SEQ ID NO. 6) to form a two-fold exo-motif "UGCGGAC" (SEQ ID NO. 32), The present invention also contemplates a three-fold or more exo-motif, i.e., an exo-motif consisted of three or more motifs of SEQ
ID NO. 5 to SEQ ID
NO. 31. Therefore, the term "exo-motif" used herein is meant to include nucleotide sequences that are able to enhance or facilitate packaging of miRNA to an exosome, including any of the single exo-motif of SEQ ID NO. 5 to SEQ ID NO. 31 and any two-fold (e.g. SEQ ID NO.
32), three-fold or more fold exo-motifs generated by the combinations of the single motifs.
In the present invention, the exo-motif is operably linked to the seed sequence of the miRNA. The term "operably linked" refers to functional linkage between a regulatory sequence (e.g. the exo-motif) and a nucleic acid sequence (e.g., the seed sequence of the miRNA) resulting in an enhance of, or facilitating the packaging of the miRNA into an exosome. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. Operably linked RNA sequences can be contiguous with each other.
In some embodiments, an exo-motif is located downstream the seed sequence of the miRNA. In some embodiments, an exo-motif is located upstream the seed sequence of the miRNA. In some embodiments, the seed sequence of the miRNA is flanked by exo-motifs. In one embodiment, an exo-motif is operably linked to the seed sequence of the miRNA. In one embodiment, an exo-motif is obtained by mutation of one or more of the nucleotide sequences of the miRNA except for the seed sequence. In one embodiment, the miRNA targeting I0P4 with exo-motif contains a nucleotide sequence of SEQ ID NO. 3 or SEQ ID NO. 4. In one embodiment, the miRNA targeting ICP4 with exo-motif is a nucleotide sequence of SEQ ID NO. 3 or SEQ ID NO. 4.
In some embodiments where an exo-motif is located downstream the seed sequence of the miRNA, the 3' end last nucleotide of the seed sequence and the 5' end first nucleotide of the exo-motif share a same nucleotide, for example, guanine nucleotide "G" or uracil nucleotide "U". For example, SEQ ID NO. 3 shows the sharing of the uracil nucleotide "U" between the exo-motif and the seed sequence. For example, SEQ ID NO. 4 shows the sharing of the guanine nucleotide "G"
between the exo-motif and the seed sequence.
In addition to the seed sequence and the exo-motif, the miRNA also includes additional nucleic acid sequence to facilitate binding to the target region of the mRNA. These additional nucleic acids are normally located downstream the exo-motif with a length of several nucleotides, e.g., 5 to 10 nucleotides. The additional nucleic acid sequences are preferably complementary to the corresponding segment of the target mRNA, but, as described above, not necessarily 100%
complementary.
Methods for transferring miRNAs into an exosome are available in the art, such as by co-transfecting a cell with a miRNA expression vector and a plasmid encoding ICP4, as described in the Example. Isolation, identification or characterization of an exosome is technically feasible in the art. Several proteins, e.g. CD9, 0D63, CD81, 0D82, Annexin, Flotillin, etc can be used as a marker of exosomes. Other methods for packaging miRNAs into exosomes may also be applicable with the present invention.
The exosome of the present invention contains an inhibitory amount of miRNA
targeting I0P4. An inhibitory amount is meant an amount sufficient for inhibiting the expression of the protein I0P4 once the miRNA in question was delivered into a cell infected with HSV virus.
The table below lists the nucleic acid sequences of miRNAs, seed sequences, and miRNA-motif used in the Example of the invention.
