CA2552996A1 - Window period vaccine small interference rnas vaccines for the prevention and treatment of viral respiratory infectious diseases - Google Patents
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Abstract
The invention may be broadly conceptualized as an approach in which a kind of window period vaccines (small interference RNAs vaccines), which specifically inhibits viruses influenza virus and foot-and-mouth disease virus (FMDV) that cause the illness by infecting respiratory tract, residing and replicating there . The invention comprises:
.cndot. Design and generation of a serial of small interference RNA molecules including siRNAs, shRNAs and microRNAs which are specifically for the genomic highly conserved regions of influenza virus and FMDV.
.cndot. Screening of the best small interference RNAs.
.cndot. Delivery of viral diseases specific small interference RNAs with cationic lipid complexes as spray vaccines during the window period of viral infection.
By imitating viruses' infectious route of entry into an organism, small interference RNAs vaccines could be directly sprayed to the infected respiratory tract during the window period of virus infection. The direct delivery of small interference RNAs into specific cells would save the usage of small interference RNAs and increase the efficiency of silencing of gene expression in the target organs or tissues and also reduce potential toxicity and other side effects. Small interference RNAs vaccines could inhibit the multiplication of influenza virus and FMDV, degrade their specific mRNAs and prevent their epidemics. Small interference RNAs vaccines as window period vaccines would be the most promising and practical vaccines.
.cndot. Design and generation of a serial of small interference RNA molecules including siRNAs, shRNAs and microRNAs which are specifically for the genomic highly conserved regions of influenza virus and FMDV.
.cndot. Screening of the best small interference RNAs.
.cndot. Delivery of viral diseases specific small interference RNAs with cationic lipid complexes as spray vaccines during the window period of viral infection.
By imitating viruses' infectious route of entry into an organism, small interference RNAs vaccines could be directly sprayed to the infected respiratory tract during the window period of virus infection. The direct delivery of small interference RNAs into specific cells would save the usage of small interference RNAs and increase the efficiency of silencing of gene expression in the target organs or tissues and also reduce potential toxicity and other side effects. Small interference RNAs vaccines could inhibit the multiplication of influenza virus and FMDV, degrade their specific mRNAs and prevent their epidemics. Small interference RNAs vaccines as window period vaccines would be the most promising and practical vaccines.
Description
WINDOW PERIOD VACCINE
Small interference RNAs vaccines for the prevention and treatment of viral respiratory infectious diseases FIELD OF INVENTION:
The invention pertains generally to biotechnological field. More specifically, the invention relates to applying RNA interference technology to anti-viruses by inhibiting viruses multiplication, degrading targets mRNA.
BACKGROUND OF THE INVENTION
Influenza virus and FMDV are two very harmful kinds of viruses which cause illness to human and livestock, respectively. Both of them cause illness through respiratory tract infection. Until now, there is still not a very effective method that can control the above two diseases.
Influenza is commonly seen in local outbreaks or epidemics throughout the world.
Epidemics may appear at any time but are usually concentrated in months of high humidity. They occur explosively with little or no warning. The number of people affected can vary from a few hundred to hundreds of thousands. Epidemics may be short-lived, lasting days or weeks but larger epidemics may last for months.
Although influenza is a mild disease in most individuals, it is life threatening to elderly or debilitated individuals. Epidemics are responsible for large losses in productivity. On the basis of their nucleocapsid and M protein antigens, the influenza viruses are divided into 3 distinct immunological types (A, B, and C). Influenza A viruses also occur in pigs, birds, and horses. However, only man is infected by influenza B and C. The antigenic differences of the haemagglutinin and the neuraminidase antigens of influenza A viruses provide the basis of their classification into subtypes.
Vaccines against influenza have been around for 50 years. The efficacy of influenza vaccines is still questioned, and the ability of vaccines to limit epidemic infection has not been proven. Although a case of the flu elicits a strong immune response against the strain that caused it, the speed with which new strains arise by antigenic drift soon leaves one susceptible to a new infection. Immunization with flu vaccines has proved moderately helpful in reducing the size and severity of new epidemics.
Current flu vaccines incorporate inactivated virus particles. Others use the purified hemagglutinin. Both types incorporate antigens from the three major strains in circulation, currently: an A strain of the H 1 N 1 subtype, an A strain of the H3N2 subtype and a B
strain. Because of antigenic drift, the strains used must be changed periodically as new strains emerge that are no longer controlled by people's residual immunity.
Foot-and-mouth disease (FMD) is a severe, highly infectious viral disease of cloven-hoofed animals. Although not usually fatal, it causes suffering and vastly reduces animals' commercial value by reducing their weight and milk output. FMD is widely believed to be the most economically devastating livestock disease in the world. The etiological agent of FMD is FMD virus (FMDV). The spreading capacity of the virus and its ability to change its antigenic identity make it a real threat to the dairy and meat industry in many countries.
