RU2409666C2 - Rnai-modulation of rsv, piv and other respiratory viruses and application thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method of decreasing the levels of protein, mRNA or titre of respiratory syncytial virus (RSV) in an airway cell provides the introduction of a siRNA agent. The siRNA agent contains a sense chain 15-23 nucleotides long and a complementary anti-sense chain 15-23 nucleotides long. The siRNA agent is introduced by inhalation or intranasal spraying. There is also presented a method of decreasing the levels of titre of respiratory syncytial virus (RSV) and parainfluenza virus (PIV). Also, the method provides combined introduction of two siRNA agent.

EFFECT: inventions can be used in medicine.

16 cl, 5 dwg, 1 tbl

Description

Technical field

The invention relates to the field of treatment of respiratory viral infections and to compositions and methods for modulating viral replication and, in particular, to reverse regulation of the expression of the gene (s) of the respiratory virus by oligonucleotides by RNA interference, with local injection into the lungs and nasal cavity by inhalation / intranasal or systemic injection / intravenously.

State of the art

Due to their natural function, the respiratory tract is exposed to a variety of pathogens coming in by airborne droplets, which cause various respiratory diseases. A viral infection of the respiratory tract is the most common cause of hospitalization of children in developed countries, it is estimated in the US that the number of hospitalizations is approximately 91,000 worth $ 300 million. The two main causative agents of respiratory diseases are the human respiratory syntial virus (RSV) and parainfluenza virus (PIV); together they infect the upper and lower respiratory tract, causing croup, pneumonia and bronchiolitis (Openshaw, PJM Respir. Res. 3 (Suppl 1), S15-S20 (2002), Easton, AJ, et al., Clin. Microbiol. Rev. 17, 390-412 (2004)). Only RSV infects up to 65% of children during the first year of life and almost all children during the first two years. RSV is also an important cause of morbidity and mortality in the elderly. Immunity after RSV infection is not complete and long-term, and therefore, repeated infections occur in all age groups. Infants who have had RSV-induced bronchiolitis have an increased risk of wheezing and asthma at an older age. The search for an effective treatment and vaccine against RSV for almost four decades has yielded modest results (Openshaw, PJM. Respir. Res. 3 (Suppl 1), S15-S20 (2002), Maggon, K. et al, Rev. Med. Virol. 14,149-168 (2004)). There are currently no clinically approved vaccines against RSV or PIV. There are also strains of both viruses that infect animals other than humans, such as cattle, goats, pigs and sheep, causing losses in agriculture and the meat and dairy industry (Easton, AJ, et al., Clin. Microbiol. Rev. 17, 390- 412 (2004)).

Both RSV and PIV contain unsegmented genomes consisting of an RNA minus chain and belong to the Paramyxoviridae family. Prevention and treatment is complicated by a number of features of these viruses. Viral genomes mutate at high speed due to the lack of a mechanism for correcting errors in the replication of RNA genomes, which creates significant difficulties for the development of a reliable vaccine or antiviral drug (Sullender, WM Clin. Microbiol. Rev. 13.1-15 (2000)). Work on the development of possible inhibitors of the RSV (F) fusion protein was terminated in part due to the fact that the virus acquires resistance due to mutations that were mapped to the F gene (Razinkov, V., et. Al., Antivir. Res. 55,189-200 (2002), Morton, CJ. Et al. Virology 311, 275-288 (2003)). Both viruses bind to cellular proteins, which creates additional difficulties for obtaining cell-free viral material for vaccination (Burke, E., et al., Virology 252,137-148 (1998), Burke, E., et al., J. Virol 74, 669-675 (2000), Gupta, S., et al., J. Virol. 72,2655-2662 (1998)). Finally, the immunology of both viruses, especially RSV, is extremely complex (Peebles, RS, Jr., et al., Viral. Immunol. 16, 25-34 (2003), Haynes, LM, et al., J. _t Virol. 77 9831-9844 (2003)). The use of denatured RSV proteins as vaccines leads to "immunopotentiation" or an increase in the severity of the disease (Polack, FP et al. J. Exp. Med. 196, 859-865 (2002)), but this phenomenon has been investigated, not excluded for PIV. The problem is generally underestimated due to the recent closure of a number of biopharmaceutical programs against RSV.