Table 2. Nucleic acid sequences of miRNAs, seed sequences, and miRNAs linked with exo-motifs Sequence ID Identity Description Nucleic Acid Sequence SEQ ID NO. 1 miR401 Seed Sequence 5 ' -AGAGGAU- 3 ' SEQ ID NO. 2 miR402 Seed Sequence 5' -ACAGCAG- 3 ' SEQ ID NO. 3 miR401 miRNA + exo-motif 5' -AAGAGGAUGCGGACGACGAGG- 3 ' SEQ ID NO. 4 miR402 miRNA + exo-motif 5' -UACAGCAGCCGCGUGAUCAGG- 3 ' SEQ ID NO. 33 miR403 Seed Sequence 5'-UCAGCAG-3' SEQ ID NO. 34 miR403 miRNA + exo-motif 5' - GUCAGCAGGAAGCCCUUCUGC- 3 ' Methods and Therapies An aspect of the disclosure provides a method for prophylaxis or treatment of HSV viral infection in a subject comprising administering to the subject in need thereof a therapeutically effective amount of an exosome of the invention or a pharmaceutical composition comprising the exosome of the invention. Equally, the disclosure provides the exosome as described above for use in a method for treating or alleviating symptoms involved with viral infection. As described above, the exosome carries a miRNA targeting I0P4 to which an exo-motif is operably linked.
In certain embodiments, the exosome or the pharmaceutical composition is administered parenterally, e.g. intravenously, intramuscularly, percutaneously or intracutaneously.
In some embodiments, it may be desirable to combine an exosome of the invention with other agents effective in the treatment of viral infection. For example, the treatment of a viral infection may be implemented with an exosome of the invention and other anti-virus therapies, such as anti-virus agents.
The therapy may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments where the other agent and exosome are applied separately to the subject, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and exosome would still be able to exert an advantageously combined effect on the subject. In such instances, it is contemplated that one may administer the subject with both modalities within about 12 to 72 hrs of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.
In certain embodiments, the methods of treating a viral infection prevent the progression of the infection and/or the onset of disease caused by the viral infection. Thus, in some embodiments, a method for preventing the progression of a viral infection and/or the onset of disease caused by the viral infection, comprises administering an effective amount of an exosome of the invention or a composition comprising the exosome to a subject in need thereof. In certain embodiments, the methods of treating a viral infection prevent the onset, progression and/or recurrence of a symptom associated with a viral infection. Thus, in some embodiments, a method for preventing a symptom associated with a viral infection in a subject, comprises administering an effective amount of an exosome of the invention or a composition comprising the exosome to a subject in need thereof.
The viral infection that are treated by the present invention is caused by HSV-1 or HSV-2 virus.
Viral infections can cause diseases or conditions including oral herpes, herpetic gingivo-stomatitis, cold sores, herpes pharyngitis, herpes keratitis, herpes whitlow, herpes gladiatorum, eczema herpeticum, neonatal herpes, genital herpes, herpetic proctitis, HSV
encephalitis, HSV meningitis, disseminated herpes simplex infection and etc.
Oral herpes also known as cold sores can be the result of an HSV-1 or an HSV-2 infection.
Because of the association of HSV-2 with sexual transmission, infections in children are usually the result of HSV-1. The main diagnosis is herpetic gingivo-stomatitis, where the typical clear lesions first develop followed by ulcers that have a white appearance. The infection, often initially on the lips, spreads to all parts of the mouth and pharynx. Reactivation from the trigeminal ganglia can result in what are known as cold sores. Herpes pharyngitis is often associated with other viral infections of the upper respiratory tract. The disease is more severe in immunosuppressed people such as AIDS patients. Herpes keratitis is an infection of the eye and is primarily caused by HSV-1.
It can be recurrent and may lead to blindness. It is a leading cause of corneal blindness in the United States. Herpes whitlow affects people who come in manual contact with herpes-infected body secretions and can be caused by either type of HSV. HSV enters the body via small wounds on the hands or wrists. It can also be caused by transfer of HSV-2 from genitals to the hands.
Herpes gladiatorum is often found in wrestlers. It apparently spreads by direct contact from skin lesions on one wrestler to his/her opponent, and usually appears in the head and neck region (which are frequently sites of contact in wrestling holds). Oddly, the lesions are more often on the right side of the body (perhaps because most wrestlers are right handed). It is also seen in other contact sports such as rugby where it is known as scrum pox.
Eczema herpeticum is a pediatric condition found in children with active eczema or preexisting atopic dermatitis. HSV can spread over the skin at the site of eczema lesions.