FMDV has the potential to cause explosive epidemics of FMD because of its diverse host range, low infectious dose requirement, high quantity of virus excretion, multiple routes of transmission, and short incubation period. FMDV occurs as seven distinct serotypes (Euroasiatic serotypes A, 0, C and Asial and South African Territories [SAT]
serotypes SATI, SAT2, and SAT3) and multiple subtypes reflecting significant genetic variability.
The measures of control are routing vaccination and slaughter policy. Current FMD
vaccines based on inactivated virus are effective in preventing the disease but present the risks of incomplete inactivation or escape of virus from vaccine production laboratories.
There are seven different types and more than 60 subtypes of FMD virus.
Vaccines for FMD must match the type and subtype present in the affected area and there is no universal vaccine against the disease. The development of recombinant peptide vaccine and chemically synthetic vaccine has achieved great success, as reported previously.
Although these vaccines are safe and effective in eliciting antiviral activity, they fail to induce immune response in a short period. However, the speed of spread of FMDV
is so rapid. For slaughter policy, if an outbreak threatened to become extensive, it is considered that this strategy alone might not be sufficient to eradicate the virus.
Although not usually fatal, it causes suffering and vastly reduces animals' commercial value by reducing their weight and milk output. Thus, developing emergency antiviral strategies is necessary in order to prevent outbreaks of FMD.
RNA interference (RNAi) is an evolutionarily conserved, genetic surveillance mechanism that permits the sequence-specific post-transcriptional down-regulation of target genes.
Post-transcriptional regulation of gene expression can be mediated by small noncoding RNAs such as short interfering RNA (siRNA), shRNA and microRNA.
siRNA are short RNA duplexes that direct the degradation of homologous mRNA
through the RNA interference (RNAi) pathway. microRNA, in contrast, are single-stranded RNAs that bind to partially complementary (50-85%) 3' untranslated regions of mRNA
leading to translational repression without target degradation. Both siRNA and miRNA
are 22 nt in length and are produced from longer double-stranded precursor RNA by the multidomain ribonuclease III enzyme Dicer.
The shRNAs bearing a fold-back, stem-loop structure of approximately 19 perfectly matched nucleotides connected by various spacer regions and ending in a 2-nucleotide 3'-overhang can be as efficient as siRNAs at inducing RNA interference. After transcription by RNA polymerase, the inverted repeats anneal and form a hairpin, which is then cleaved by Dicer to form a siRNA. These siRNAs are incorporated into an RNAi targeting complex known as RISC (RNA-induced silencing complex), which destroys mRNAs homologous to the integral siRNA. The target mRNA is cleaved in the center of the region complementary to the siRNA, with the net result being rapid degradation of the target mRNA and decreased protein expression.
The discovery of RNAi as a biological response to double-stranded RNA (dsRNA) was first made in the nematode C. elegans. Injecting dsRNAs into the worm was found to silence genes whose sequences were complementary to those of the introduced dsRNAs.
Since then, an RNAi pathway has been shown to be present in many eukaryotes.
There have been several publications demonstrating the silencing of various genes by siRNA in mammalian cells. Furthermore, siRNA has been shown to be active in controlling virus replication, including that of human immunodeficiency virus, poliovirus and hepatitis B
virus. The antiviral potential of small interfering RNA (siRNA) targeting VP 1 of foot-and-mouth disease virus (FMDV) in BHK-21 cells and suckling mice gave an 80 to 90%
reduction in the expression of FMDV VPl in BHK-21 cells. These observations open the field for further studies toward novel therapy approaches for anti-viral treatments using small interference RNAs. RNAi may provide a viable therapeutic approach to treat Influenza virus and FMDV.
However, application of small interference RNAs as clinical drugs for effective antiviral therapies, although it has a tremendous potential, has no any breaking-through progress due to some technical difficulties. The major obstacle for using small interference RNAs as drugs involve in their delivery directly to the blood circulatory system.
First of all, the specific small interference RNAs still cannot directionally attack the target cells.
Secondly, their effects are transient which decreases the bioavailability.
Increasing dosage could result in potential toxicity and other side effects. Finding another treatment method is the best way to break through the standstill of small interference RNAs application.
To treat the viral respiratory infectious disease, small interference RNAs can be made as a spray and administered direct to the infected part-the mucous membrane of the upper respiratory tract during the window period of virus infection. It enhances efficiency of silencing of virus gene expression in the target organ or tissue, largely decreases the side effect and easily solves the cell targeting problem at the same time.
Therefore, small interference RNAs as vaccines have very practical values in effectively inhibiting virus replication and further controlling the spread of the epidemic disease and helping in disease treatment.
PURPOSE OF THIS INVENTION:
This invention aims to screen and produce specific small interference RNAs as vaccines which specifically target genomic highly conserved regions of influenza virus and FMDV, respectively. Imitating viruses' infectious route of entry into an organism, applying small interference RNAs with cationic lipid transfection reagents as spray vaccines to the infectious districts during window period of virus infection, therapies are most appropriately delivered throught respiratory tract by inhalation. Vaccines could inhibit Influenza virus and FMDV viruses' multiplication and prevent their epidemics.