The RSV genome consists of single-stranded minus RNA with a length of 15,222 nucleotides and this chain yields eleven major proteins (Falsey, A. R., and Ε. Ε. Walsh, 2000, Clinical Microbiological Reviews 13: 371-84). Two of these proteins, glycoproteins F (fusion) and G (attachment), are the main surface proteins and are most important for stimulating protective immunity. Protein SH (small hydrophobic), protein Μ (matrix) and protein M2 (22 kDa) are associated with the envelope of the virus, but do not cause a protective immune response. Proteins Ν (the main protein bound to the nucleocapsid), Ρ (phosphoprotein), and L (the main polymerase protein) were found to be associated with virion RNA. Two non-structural proteins, NS1 and NS2, are apparently involved in the interaction of the body and the virus, but are not present in the infectious virions.

Human RSV strains are divided into two main groups, A and B. It is shown that the most divergent among RSV proteins is glycoprotein G. It is believed that the variability of RSV glycoprotein G between groups and within two RSV groups plays an important role in the ability of RSV to cause disease outbreaks annually. Glycoprotein G consists of 289-299 amino acids (depending on the RSV strain) and contains an intracellular, transmembrane and high glycosylated skeleton mass of 90 kDa, as well as heparin binding domains. The glycoprotein exists in secreted and membrane-bound forms.

Successful treatments for RSV infection are currently unknown (Maggon and Barik, 2004, Reviews in Medical Virology 14: 149-68). RSV lower respiratory tract infection in most cases goes away by itself. There are no definitive guidelines or criteria regarding the treatment, hospitalization and discharge of children with this infection. To eliminate hypoxia that may occur in the presence of RSV infection, oxygen can be used through the nasal cannula. Mechanical ventilation in children with respiratory failure, shock, or recurrent asphyxia can reduce mortality. Some doctors prescribe steroids. However, several studies have shown that in children hospitalized with bronchiolitis, steroid treatment does not affect the course of the disease. Thus, corticosteroids, alone or in combination with bronchodilators, are ineffective in the treatment of bronchiolitis in patients who do not have concomitant diseases and who are not on mechanical ventilation. Steroids have also been used in children with concomitant heart and lung diseases, such as bronchopulmonary dysphasia and asthma.

Since the mid-1980s, ribavirin, an analogue of guanosine with an antiviral effect, has been used to treat children with brochiolitis caused by RSV, but many studies that evaluated the use of ribavirin have yielded conflicting results. In most centers, ribavirin is now used only in immunocompromised and critically ill patients.

With severe bronchiolitis caused by RSV, the serum concentration of retinol is low, however, tests conducted on hospitalized children with bronchiolitis caused by RSV showed that the administration of vitamin A does not give a positive effect. In therapeutic trials in which patients with lower respiratory tract infection were injected with 1500 mg / kg of anti-RSV immunoglobulin intravenously or 100 mg / kg of anti-RSV immunoglobulin by inhalation, no significant beneficial effects were recorded.

In developed countries, treatment for lower respiratory tract infections caused by RSV is usually limited to symptomatic treatment. Antiviral therapy is usually given only in life-threatening situations, due to its high cost and lack of consensus on effectiveness. In developing countries, oxygen is the primary treatment (where available) and prevention is the only way to reduce mortality.

RNA interference, or “RNAi,” is a term coined by Fire and colleagues to describe the observation that double-stranded RNA (dsRNA) when introduced to worms can block gene expression (Fire et al, Nature 391: 806-811.1998). Short dsRNA controls gene-specific post-transcriptional silencing in many organisms, including vertebrates, and provides a new tool for studying gene function. RNAi has been proposed as a way to create a new class of therapeutic agents. However, to date, most of these assumptions remain mere proposals and it has not been proven that RNAi can be used for medicinal purposes.

Thus, there is a need for safe and effective RSV vaccines, especially for children. There is also a need for therapeutic agents and methods for treating RSV infection at any age and in people with immunodeficiency. There is also a need for scientific methods to characterize the protective immune response to RSV in order to study the pathogenesis of the disease and facilitate the development of therapeutic agents and vaccines. The present invention overcomes the disadvantages of the prior art by providing methods and compositions that effectively modulate or prevent infections caused by RSV and PIV, which can be spread to other respiratory viruses. Specifically, the present invention contributes to the development of the art by providing iRNA agents that are shown to reduce RSV and PIV levels in vivo and by demonstrating the therapeutic effect of this class of molecules. In addition, the possibility of simultaneous treatment of more than one virus has been shown.

findings

The present invention is based on in vivo evidence of the possibility of inhibiting RSV and PIV with intranasal administration of RNAi agents, as well as parenteral administration of such agents. In addition, it has been shown that an effective reduction in virus titer with the simultaneous use of two different iRNA agents can be achieved for more than one virus. Based on these data, the present invention relates to general and specific compositions and methods that can be used to lower RSV or PIV mRNA levels, RSV or PIV protein levels, and RSV and PIV virus titers in an individual, such as a mammal, such as a human. This data can be applied to other respiratory viruses.