The virus can spread to other organs such as liver. Neonatal herpes is a severe disease from HSV-2 and is often fatal, although such infection is rare. Infection is especially possible if the mother is shedding virus at the time of delivery. The virus can be contracted either in utero or during birth.
Because the neonate has an underdeveloped immune system, the virus can spread rapidly to many peripheral organs (e.g. lungs and liver) and can infect the central nervous system. Genital herpes and herpetic proctitis are usually the result of an HSV-2 infection with about 10% of cases being the result of HSV-1. Primary infection is often asymptomatic but many painful lesions can develop on the glans or shaft of the penis in men and on the vulva, vagina, cervix and perianal region of women where it may be accompanied by vaginal discharge. A variety of the infections also cause proctitis.
Secondary episodes of genital herpes, a result of reactivation of virus in the sacral ganglion, are frequently less severe (and last a shorter time) than the first episode.
Recurrent episodes seem usually to result from a primary HSV-2 infection. Whether there is an apparent active disease or not, an infected patient remains infectious without overt symptoms. HSV
encephalitis is the result of an HSV-1 infection and is the most common sporadic viral encephalitis. HSV
encephalitis is febrile and may result in damage to one of the temporal lobes, clinically marked by blood in the spinal fluid and seizures. The disease can be fatal and, in the US, fewer than 1000 cases per year are described. HSV meningitis is the result of an HSV-2 infection.
Disseminated herpes simplex infection is the spread of the infection throughout the body. This is a serious and life-threatening complication of HSV in patients with an impaired immune system.
Pharmaceutical Compositions An aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an exosome as described above and a pharmaceutically acceptable carrier. The pharmaceutical composition is useful for prophylaxis or treatment of HSV
infection in a subject. The exosome may be prepared in a suitable pharmaceutically acceptable carrier or excipient. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and 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 will be preferable to include isotonic agents, for example, sugars 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.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intratumoral and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 mL of isotonic NaCI
solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580).
Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The compositions disclosed herein may be formulated in a neutral or salt form.
Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
Examples In principle, controlled diminution of virus production in susceptible cells or accumulation of a specific viral protein can be done in only three ways, i.e. by the use of inhibitors of viral or cellular functions, by mutagenesis of the promoter regulating the expression of a key gene or by delivering to the susceptible cells a miRNA targeting the mRNA encoding the gene product.
In the studies described in this report the desired exosome payload was a miRNA, miRNAs are potent tools that in principle can be used to control the replication of infectious agents.
The objectives were to design miRNAs that can block the replication of herpes simplex virus 1 and which could be delivered to infected cells via exosomes. We designed 3 miRNAs targeting the mRNA encoding ICP4, an essential viral regulatory protein. Of the 3 miRNAs, one miRNA401 effectively blocked I0P4 accumulation and viral replication on transfection into susceptible cells. To facilitate packaging of the miRNA into exosomes we incorporated into the sequence of miRNA401 an exosome packaging motif. miRNA401 was shown to be packaged into exosomes and successfully delivered by exosomes to susceptible cells where it remained stable for at least 72 hrs. Finally, the results show that miRNA401 delivered to cells via exosomes effectively reduced virus yields in a miRNA401 dose dependent fashion. The protocol described in this report can be applied to study viral gene functions without actually deleting or mutagenizing the gene.
Incorporation of RNAs into exosomes is sequence dependent and facilitated by hnRNPA2B1, a component of exosomes. hnRNPA2B1 sorts into exosomes RNAs containing one of two known exosome-packaging motifs (EXO-motifs). A key function of hnRNPA2B1 is to regulate mRNA
trafficking to axons in neural cells that is mediated by binding a 21-nt RNA
sequence called RNA
trafficking sequence (RTS). This sequence contains both of the EXO-motifs.
The Examples show that HSV-1 replication can be reduced in a dose dependent manner by delivering to the cells via exosomes a miRNA designed to target mRNA encoding I0P4, a major regulatory protein of HSV-1.