Efficient small interference RNAs delivery could also be achieved by directly spraying the small interference RNAs lipid complexes into the infected respiratory tract during window period of virus infection. The direct delivery of small interference RNAs into specific cells would save the usage of small interference RNAs and increase the efficiency of silencing of gene expression in the target organs or tissues and also reduce potential toxicity and other side effects. Small interference RNA vaccines could inhibit multiplication of influenza virus and FMDV and prevent their epidemics. Small interference RNA vaccines would be the most promising and practical vaccines.
SUMMARY OF THE INVENTION
The main technology for this invention comprises:
1, target choice: computer homology analysis by using the PILEUP and PRETTY, homology searches in GenBank by TASTA.
Small interference RNAs vaccines for the prevention and treatment of viral respiratory infectious diseases FIELD OF INVENTION:
The invention pertains generally to biotechnological field. More specifically, the invention relates to applying RNA interference technology to anti-viruses by inhibiting viruses multiplication, degrading targets mRNA.
BACKGROUND OF THE INVENTION
Influenza virus and FMDV are two very harmful kinds of viruses which cause illness to human and livestock, respectively. Both of them cause illness through respiratory tract infection. Until now, there is still not a very effective method that can control the above two diseases.
Influenza is commonly seen in local outbreaks or epidemics throughout the world.
Epidemics may appear at any time but are usually concentrated in months of high humidity. They occur explosively with little or no warning. The number of people affected can vary from a few hundred to hundreds of thousands. Epidemics may be short-lived, lasting days or weeks but larger epidemics may last for months.
Although influenza is a mild disease in most individuals, it is life threatening to elderly or debilitated individuals. Epidemics are responsible for large losses in productivity. On the basis of their nucleocapsid and M protein antigens, the influenza viruses are divided into 3 distinct immunological types (A, B, and C). Influenza A viruses also occur in pigs, birds, and horses. However, only man is infected by influenza B and C. The antigenic differences of the haemagglutinin and the neuraminidase antigens of influenza A viruses provide the basis of their classification into subtypes.
Vaccines against influenza have been around for 50 years. The efficacy of influenza vaccines is still questioned, and the ability of vaccines to limit epidemic infection has not been proven. Although a case of the flu elicits a strong immune response against the strain that caused it, the speed with which new strains arise by antigenic drift soon leaves one susceptible to a new infection. Immunization with flu vaccines has proved moderately helpful in reducing the size and severity of new epidemics.
Current flu vaccines incorporate inactivated virus particles. Others use the purified hemagglutinin. Both types incorporate antigens from the three major strains in circulation, currently: an A strain of the H 1 N 1 subtype, an A strain of the H3N2 subtype and a B
strain. Because of antigenic drift, the strains used must be changed periodically as new strains emerge that are no longer controlled by people's residual immunity.
Foot-and-mouth disease (FMD) is a severe, highly infectious viral disease of cloven-hoofed animals. Although not usually fatal, it causes suffering and vastly reduces animals' commercial value by reducing their weight and milk output. FMD is widely believed to be the most economically devastating livestock disease in the world. The etiological agent of FMD is FMD virus (FMDV). The spreading capacity of the virus and its ability to change its antigenic identity make it a real threat to the dairy and meat industry in many countries.
FMDV has the potential to cause explosive epidemics of FMD because of its diverse host range, low infectious dose requirement, high quantity of virus excretion, multiple routes of transmission, and short incubation period. FMDV occurs as seven distinct serotypes (Euroasiatic serotypes A, 0, C and Asial and South African Territories [SAT]
serotypes SATI, SAT2, and SAT3) and multiple subtypes reflecting significant genetic variability.
The measures of control are routing vaccination and slaughter policy. Current FMD
vaccines based on inactivated virus are effective in preventing the disease but present the risks of incomplete inactivation or escape of virus from vaccine production laboratories.
There are seven different types and more than 60 subtypes of FMD virus.
Vaccines for FMD must match the type and subtype present in the affected area and there is no universal vaccine against the disease. The development of recombinant peptide vaccine and chemically synthetic vaccine has achieved great success, as reported previously.
Although these vaccines are safe and effective in eliciting antiviral activity, they fail to induce immune response in a short period. However, the speed of spread of FMDV
is so rapid. For slaughter policy, if an outbreak threatened to become extensive, it is considered that this strategy alone might not be sufficient to eradicate the virus.
Although not usually fatal, it causes suffering and vastly reduces animals' commercial value by reducing their weight and milk output. Thus, developing emergency antiviral strategies is necessary in order to prevent outbreaks of FMD.
RNA interference (RNAi) is an evolutionarily conserved, genetic surveillance mechanism that permits the sequence-specific post-transcriptional down-regulation of target genes.