The present invention specifically relates to iRNA agents consisting of or containing at least 15 consecutive nucleotides of one of the RSV, PIV or other respiratory virus genes, especially the Ρ RSV or PIV gene and the Ν, G, F, SH, M and L genes RSV. The iRNA agent preferably contains less than 30 nucleotides in the chain, for example 21-23 nucleotides. A double-stranded iRNA agent can either have blunt ends or, more preferably, sticky ends of 1-4 nucleotides at one or both of the 3'-ends of the agent.

In addition, the iRNA agent may contain only natural ribonucleotide subunits or may be synthesized and thus contain one or more sugar or base modifications of one or more ribonucleotide subunits that are included in the agent. The iRNA agent can be further modified to combine with a ligand, such as cholesterol, which is selected to improve the stability, distribution, or uptake of the agent by cells. The iRNA agents may additionally be in isolated form or may be part of a pharmaceutical composition used in the methods described herein, in particular a pharmaceutical composition prepared for delivery to the lungs or nasal cavity or for parenteral administration. The pharmaceutical compositions may contain one or more iRNA agents and, in some embodiments, will contain two or more iRNA agents, each of which is directed against a different respiratory virus, such as RSV and PIV.

The present invention also relates to a method for reducing the level of RSV, PIV or mRNA of another respiratory virus in a cell. The methods of the present invention utilize cellular mechanisms involved in RNA interference that selectively destroy the viral mRNA in the cell and include the step of contacting the cell with one of the antiviral iRNA agents of the present invention. Such methods can be performed directly in the cell or can be performed on an individual mammal by administering to the individual one of the iRNA agents / pharmaceutical compositions of the present invention. A decrease in viral mRNA in cells leads to a decrease in the amount of viral proteins formed and in the body leads to a decrease in the titer of the replicating virus. In the examples section, these methods are demonstrated for PIV and RSV, but these methods can be extended to other respiratory viruses.

In addition, the present invention relates to methods for reducing mRNA levels of two or more respiratory viruses in a cell, where each mRNA comes from a different virus. The present methods utilize cellular mechanisms involved in RNA interference that achieve the selective destruction of the viral mRNA of two different viruses in the cell, and which involve the step of contacting the cell with the two antiviral iRNA agents of the present invention. Such methods can be carried out directly in the cell or can be carried out on an individual mammal by administering to an individual two iRNA agents of the present invention. The decrease in the cells of the viral mRNA of two different viruses leads to a decrease in the amount of viral proteins formed and in the body leads to a decrease in the titer of both replicating viruses. In the examples section, this is proven for PIV and RSV and the simultaneous administration of iRNA agents. This embodiment of the present invention can be applied to any two respiratory viruses.

The methods and compositions of the invention, for example iRNA methods and compositions, can be used with any dosage and / or composition described herein, as well as with any route of administration described herein. It is particularly important that the intranasal administration of an iRNA agent and its ability to inhibit virus replication in respiratory tissues has been demonstrated herein. This data can be applied to another respiratory virus, such as PIV, as shown in the examples, and to other local delivery routes to the lungs, such as inhalation / nebulization.

Details of one or more embodiments of the invention are set forth in the accompanying drawings and description below. Other features, objects, and advantages of the invention will be apparent from the present description, drawings, and claims. The application covers all cited references, patents and patent applications by including them as a reference in full for all purposes.

Brief Description of the Drawings

Figure 1. Titration of antiviral siRNA ex vivo. (a) Immunoblotting of all proteins of A549 cells infected with RSV (ex vivo), with profilin as an internal control. The numbers in the box represent the target mRNA levels Ρ after siRNA treatment, expressed as a percentage of untreated levels. In the following three groups, the virus was introduced 4 hours after siRNA. (b) Pulmonary infectious virus in mice infected with RSV (n = 8 for each result). (c) Pulmonary infectious virus in mice infected with HPIV3 (n = 8 for each result). (d) Same as in (b), except that a naked siRNA without transfection reagent was introduced. Asterisks indicate statistically significant inhibition (P <0.05). siRNAs are described in table 1.