Materials and Methods Exosome Isolation and Quantification. Cells seeded in 1150 flask for 24 h were extensively rinsed with PBS and then incubated n serum free medium. After 18 h the cell-free extracellular medium was centrifuged at 2000g for 30 min, The supernatant fluid was harvested mixed with recommended dose of Total Exosome Isolation kit reagent (Thermo Fisher Cat No. 4478359), stored overnight at 4 C and then centrifuged for 1 h. The pelleted exosomes were then resuspended in 200 pl of PBS or were lysed in RIPA buffer and then quantified by a BCA assay using the Enhanced BCA
Protein Assay Kit (Beyotime Biotechnology, China) according to manufacturer's instructions.
Exosome protein content was determined by calibration against standard curve, which was prepared by plotting the absorbance at 562 nm versus bovine serum albumin standard concentration.
Exosome Size Analysis. Exosome size distribution analysis was done using the qNano system (Izon, Christchurch, New Zealand). lzon's qNano technology (www.izon.com) was employed to detect extracellular vesicles passing through a nanopore by way of a single-molecule electrophoresis. In practice it enables accurate particle-by-particle characterization of vesicles from 75 to 150 nm in size of exosomes, without averaging the particle sizes. Purified exosomes were diluted to 1:10 in PBS with 0.05% Tween-20, vigorously shaken, and measured by using an NP150 (A45540) nanopore aperture according to the manufacturer's instructions. Data processing and analysis were carried out on the Izon Control Suite software v3.3 (Izon Science).
Quantitative RT-PCR for miRNA. Total RNAs from cells and liquid exosomes were isolated using TRIzol reagent (Thermo Fisher Scientific) and TRIzol LS reagent (Thermo Fisher Scientific) according to the respective manufacturer's instructions. The procedure was performed as described. The miRNA
tested were reverse-transcribed from 50 ng total RNA in duplicate by specific stem-loop primer as described in the TaqMan miRNA reverse transcription kit (Applied Biosystems, Inc.). The expression of miRNA was determined by real-time PCR using TaqMan Universal Master Mix II kit purchased from Applied Biosystems, Inc. miRNA copy number was normalized by comparison with cellular 18s rRNA.
The primers of miR401 were designed according to Chen et al. and synthesized by Ige Biotechnology.
The sequences are as follows: miR401 stem loop primer, 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCTCGT-3' (SEQ ID NO. 35);
forward primer, 5'-TGCTGGCGAAGAGGATGC-3' (SEQ ID NO, 36); reverse primer, 5'-CCAGTGCAGGGTCCGAGGTA-3' (SEQ ID NO. 37); probe, 5'-(6-FAM) CTGGATACGA000TCGTC(MGB)-3 (SEQ ID NO. 38).
Results Design and construction of a miRNA capable of suppressing the replication of HSV-1.
The objective of the first series of experiments was to design a miRNA
targeting I0P4, the major regulatory protein of HSV-1. To this end we have constructed 3 miRNAs designated miR401, mi402 and miR403. The sequence of each of the miRNAs shown in Figure 1 Panel A
contains downstream of miRNA seed sequence additional sequences embodying exosome-packaging-associated motifs (exo-motifs). As illustrated in Panel A, the miRNAs were cloned downstream of an open reading frame encoding EGFP into a miRNA expression vector named "pcDNA6,2-GW/EmGFP-miR-neg control plasmid" as described in Materials and Methods.
To test the miRNAs HEp-2 cells were co-transfected with the miRNA expression vectors (pmiR401, pmiR402, pmiR403) described above and a plasmid encoding ICP4 tagged at the C
terminus with His (pICP4-His). As shown in Figure 1 panel B miR401 was the most effective of the 3 constructs in suppressing the accumulation of CP4. The results show that the accumulation of ICP4 is repressed by miR401 at higher efficiency. miR402 showed moderate effect, whereas the non-targeting (NT) and miR403 plasmids had no effect on accumulation of ICP4 (Figure 1B). Figure 1 panel B shows the expression of EGFP in cells transfected with the construct containing miR401 suggesting that that the plasmid is efficiently transfected into HEp-2 cells.
Therefore, miR401 was selected for further studies.