Post-transcriptional regulation of gene expression can be mediated by small noncoding RNAs such as short interfering RNA (siRNA), shRNA and microRNA.
siRNA are short RNA duplexes that direct the degradation of homologous mRNA
through the RNA interference (RNAi) pathway. microRNA, in contrast, are single-stranded RNAs that bind to partially complementary (50-85%) 3' untranslated regions of mRNA
leading to translational repression without target degradation. Both siRNA and miRNA
are 22 nt in length and are produced from longer double-stranded precursor RNA by the multidomain ribonuclease III enzyme Dicer.
The shRNAs bearing a fold-back, stem-loop structure of approximately 19 perfectly matched nucleotides connected by various spacer regions and ending in a 2-nucleotide 3'-overhang can be as efficient as siRNAs at inducing RNA interference. After transcription by RNA polymerase, the inverted repeats anneal and form a hairpin, which is then cleaved by Dicer to form a siRNA. These siRNAs are incorporated into an RNAi targeting complex known as RISC (RNA-induced silencing complex), which destroys mRNAs homologous to the integral siRNA. The target mRNA is cleaved in the center of the region complementary to the siRNA, with the net result being rapid degradation of the target mRNA and decreased protein expression.
The discovery of RNAi as a biological response to double-stranded RNA (dsRNA) was first made in the nematode C. elegans. Injecting dsRNAs into the worm was found to silence genes whose sequences were complementary to those of the introduced dsRNAs.
Since then, an RNAi pathway has been shown to be present in many eukaryotes.
There have been several publications demonstrating the silencing of various genes by siRNA in mammalian cells. Furthermore, siRNA has been shown to be active in controlling virus replication, including that of human immunodeficiency virus, poliovirus and hepatitis B
virus. The antiviral potential of small interfering RNA (siRNA) targeting VP 1 of foot-and-mouth disease virus (FMDV) in BHK-21 cells and suckling mice gave an 80 to 90%
reduction in the expression of FMDV VPl in BHK-21 cells. These observations open the field for further studies toward novel therapy approaches for anti-viral treatments using small interference RNAs. RNAi may provide a viable therapeutic approach to treat Influenza virus and FMDV.
However, application of small interference RNAs as clinical drugs for effective antiviral therapies, although it has a tremendous potential, has no any breaking-through progress due to some technical difficulties. The major obstacle for using small interference RNAs as drugs involve in their delivery directly to the blood circulatory system.
First of all, the specific small interference RNAs still cannot directionally attack the target cells.
Secondly, their effects are transient which decreases the bioavailability.
Increasing dosage could result in potential toxicity and other side effects. Finding another treatment method is the best way to break through the standstill of small interference RNAs application.
To treat the viral respiratory infectious disease, small interference RNAs can be made as a spray and administered direct to the infected part-the mucous membrane of the upper respiratory tract during the window period of virus infection. It enhances efficiency of silencing of virus gene expression in the target organ or tissue, largely decreases the side effect and easily solves the cell targeting problem at the same time.
Therefore, small interference RNAs as vaccines have very practical values in effectively inhibiting virus replication and further controlling the spread of the epidemic disease and helping in disease treatment.
PURPOSE OF THIS INVENTION:
This invention aims to screen and produce specific small interference RNAs as vaccines which specifically target genomic highly conserved regions of influenza virus and FMDV, respectively. Imitating viruses' infectious route of entry into an organism, applying small interference RNAs with cationic lipid transfection reagents as spray vaccines to the infectious districts during window period of virus infection, therapies are most appropriately delivered throught respiratory tract by inhalation. Vaccines could inhibit Influenza virus and FMDV viruses' multiplication and prevent their epidemics.
Efficient small interference RNAs delivery could also be achieved by directly spraying the small interference RNAs lipid complexes into the infected respiratory tract during window period of virus infection. The direct delivery of small interference RNAs into specific cells would save the usage of small interference RNAs and increase the efficiency of silencing of gene expression in the target organs or tissues and also reduce potential toxicity and other side effects. Small interference RNA vaccines could inhibit multiplication of influenza virus and FMDV and prevent their epidemics. Small interference RNA vaccines would be the most promising and practical vaccines.
SUMMARY OF THE INVENTION
The main technology for this invention comprises:
1, target choice: computer homology analysis by using the PILEUP and PRETTY, homology searches in GenBank by TASTA.
2, small interference RNAs screening (siRNA, shRNAs and microRNA design and preparation, initial assessment of particular small interference RNA
suppressive effects) 3, small interference RNAs production 4, small interference RNAs delivery 5, confirmation of RNA interfering effects 6, timing for applying window period vaccine DETAILED DESCRIPTION OF THE INVENTION
1, Viral target choice Influenza virus belongs to the genus orthomyxovirus in the family of Orthomyxoviridae.
The genome is segmented, with 8 RNA fragments (7 for influenza C). There are 4 antigens present, the haemagglutinin (HA), neuraminidase (NA), nucleocapsid (NA), the matrix (M) and the nucleocapsid proteins. The NP is a type-specific antigen which occurs in 3 forms, A, B and C, which provides the basis for the classification of human influenza viruses. The matrix protein (M protein) surrounds the nucleocapsid and makes up 35-45% of the particle mass. 2 surface glycoproteins are seen on the surface as rod-shaped projections. The haemagglutinin (HA) is made up of 2 subunits, HA1 and HA2. HA
mediates the attachment of the virus to the cellular receptor. Neuraminidase molecules are present in lesser quantities in the envelope.