Figure 2. Decreased viral antigens in murine lungs treated with siRNA without IFN activation. ( a ) The virus was introduced 4 hours after siRNA, and viral antigens were detected by indirect immunohistochemistry 4 days after infection (green — RSV; red — HPIV3). (1) imitation of infection, treatment with antibodies to P RSV; (2-6) infected with RSV, treatment with antibodies to P RSV; (7, 8) infected with HPIV3, treatment with antibodies to HPIV3. The following siRNAs (5 nmol - 70 μg) were used: (1, 2) nothing; (3) siRNA # 1 plus TransIT-TKO reagent; (4) siRNA # 1 without reagent; (5) negative control: siRNA plus TransIT-TKO; (6) luc-siRNA plus TransIT-TKO; (7) nothing; (8) siRNA # 4 plus TransIT-TKO. Characteristic lung tissue was removed 5 days after infection. Band = 400 μm. (b) The siRNA # 1 antisense strand was detected by Northern blotting of various amounts of total lung RNA 2 days after the introduction of siRNA using labeled Ρ RSV DNA as a probe. The probe against NS1 RSV did not respond, proving detection specificity. ( c ) Intranasally administered siRNA (10 nmol or 140 μg per mouse) did not activate pulmonary IFN type I (IFN-α) or type II (IFN-γ) above the detection threshold (~ 10 pg / ml), whereas in control lungs RSV infection activated type Π and low levels of type I. Lanes: 1 - siRNA # 1; 2 - siRNA # 4; 3 - Luc siRNA; 4 - absence of siRNA, but RSV infection (error value is shown). Lungs were removed 2 days after siRNA injection and 4 days after infection (n = 4 for each schedule), ( d ) siRNA-mediated inhibition of mixed infection caused by RSV and HPIV3, determined by indirect immunohistochemistry (green - RSV; red - HPIV3). (1, 5) lack of siRNA; (2, 6) siRNA # 1, 5 nanomoles (70 μg); (3, 7) siRNA # 4, 5 nanomoles (70 μg); (4, 8) siRNA # 1 and siRNA # 4, 5 nanomoles of each. (1-4) treated with anti-RSV antibodies; (5-8) treated with antibodies to HPIV3. The virus was introduced 4 hours after siRNA, and lung tissue was examined 4 days after infection. Band = 400 μm.

Figure 3. Competitive inhibition of the virus by a high concentration of siRNA in mixed infection caused by RSV and HPIV3. (a) real-time PCR (ex vivo ); (b) immunoblotting ( ex vivo); (c) pulmonary immunoblotting with goat anti-virus antibodies. The intensity of the band corresponding to the viral protein Ν was quantified and expressed as a percentage in lung samples untreated with siRNA. The virus was introduced 4 hours after siRNA, and lung tissue was removed 5 days after infection (n = 4 for each result). Black graph - RSV; white chart is HPIV3. Standard errors as shown.

Figure 4. Weakening lung pathology and reducing the asthma marker in mice treated with siRNA # 1. (a) respiratory rate; (b) Pulmonary histopathology; (c) Leukotriene. Ρ < 0.002 in all analyzes; n = 4 for each result; The standard error value is shown. The virus was introduced 4 hours after siRNA (70 μg). Mice treated with negative siRNA control were indistinguishable from untreated siRNA (data not shown).

Figure 5. The therapeutic effect of siRNA in RSV disease. Changes in (a) body weight and (b) lung virus titer during RSV infection in mice. The standard error value is shown; n = 6 for each result. Arrows indicate the day of siRNA administration (70 mcg).

DETAILED DESCRIPTION

For ease of description, the term “nucleotide” or “ribonucleotide” is sometimes used to refer to one or more monomeric subunits of an RNA agent. It is understood that the use of the term “ribonucleotide” or “nucleotide” herein may, in the case of a modified RNA or nucleotide substituent, also refer to a modified nucleotide or substitution residue, as described below, in one or more positions.

An “RNA agent” as used herein is unmodified RNA, modified RNA, or nucleoside substituents, all of which are described herein or are known in the art of synthetic RNA. Although many modified RNAs and nucleoside substituents have been described, preferred examples include those that are more resistant to nuclease cleavage than unmodified RNAs. Preferred examples include those that have a 2'-sugar modification, a single-chain protrusion modification, preferably a single-chain 3'-protrusion, or, especially in single chain, a 5'-modification that affects one or more phosphate groups or one and more analogues of the phosphate group.