-The first step in this process was the construction of a stable cell line by transfection of the plasmid into HEp-2 cell under selection of antibiotic of Blasticidin. Single cell clone from EGPF positive cells was selected, amplified and designated HEp-miR401 (Figure 1C), Figure 1D shows expression of mature miR401 by qPCR analysis. As expected the parental HEp-2 cells failed to express detectable expression of EGFP and miR401 (Figure 10 and 1D, respectively).
Accumulation of viral proteins is reduced in HEp-miR401 cells.
In this series of experiments replicate cultures of HEp-2 or HEp-miR401 cells were exposed to 0.1, 1, or 10 PFU of HSV-1(F) per cell. The cultures were harvested at indicated times after infection and then solubilized, subjected to electrophoresis in denaturing gels, reacted with antibodies to ICP27, ICP8 or VP16 representing different kinetic classes of virus replication. GFP is positive indicator of HEp-miR401 stable cell line and GAPDH served as a loading control (Figure 2). The protein bands were scanned with the aid of ImageJ scanner. The optical density of the bands was normalized with respect to the optical density of corresponding bands generated from HEp-2 cells at 24 h (1.0, 10 PFU/cell) 0r48 h (0.1 PFU/cell) after infection. The results show that in cells exposed to 0.1 PFU or 1.0 PFU/cell the amounts of the viral proteins decreased to undetected level. Accumulation of viral proteins in cells exposed to 10 PFU/cell was significantly lower than those detected in the HEp-2 cells (Figure 2).
miR401 inhibits the replication of HSV-1(F) virus.
In this series of experiments replicate cultures of HEp-2 or HEp-miR401 cells were exposed for 0.1 or 0.01 PFU of HSV-1(F) per cell. The cells harvested at the times shown in Figure 3 and viral progeny were titrated on Vero cells. The results shown indicate that the accumulations of virus in HEp-miR401 cells were significantly lower than those obtained from infected HEp-2 cells.
Characterization of miR401 packaged in exosomes.
Several experiments were carried out to test the hypothesis that miR401 produced in HEp-miR401 cells is packaged into exosomes. First, exosomes produced in HEp-2 or HEp-miR401 were purified as described in Materials and Methods. Next, 200 pl of purified exosomes obtained from cultures grown in T150 cells were then measured with respect to size by nanoparticle tracking analysis using lzon's qNano technology. The results (Figure 4A) show that the exosomes purified from HEp-2 cells or HEp-miR401 cells over lapped in size and formed a single band ranging from 75 and 150 nm in size.
Next, the purified exosomes were tested for the presence of proteins associated with exosomes that is 009, Annexin V, and Flotillin-1. In brief, purified exosomes were lysed by RIPA lysis buffer, 45 pg of solubilized exosome protein subjected to electrophoresis in denaturing gels, and reacted with antibodies to 009, AnnexinV, or Flotillin-1 (Figure 4B). The results indicate that the exosome proteins were present in purified exosomes derived from HEp-2 or HEp-m1R401 cell line, To quantify the amounts of miRNA packaged in exosomes we selected CD9 as an indicator of exosome concentration. To quantify CD9 protein, we used an 11 kDa 009 fragment purchased from Sino Biological Inc as a substitute for the full size CD9 protein. In brief, serial dilutions of 009 protein fragment were subjected to electrophoresis in denaturing gels, reacted with antibodies to CD9 (Figure 5A), the bands intensities were quantified using ImageJ to construct a standard curve.
Equivalence of full length of CD9 protein (28 KDa) was calculated accordingly (Figure 5B), Next, 200 pl of exosomes preparations purified from either T150 of HEp-miR401 or T150 of parental HEp-2 cells in three independent experiments. Fifty microliters of preparation from each was subjected to electrophoresis in denaturing gels, reacted with antibodies to 009 (Figure 50) and the bands intensities were quantified using ImageJ. The results showed that on average, 50 pl of purified exosomes equaled 283 ng of 0D9 obtained from HEp-miR401 and 251 ng of 009 obtained from HEp-2 parental cells (Figure 50). We measured the amounts of miR401 packaged in exosomes produced in the two cell lines. The results were that in exosomes produced in HEp-miR401 cell 1 ng of 009 corresponded to 1X106 miR401. miR401 was not detected in exosomes produced in HEp-2 cells (Figure 5E).