FMDV belongs to the genusAphthovirus of the family Picornaviridae. FMDV is a small nonenveloped virus with a pseudo T=3 icosahedral capsid made up of 60 copies each of four structural proteins. The four capsid proteins, 1 A, 1 B, 1 C, and 1 D (also known as VP4, VP2, VP3, and VP I, respectively), are encoded by the N-terminal half of the ORF, and with the exception of I A, which is excluded from the virion surface, are involved in antigenicity and binding to a subset of RGD-dependent integrins and heparan sulfate proteoglycan receptors on the cell surface. Nonstructural proteins represent about two-thirds of the ORF-encoded proteins and include Lp' , 2A, 2B, 2C, 3A, 3B, 3Cp' , and 3DP '. FMDV polyprotein processing is mediated by Lp' , 3CAr , and 2A. Lpr is a protease that, in addition to excising itself from the polyprotein, cleaves the cellular translation initiation factor eIF4G, resulting in a shutoff of host cap-dependent translation. 3CP' , a member of the trypsin family of serine proteinases, performs all but three of the cleavages leading to mature viral proteins and also cleaves host cell proteins. FMDV 2A
mediates autocleavage at its C terminus, apparently by inducing a ribosomal skip during polyprotein synthesis. Although the functions of the FMDV 2B and 2C proteins are unknown, preliminary work suggests that, similar to those of other picornaviruses, they localize to endoplasmic reticulum (ER)-derived vesicles, the sites of viral genome replication. 3A is thought to be a multifunctional integral membrane protein that enhances viral RNA synthesis by 3DP" and stimulates cleavage of the 3CD
precursor.
FMDV encodes three non-identical copies of genome-linked 3B, a protein required for viral RNA replication. The 3D gene encodes the viral RNA-dependent RNA
polymerase, and it and 3A co-localize with ER membrane-associated replication complexes.
The small interference RNAs mainly target highly conserved regions. For FMDV:
the 1 A
capsid region, 2B, 3B functional region and the 3D polymerase gene, For Influenza virus:
fragment 5 encoding nucleoprotein region, fragment 7 encoding plasma-protein region and fragment 8 encoding un-structural protein region.
The basic principles for the viral target choice are:
= Beginning with the AUG starting codon of the target gene transcript, scan downstream for AA dinucleotide sequences; each AA and the 3 adjacent 19 nucleotides are potential siRNA targets.
= Compare the sequences of the potential target sequences to sequences in the species-appropriate genome database and eliminate from consideration any target sequences that are homologous to other coding sequences.
= Select 3-4 target sequences along the length of the gene for production of siRNAs.
Of course it is important for all siRNA experiments to include negative control siRNAs with the same nucleotide composition but a scrambled sequence.Select highly conserved regions which encode viruses' functional proteins, mainly RNA
polymerase, and capsid proteins.
= Computer homology analysis by using the PILEUP and PRETTY, homology searches in GenBank by TASTA.
2, Small interference RNAs screening There are five producing RNAi molecule methods: Chemical synthesis, In vitro transcription, Digestion of long dsRNA by an RNase III farnily enzyme (e.g.
Dicer, RNase Iti), Expression in cells from an siRNA expression plasmid or viral vector, Expression in cells from a PCR-derived siRNA expression cassette. All the methods, except digestion of long dsRNA by RNase 111, require the design of individual siRNA, shRNA and microRNA sequences prior to preparation.
A, Small interference RNAs design Based on following guidelines to design small interference RNAs:
= Find occurrence with an mRNA with "AA"
= Capture following 19 nucleotides = GC content 30%--50%
= G/C at the 5' end of the sense strand = A/U at the 5' end of the antisense strand = At least 5 A/U residues in the first 7 bases of the 5' terminal of the antisense strand = No runs of more than 9 G/C residues = Blast search to find sequences with low homology to other genes B, Generation of small interference RNAs by expression vectors After choosing the targets, designing small interference RNAs, generation of siRNAs, shRNA. and microRNA could be achieved almost no efPoi-ts by small interference RNAs expression vectors method.
C, Initial assessment of particular small interference RNAs suppressive effects After delivery of small interference RNAs with or without cationic lipid complex cells or individuals, reduction of the targeted gene expression can be measured.