An “iRNA agent”, sometimes referred to as an “RNAi agent” (short for “interfering RNA agent”), as used herein, is an RNA agent that can reverse regulate the expression of a target gene, for example, RSV. Not wishing to be bound by theory, an iRNA agent can act according to one or more of many mechanisms, including post-transcriptional cleavage of the target mRNA, sometimes referred to in the art as RNAi, or pre-transcriptional or pre-translational mechanisms. An iRNA agent may contain one chain or may contain more than one chain, for example, an iRNA agent may be a double-stranded (ds) iRNA agent. If the iRNA agent is single-stranded, it is particularly preferred that it contains a 5'-modification that affects one or more phosphate groups or one or more analogues of the phosphate group.

A “single-stranded iRNA agent,” as used herein, is an iRNA agent that consists of a single molecule. It may contain a double region that has arisen due to the formation of pairs within the chain, for example, it may be or contain a hairpin structure or a structure in the form of a handle of a pan. Single stranded iRNA agents are preferably antisense with respect to the target molecule.

“Ds iRNA agent” (short for “double-stranded iRNA agent”), as used herein, is an iRNA agent that contains more than one and, preferably, two chains in which interchain hybridization can form a region with a duplex structure .

Isolated iRNA agents described herein, including ds iRNA agents and siRNA agents, can mediate silencing of a target gene, for example, through RNA degradation. For convenience, such RNA is also referred to herein as RNA undergoing silencing. Such a gene is also referred to as a target gene. Preferably, the RNA subjected to silencing is a gene product of the endogenous RSV gene.

As used herein, the phrase “mediates RNAi” refers to the ability of an agent to induce, in a sequence-specific manner, silencing of a target gene. “Target gene silencing” means the process by which a cell, in the absence of contact with an agent containing and / or secreting a specific product of a target gene, after contact with the agent will contain and / or secrete at least 10%, 20%, 30 %, 40%, 50%, 60%, 70%, 80% or 90% less of such a gene product, compared with a similar cell that has not been in contact with the agent. Such a target gene product may, for example, be messenger RNA (mRNA), a protein, or a regulatory element.

In the antiviral applications of the present invention, silencing the target gene will result in a reduction in the “virus titer” in the cell. As used herein, “reduction in virus titer” refers to a decrease in the amount of viable virus produced by a cell or found in the body that undergo silencing of a target gene of a virus. A decrease in the cellular amount of the resulting virus will preferably lead to a decrease in the amount of measurable virus produced in the tissues of the treated object and a decrease in the severity of the symptoms of the viral infection. The iRNA agents of the present invention are also referred to as “antiviral iRNA agents”.

As used herein, an “RSV gene” refers to any of the genes identified in the viral RSV genome (see Falsey, A. R., and Ε. Walsh, 2000, Clinical Microbiological Reviews 13: 371-84). These genes are known in the art and include the genes F, G, SH, Μ, Ν, Ρ and L.

As used herein, a “PIV gene" refers to any of the genes identified in the PIV viral genome (See GenBank Accession # NC_001796). These genes are known in the art and include the genes N, P, C, D, V, M, F, HN and L.

As used herein, “respiratory virus” refers to viruses that replicate in cells of the respiratory system. Such viruses include, but are not limited to RSV, PIV, influenza virus, metapneumovirus, adenovirus and coronavirus (such as SARS).

As used herein, the term “complementary” is used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between the compound of the invention and the target RNA molecule, for example RSV, PIV or another respiratory virus mRNA molecule. Specific binding needs a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to sequences other than the target, under conditions in which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays , under the conditions under which the analyzes are performed. Sequences different from the targets typically differ by at least 4 nucleotides.

As used herein, an iRNA agent is “sufficiently complementary” to an RNA target, such as an mRNA target (eg, an RSV or PIV mRNA target), if the iRNA agent reduces the formation of the protein encoded by the target RNA in the cell. An iRNA agent can also be "strictly complementary" (excluding SRMS containing subunit (s)), for example, an RNA target and an iRNA agent are annealed, preferably to form a hybrid made exclusively from Watson-Crick base pairs in the strict region complementarity. A “sufficiently complementary” iRNA agent may contain an internal region (for example, at least 10 nucleotides) that is strictly complementary to the viral target RNA. In addition, in some embodiments, the iRNA agent specifically recognizes a single nucleotide difference. In this case, the iRNA agent mediates RNAi only if the region ( for example, within 7 nucleotides), which differs by one nucleotide, has strict complementarity. Preferred iRNA agents will be based on, or composed of, or comprised of, the sense and antisense sequences presented in the examples.