Exosome-delivered miR401 inhibits HSV-1 replication in a dose dependent manner.
In this series of experiments, we examined whether miR401 delivered via exosome is effective in diminishing the replication of HSV-1. We report 2 series of experiments.
In the first replicate cultures each containing 2.5x105 HEp-2 cells were exposed for 12 h to purified exosomes produced in HEp-miR401 cells or HEp-2 cells. The amounts of miR401 contained in the exosomes ranged from 360 to 10,000 copies per cell. The culture exposed to exosomes produced in HEp-2 cell was equivalent in amount to the highest concentration of exosomes containing miRNA401. After 12 h of incubation the cells were rinsed, exposed to 0.1 PFU
of HSV-1 (F) per cell and harvested at 24 h. The cell lysate was subjected to an electrophoresis in denaturing gels and reacted with antibodies to I0P4, I0P27, I0P8 or US11. Figure 6A shows that the accumulations of viral proteins ICP27, I0P8 and US11 decreased in HEp-2 exposed to exosomes containing miR401 in a dose dependent manner.
The second series of experiments was a replica of the first except that the infected cells were harvested at 48 h after infection and the virus produced in the infected cells was titered in Vero cells. The results shown in Figure 6B indicate that the accumulation of virus in cells exposed to exosome containing miR401 decreased significantly in a dose-dependent manner as predicted by the effects of miR401 on the accumulation of viral proteins.
The results show that miRNA401 is packaged in exosomes and is readily delivered to susceptible cells. We have also shown that the miRNA delivered via exosomes persists in recipient cells for at least 72 h. We have quantified the packaged miRNA in terms of miRNA copies per ng of CD9, a key protein component of the exosomes. Lastly, we have shown that miRNA401 delivered to cells in exosomes reduces viral yields in a dose dependent manner. The results presented herein also show that miRNAs can be used to define the targeted gene function as well as block viral replication if the targeted gene plays an essential role in viral replication.
In many instances it may obviate the cumbersome process of deleting the targeted gene to define its function.
It should be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the disclosures embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure.
All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.
The disclosures illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising," "including," containing," etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed.
Appendix A lists the sequences as described herein.
APPENDIX A
<110> IMMVIRA CO., LIMITED
<120> EXOSOMES CARRYING ICP4-TARGETING miRNA, PHARMACEUTICAL
COMPOSITIONS AND METHODS FOR TREATING HSV INFECTION
<130> PAT 104740-1 <160> 39 <170> PatentIn version 3.5 <210> 1 <211> 7 <212> RNA
<213> Artificial Sequence <220>
<223> miR401 seed sequence <400> 1 agaggau 7 <210> 2 <211> 7 <212> RNA
<213> Artificial Sequence <220>
<223> miR402 seed sequence <400> 2 acagcag 7 <210> 3 <211> 21 <212> RNA
<213> Artificial Sequence <220>
<223> miRNA401 + exo-motif <400> 3 aagaggaugc ggacgacgag g 21 <210> 4 <211> 21 <212> RNA
<213> Artificial Sequence <220>
<223> miRNA402 + exo-motif <400> 4 uacagcagcc gcgugaucag g 21 <210> 5 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 5 ggag 4 <210> 6 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 6 ggac 4 <210> 7 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 7 ggcg 4 <210> 8 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 8 ggcc 4 <210> 9 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 9 gggg 4 <210> 10 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 10 gggc 4 <210> 11 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 11 ugag 4 <210> 12 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 12 ugac 4 <210> 13 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 13 ugcg 4 <210> 14 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 14 ugcc 4