= Monitoring phenotypic changes of the cell = Measuring changes in mRNA levels using RT-PCR
= Detecting changes in protein levels by immunocytochemistry or Western blot analysis 3, siRNAs, shRNAs and microRNAs production To highly efficiently produce small interface RNA vaccine, using small interference RNA
expression vectors, two oligo deoxynucleotides encoding the highly effective shRNAs, siRNAs and microRNAs sequence which are chosen by screening steps, annealed, and cloned into the vector downstream of the promoter. Small interference RNA
expression vectors can rely on an RNA polymerase III (pol III) promoter to drive the expression of small interference RNA siRNA, shRNA and microRNA. RNA pol III was chosen to drive siRNA expression because it naturally expresses relatively large amounts of small RNAs, it terminates transcription upon incorporating a string of 1-4 uridines, and its transcripts lack poly(A) tails.
= Annealing of the targeted oligonucleotides = Ligation of the annealed oligonucleotides into the vectors = Transformation of expression vectors into E. coli and bacteria fermentation = siRNA, shRNA and microRNA extraction and purification 4, Small interference RNAs delivery Delivery focuses to tissues and cells where the target viruses reside and replicate, somewhat analogous to targeting vaccine delivery to focus immunity in tissues and cells that lie in the viruses' infectious route of entry into an organism. Influenza virus causes disease by infecting respiratory tract and replicating there and FMDV cause disease mainly by infecting respiratory tract and replicating there. Therapies are most appropriately delivered throught respiratory tract by inhalation. Therefore, efficient small interference RNAs delivery and silencing can be achieved by spraying mixed small interference RNAs with cationic lipid based complexes as window period vaccines to the infectious district and respiratory tract of infected, suspicious individuals during window period of virus infection.
5, Confirmation of vaccine effects in RNA interference Suppression can be confirmed at both the mRNA and protein levels.
= The mRNA level: Northern blotting and quantitative, real-time RT-PCR can be used to demonstrate reduction of expression.
= The protein level: Quantitative Western blotting, phenotypic and functional assays are some of the options available to show protein knockdown.
= Negative and positive controls: siRNAs, shRNAs and microRNAs containing a single mismatch can be used as negative controls. Positive controls with small interference RNAs known to exhibit an RNAi effect may also be useful.
6, Timing for applying window period vaccine Timing is very important for applying this kind of vaccines. Applying mixed small interference RNA with cationic lipid based complexes as window period vaccine to the infectious district and infected, suspicious individuals during window period of virus infection could inhibit viruses' multiplication and prevent their epidemics.
THE ADVANGAGE OF THIS INVENTION
Strategies aimed at conferring rapid and efficient protection against Influenza virus and FMDV face some challenges:
= Tremendous harms = Rapid spread = Highly genetic variation The traditional vaccines based on virus inactivation could be effective in preventing disease within 4 to 5 days post-vaccination, due to a critical role for innate immune defenses. Window period vaccines could be as emergency vaccines to prevent and treat these viruses. By imitating viruses' infectious route of entry into an organism, window period vaccines could be sprayed to the respiratory tract of infected and suspicious individuals during window period of virus infection. The direct delivery of window period vaccines into specific cells would save the usage of small interference RNAs and increase the efficiency of silencing of gene expression in the target organ or tissue without activating nonspecific cellular responses, hence presumably minimizing potential toxicity and undesirable side effects. Window period vaccines could also be sprayed in infectious districts. Therapies are most appropriately delivered throught respiratory tract by inhalation. Thus, antiviral strategies based on the specific and rapid inhibition of Influenza virus and FMDV infection could complement and improve the traditional tools.
suppressive effects) 3, small interference RNAs production 4, small interference RNAs delivery 5, confirmation of RNA interfering effects 6, timing for applying window period vaccine DETAILED DESCRIPTION OF THE INVENTION
1, Viral target choice Influenza virus belongs to the genus orthomyxovirus in the family of Orthomyxoviridae.
The genome is segmented, with 8 RNA fragments (7 for influenza C). There are 4 antigens present, the haemagglutinin (HA), neuraminidase (NA), nucleocapsid (NA), the matrix (M) and the nucleocapsid proteins. The NP is a type-specific antigen which occurs in 3 forms, A, B and C, which provides the basis for the classification of human influenza viruses. The matrix protein (M protein) surrounds the nucleocapsid and makes up 35-45% of the particle mass. 2 surface glycoproteins are seen on the surface as rod-shaped projections. The haemagglutinin (HA) is made up of 2 subunits, HA1 and HA2. HA
mediates the attachment of the virus to the cellular receptor. Neuraminidase molecules are present in lesser quantities in the envelope.