5/14 <210> 15 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 15 uggg 4 <210> 16 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 16 uggc 4 <210> 17 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 17 cgag 4 <210> 18 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 18 cgac 4 <210> 19 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 19 cgcg 4 <210> 20 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 20 cgcc 4 <210> 21 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 21 cggg 4 <210> 22 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 22 cggc 4 <210> 23 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 23 cccu 4 <210> 24 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 24 cccg 4 <210> 25 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 25 ccca 4 <210> 26 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 26 uccu 4 <210> 27 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 27 uccg 4 <210> 28 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 28 ucca 4 <210> 29 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 29 gccu 4 <210> 30 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 30 gccg 4 <210> 31 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 31 gcca 4 <210> 32 <211> 7 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 32 ugacgac 7 <210> 33 <211> 7 <212> RNA
<213> Artificial Sequence <220>
<223> miR403 seed sequence <400> 33 ucagcag 7 <210> 34 <211> 21 <212> RNA
<213> Artificial Sequence <220>
<223> miRNA403 + exo-motif <400> 34 gucagcagga agcccuucug c 21 <210> 35 <211> 50 <212> DNA
<213> Artificial Sequence <220>
<223> miR401 stem loop primer <400> 35 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgaccctcgt <210> 36 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> miR401 forward primer <400> 36 tgctggcgaa gaggatgc 18 <210> 37 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> miR401 reverse primer <400> 37 ccagtgcagg gtccgaggta 20 <210> 38 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> probe <220>
<221> misc feature <222> (1)..(18) <223> 6-Carboxy-fluorescein(6-FAM) is attached to 5' end and Minor Groove Binder(MGB) is attached to 3' end <400> 38 ctggatacga ccctcgtc 18 <210> 39 <211> 15 <212> DNA
<213> herpes simplex virus <220>
<221> misc feature <222> (7)..(10) <223> n is a, c, g, t or u <220>
<221> y <222> (11)..(11) <223> y is c, u or t <220>
<221> r <222> (14)..(14) <223> r is a or g <400> 39 atcgtcnnnn ycgrc 15
<213> Artificial Sequence <220>
<223> exo-motif <400> 15 uggg 4 <210> 16 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 16 uggc 4 <210> 17 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 17 cgag 4 <210> 18 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 18 cgac 4 <210> 19 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 19 cgcg 4 <210> 20 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 20 cgcc 4 <210> 21 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 21 cggg 4 <210> 22 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 22 cggc 4 <210> 23 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 23 cccu 4 <210> 24 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 24 cccg 4 <210> 25 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 25 ccca 4 <210> 26 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 26 uccu 4 <210> 27 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 27 uccg 4 <210> 28 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 28 ucca 4 <210> 29 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 29 gccu 4 <210> 30 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 30 gccg 4 <210> 31 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 31 gcca 4 <210> 32 <211> 7 <212> RNA
<213> Artificial Sequence <220>
<223> exo-motif <400> 32 ugacgac 7 <210> 33 <211> 7 <212> RNA
<213> Artificial Sequence <220>
<223> miR403 seed sequence <400> 33 ucagcag 7 <210> 34 <211> 21 <212> RNA
<213> Artificial Sequence <220>
<223> miRNA403 + exo-motif <400> 34 gucagcagga agcccuucug c 21 <210> 35 <211> 50 <212> DNA
<213> Artificial Sequence <220>
<223> miR401 stem loop primer <400> 35 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgaccctcgt <210> 36 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> miR401 forward primer <400> 36 tgctggcgaa gaggatgc 18 <210> 37 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> miR401 reverse primer <400> 37 ccagtgcagg gtccgaggta 20 <210> 38 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> probe <220>
<221> misc feature <222> (1)..(18) <223> 6-Carboxy-fluorescein(6-FAM) is attached to 5' end and Minor Groove Binder(MGB) is attached to 3' end <400> 38 ctggatacga ccctcgtc 18 <210> 39 <211> 15 <212> DNA
<213> herpes simplex virus <220>
<221> misc feature <222> (7)..(10) <223> n is a, c, g, t or u <220>
<221> y <222> (11)..(11) <223> y is c, u or t <220>
<221> r <222> (14)..(14) <223> r is a or g <400> 39 atcgtcnnnn ycgrc 15
Claims (13)
1. An exosome comprising:
an inhibitory amount of ICP4-targeting miRNA, wherein the ICP4-targeting miRNA
has a seed sequence binding to mRNA of ICP-4; and an exo-motif operably linked to the seed sequence of the ICP4-targeting miRNA
to enhance the packaging of the ICP4-targeting miRNA into the exosome, wherein the seed sequence of the ICP4-targeting miRNA contains a nucleic acid sequence of SEQ ID NO.