FMDV belongs to the genusAphthovirus of the family Picornaviridae. FMDV is a small nonenveloped virus with a pseudo T=3 icosahedral capsid made up of 60 copies each of four structural proteins. The four capsid proteins, 1 A, 1 B, 1 C, and 1 D (also known as VP4, VP2, VP3, and VP I, respectively), are encoded by the N-terminal half of the ORF, and with the exception of I A, which is excluded from the virion surface, are involved in antigenicity and binding to a subset of RGD-dependent integrins and heparan sulfate proteoglycan receptors on the cell surface. Nonstructural proteins represent about two-thirds of the ORF-encoded proteins and include Lp' , 2A, 2B, 2C, 3A, 3B, 3Cp' , and 3DP '. FMDV polyprotein processing is mediated by Lp' , 3CAr , and 2A. Lpr is a protease that, in addition to excising itself from the polyprotein, cleaves the cellular translation initiation factor eIF4G, resulting in a shutoff of host cap-dependent translation. 3CP' , a member of the trypsin family of serine proteinases, performs all but three of the cleavages leading to mature viral proteins and also cleaves host cell proteins. FMDV 2A
mediates autocleavage at its C terminus, apparently by inducing a ribosomal skip during polyprotein synthesis. Although the functions of the FMDV 2B and 2C proteins are unknown, preliminary work suggests that, similar to those of other picornaviruses, they localize to endoplasmic reticulum (ER)-derived vesicles, the sites of viral genome replication. 3A is thought to be a multifunctional integral membrane protein that enhances viral RNA synthesis by 3DP" and stimulates cleavage of the 3CD
precursor.
FMDV encodes three non-identical copies of genome-linked 3B, a protein required for viral RNA replication. The 3D gene encodes the viral RNA-dependent RNA
polymerase, and it and 3A co-localize with ER membrane-associated replication complexes.
The small interference RNAs mainly target highly conserved regions. For FMDV:
the 1 A
capsid region, 2B, 3B functional region and the 3D polymerase gene, For Influenza virus:
fragment 5 encoding nucleoprotein region, fragment 7 encoding plasma-protein region and fragment 8 encoding un-structural protein region.
The basic principles for the viral target choice are:
= Beginning with the AUG starting codon of the target gene transcript, scan downstream for AA dinucleotide sequences; each AA and the 3 adjacent 19 nucleotides are potential siRNA targets.
= Compare the sequences of the potential target sequences to sequences in the species-appropriate genome database and eliminate from consideration any target sequences that are homologous to other coding sequences.
= Select 3-4 target sequences along the length of the gene for production of siRNAs.
Of course it is important for all siRNA experiments to include negative control siRNAs with the same nucleotide composition but a scrambled sequence.Select highly conserved regions which encode viruses' functional proteins, mainly RNA
polymerase, and capsid proteins.
= Computer homology analysis by using the PILEUP and PRETTY, homology searches in GenBank by TASTA.
2, Small interference RNAs screening There are five producing RNAi molecule methods: Chemical synthesis, In vitro transcription, Digestion of long dsRNA by an RNase III farnily enzyme (e.g.
Dicer, RNase Iti), Expression in cells from an siRNA expression plasmid or viral vector, Expression in cells from a PCR-derived siRNA expression cassette. All the methods, except digestion of long dsRNA by RNase 111, require the design of individual siRNA, shRNA and microRNA sequences prior to preparation.
A, Small interference RNAs design Based on following guidelines to design small interference RNAs:
= Find occurrence with an mRNA with "AA"
= Capture following 19 nucleotides = GC content 30%--50%
= G/C at the 5' end of the sense strand = A/U at the 5' end of the antisense strand = At least 5 A/U residues in the first 7 bases of the 5' terminal of the antisense strand = No runs of more than 9 G/C residues = Blast search to find sequences with low homology to other genes B, Generation of small interference RNAs by expression vectors After choosing the targets, designing small interference RNAs, generation of siRNAs, shRNA. and microRNA could be achieved almost no efPoi-ts by small interference RNAs expression vectors method.
C, Initial assessment of particular small interference RNAs suppressive effects After delivery of small interference RNAs with or without cationic lipid complex cells or individuals, reduction of the targeted gene expression can be measured.
= Monitoring phenotypic changes of the cell = Measuring changes in mRNA levels using RT-PCR
= Detecting changes in protein levels by immunocytochemistry or Western blot analysis 3, siRNAs, shRNAs and microRNAs production To highly efficiently produce small interface RNA vaccine, using small interference RNA
expression vectors, two oligo deoxynucleotides encoding the highly effective shRNAs, siRNAs and microRNAs sequence which are chosen by screening steps, annealed, and cloned into the vector downstream of the promoter. Small interference RNA
expression vectors can rely on an RNA polymerase III (pol III) promoter to drive the expression of small interference RNA siRNA, shRNA and microRNA. RNA pol III was chosen to drive siRNA expression because it naturally expresses relatively large amounts of small RNAs, it terminates transcription upon incorporating a string of 1-4 uridines, and its transcripts lack poly(A) tails.
= Annealing of the targeted oligonucleotides = Ligation of the annealed oligonucleotides into the vectors = Transformation of expression vectors into E. coli and bacteria fermentation = siRNA, shRNA and microRNA extraction and purification 4, Small interference RNAs delivery Delivery focuses to tissues and cells where the target viruses reside and replicate, somewhat analogous to targeting vaccine delivery to focus immunity in tissues and cells that lie in the viruses' infectious route of entry into an organism. Influenza virus causes disease by infecting respiratory tract and replicating there and FMDV cause disease mainly by infecting respiratory tract and replicating there. Therapies are most appropriately delivered throught respiratory tract by inhalation. Therefore, efficient small interference RNAs delivery and silencing can be achieved by spraying mixed small interference RNAs with cationic lipid based complexes as window period vaccines to the infectious district and respiratory tract of infected, suspicious individuals during window period of virus infection.
5, Confirmation of vaccine effects in RNA interference Suppression can be confirmed at both the mRNA and protein levels.
= The mRNA level: Northern blotting and quantitative, real-time RT-PCR can be used to demonstrate reduction of expression.
= The protein level: Quantitative Western blotting, phenotypic and functional assays are some of the options available to show protein knockdown.
= Negative and positive controls: siRNAs, shRNAs and microRNAs containing a single mismatch can be used as negative controls. Positive controls with small interference RNAs known to exhibit an RNAi effect may also be useful.
6, Timing for applying window period vaccine Timing is very important for applying this kind of vaccines. Applying mixed small interference RNA with cationic lipid based complexes as window period vaccine to the infectious district and infected, suspicious individuals during window period of virus infection could inhibit viruses' multiplication and prevent their epidemics.
THE ADVANGAGE OF THIS INVENTION
Strategies aimed at conferring rapid and efficient protection against Influenza virus and FMDV face some challenges:
= Tremendous harms = Rapid spread = Highly genetic variation The traditional vaccines based on virus inactivation could be effective in preventing disease within 4 to 5 days post-vaccination, due to a critical role for innate immune defenses. Window period vaccines could be as emergency vaccines to prevent and treat these viruses. By imitating viruses' infectious route of entry into an organism, window period vaccines could be sprayed to the respiratory tract of infected and suspicious individuals during window period of virus infection. The direct delivery of window period vaccines into specific cells would save the usage of small interference RNAs and increase the efficiency of silencing of gene expression in the target organ or tissue without activating nonspecific cellular responses, hence presumably minimizing potential toxicity and undesirable side effects. Window period vaccines could also be sprayed in infectious districts. Therapies are most appropriately delivered throught respiratory tract by inhalation. Thus, antiviral strategies based on the specific and rapid inhibition of Influenza virus and FMDV infection could complement and improve the traditional tools.
Claims (9)
1. A kind of vaccines which is applied during window period of virus infection. We name it "window period vaccine".
2. The vaccines of claim 1 wherein said for specifically inhibiting viruses which cause diseases through infecting respiratory tract.
3. The vaccines of claim 1 further comprising Influenza and FMD window period vaccines.
4. The vaccines of claim 1 wherein said applying to the infectious districts, therapies are most appropriately delivered throught respiratory tract by inhalation.
Vaccines could inhibit Influenza virus and FMDV viruses' multiplication and prevent their epidemics.
Vaccines could inhibit Influenza virus and FMDV viruses' multiplication and prevent their epidemics.
5. The vaccines of claim 1 wherein said applying to the infected and suspicious individuals by spraying into respiratory tract would decrease the usage of small interference RNAs and increase the efficiency of silencing of gene expression in the target organ or tissue and would reduce potential toxicity and other side effects.
6. The vaccines of claim 3 consisting of the small interference RNAs with cationic lipid complexes.
7. The vaccines of claim 6 wherein said the small interference RNA molecules including siRNAs, shRNAs and miRNAs which are specifically for the genomic highly conserved regions of influenza virus and FMDV.
8. The vaccines of claim 6 wherein said the small interference RNAs choosing multiple functional conserved regions of influenza virus and FMDV as targets to prevent cross-infection and co-infection of subtypes of viruses.
9. The vaccines of claim 6 wherein said generation of small interference RNAs by transforming small interference RNAs expression vectors into E. coli and bacteria fermentation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2552996 CA2552996A1 (en) | 2006-06-23 | 2006-06-23 | Window period vaccine small interference rnas vaccines for the prevention and treatment of viral respiratory infectious diseases |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2552996 CA2552996A1 (en) | 2006-06-23 | 2006-06-23 | Window period vaccine small interference rnas vaccines for the prevention and treatment of viral respiratory infectious diseases |
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| CA2552996A1 true CA2552996A1 (en) | 2007-12-23 |
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| CA 2552996 Abandoned CA2552996A1 (en) | 2006-06-23 | 2006-06-23 | Window period vaccine small interference rnas vaccines for the prevention and treatment of viral respiratory infectious diseases |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104774856A (en) * | 2011-04-25 | 2015-07-15 | 中国农业科学院兰州兽医研究所 | Interference target sequence with inhibiting effect on foot and mouth disease virus |
-
2006
- 2006-06-23 CA CA 2552996 patent/CA2552996A1/en not_active Abandoned
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104774856A (en) * | 2011-04-25 | 2015-07-15 | 中国农业科学院兰州兽医研究所 | Interference target sequence with inhibiting effect on foot and mouth disease virus |
| CN104774856B (en) * | 2011-04-25 | 2017-12-08 | 中国农业科学院兰州兽医研究所 | There is the interference target sequence of inhibitory action to foot and mouth disease virus |
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