1 or 2.
an inhibitory amount of ICP4-targeting miRNA, wherein the ICP4-targeting miRNA
has a seed sequence binding to mRNA of ICP-4; and an exo-motif operably linked to the seed sequence of the ICP4-targeting miRNA
to enhance the packaging of the ICP4-targeting miRNA into the exosome, wherein the seed sequence of the ICP4-targeting miRNA contains a nucleic acid sequence of SEQ ID NO.
1 or 2.
2. The exosome of claim 1, wherein the exo-motif is selected from a group consisting of nucleic acid sequence of SEQ ID NO. 5 to SEQ ID NO. 31.
3. The exosome of claim 1, wherein the exo-motif is located downstream and covalently linked to the seed sequence of the ICP4-targeting miRNA.
4. The exosome of claim 1, wherein the exo-motif is obtained by mutation of one or more nucleic acids of the ICP-4 targeting miRNA except for the seed sequence.
5. The exosome of claim 1, wherein the exo-motif is a two-fold motif generated through combination of two single exo-motifs, wherein any of the two single exo-motifs is selected from a group consisting of nucleic acid sequence of SEQ ID NO. 5 to SEQ ID NO. 31.
6. The exosome of claim 1, wherein the two-fold motif has a nucleic acid sequence of SEQ ID NO.
32.
32.
7. The exosome of claim 1, wherein the ICP4-targeting miRNA and the exo-motif, when operably linked, share at least one nucleotide.
8. The exosome of claim 1, wherein the ICP4-targeting miRNA and the exo-motif, when operably linked, has a nucleic acid sequence of SEQ ID NO. 3 or SEQ ID NO. 4.
9. A pharmaceutical composition comprising a therapeutically effective amount of an exosome of any of claims 1 to 7 and a pharmaceutically acceptable carrier.
10. A cell comprising an exosome of any of claims 1 to 8.
11. The cell of claim 10, wherein the cell is infected with HSV-1, HSV-2, or both.
12. The cell of claim 10, wherein the cell is a mammal cell.
13. The cell of claim 12, wherein the mammal cell is a human somatic cell.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA3020243A CA3020243A1 (en) | 2018-10-09 | 2018-10-09 | Exosomes carrying icp4-targeting mirna, pharmaceutical compositions and methods for treating hsv infection |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA3020243A CA3020243A1 (en) | 2018-10-09 | 2018-10-09 | Exosomes carrying icp4-targeting mirna, pharmaceutical compositions and methods for treating hsv infection |
Publications (1)
Publication Number | Publication Date |
---|---|
CA3020243A1 true CA3020243A1 (en) | 2020-04-09 |
Family
ID=70155979
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA3020243A Abandoned CA3020243A1 (en) | 2018-10-09 | 2018-10-09 | Exosomes carrying icp4-targeting mirna, pharmaceutical compositions and methods for treating hsv infection |
Country Status (1)
Country | Link |
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CA (1) | CA3020243A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2022089382A1 (en) * | 2020-10-26 | 2022-05-05 | Immvira Co., Limited | Exosomes containing mirnas targeting her2 synthesis and pharmaceutical compositions |
CN114703271A (en) * | 2022-01-26 | 2022-07-05 | 无锡市妇幼保健院 | LncRNA for regulating apoptosis and proliferation of placental trophoblasts in ICP (inductively coupled plasma) process and application thereof |
-
2018
- 2018-10-09 CA CA3020243A patent/CA3020243A1/en not_active Abandoned
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2022089382A1 (en) * | 2020-10-26 | 2022-05-05 | Immvira Co., Limited | Exosomes containing mirnas targeting her2 synthesis and pharmaceutical compositions |
CN114703271A (en) * | 2022-01-26 | 2022-07-05 | 无锡市妇幼保健院 | LncRNA for regulating apoptosis and proliferation of placental trophoblasts in ICP (inductively coupled plasma) process and application thereof |
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