As used herein, “substantially identical” if reference is made to the first nucleotide sequence compared to the second nucleotide sequence, means that the first nucleotide sequence is identical to the second nucleotide sequence, with the exception of one, two or three nucleotide substitutions (for example, substitution adenosine to uracil).

As used herein, “individual” refers to a mammalian organism being treated for a disorder mediated by expression of a virus, such as RSV or PIV infection, or being treated prophylactically to prevent a viral infection. An individual may be any mammal, such as a primate, cow, horse, mouse, rat, dog, pig, goat. In a preferred embodiment, the individual is a human.

As used herein, treatment of RSV infection, PIV infection, or other infection caused by a respiratory virus refers to the improvement of any biological or pathological parameters that are 1) partially mediated by the presence of the virus in the individual and 2) the result of which can be influenced by lower levels of viral protein present.

As used herein, “co-administration” refers to the administration to an individual of two or more agents, and in particular two or more iRNA agents. The agents may be contained in a single pharmaceutical composition and administered at the same time, or the agents may be contained in separate compositions and administered to an individual sequentially. While these two agents can be detected in the individual at the same time, these two agents are called applied together.

Since silencing is mediated by an iRNA agent, after application of the composition, the iRNA agent can last for several days, in many cases the composition can be administered with a frequency of less than once a day, or in some cases only once for the entire therapeutic regimen.

Design and selection of iRNA tools

The present invention is based on the demonstration of targeted gene silencing of a respiratory virus gene in vivo after local injection of an iRNA agent into the lungs and nasal cavity either by intranasal administration / inhalation or systemically / parenterally by injection and resulting treatment of viral infection. The present invention further extends to the use of iRNA agents for more than one respiratory virus and the treatment of both viral infections by co-administration of two or more iRNA agents.

Based on these results, the invention specifically relates to an iRNA agent that can be used in the treatment of a viral infection, in particular caused by respiratory viruses, and in particular, RSV or PIV infection, in an isolated form and in the form of a pharmaceutical composition described below. Such agents will comprise a sense strand having at least 15 consecutive nucleotides that are complementary to the viral gene, and an antisense strand having at least 15 consecutive nucleotides that are complementary to the sense strand sequences. Especially applicable are iRNA agents that contain the nucleotide sequence of the белка RSV or PIV protein gene. Other RSV target genes include the F, G, SH, Μ, Ν, and L genes. Other PIV genes include the N, P, C, D, V, M, F, HN, and L genes. The exemplary agents are shown in the table. one.

Candidates for iRNA agents can be constructed by performing, for example, stepwise gene analysis of viral genes that will serve as an iRNA target. Candidate overlapping, adjoining, or proximal means corresponding to all or some part of the transcribed region can be created and tested. Each of the iRNA agents can be tested and evaluated for their ability to reverse regulate the expression of the target gene (see below, “Evaluation of the candidate for iRNA agents”).

An iRNA agent can be rationally designed based on sequence information and desired characteristics. For example, an iRNA agent can be designed according to the relative melting temperature of the candidate duplex. In general, a duplex should have a lower melting temperature of the 5'-end of the antisense chain than the 3'-end of the antisense chain.

Accordingly, the present invention relates to iRNA agents comprising a sense strand and an antisense strand, each of which contains a sequence of at least 15, 16, 17, 18, 19, 20, 21 or 23 nucleotides in length, which is essentially identical as defined above, portions of the respiratory virus gene, especially the Ρ RSV or PIV protein genes. Examples are iRNA agents, which include those that contain 15 consecutive nucleotides of one of the agents provided in Table 1.

The length of the antisense chain of the iRNA agent should be at least 15, 16, 17, 18, 19, 25, 29, 40, or 50 nucleotides. Its length should be no more than 50, 40 or 30 nucleotides. Preferred length ranges are 15-30, 17-25, 19-23 and 19-21 nucleotides. In several embodiments, the implementation of the tool will contain 15 nucleotides of one of the funds shown in table 1.

The length of the sense chain of an iRNA agent must be equal to or equal to at least 15, 16, 17, 18, 19, 25, 29, 40, or 50 nucleotides. Its length should be equal to or be no more than 50, 40 or 30 nucleotides. Preferred length ranges are 15-30, 17-25, 19-23 and 19-21 nucleotides. In several embodiments, the implementation of the tool will contain 15 nucleotides of one of the funds shown in table 1.

The length of the double-stranded part of the iRNA agent must be equal to or equal to at least 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 50 nucleotide pairs. Its length should be equal to or be no more than 50, 40 or 30 pairs of nucleotides. Preferred length ranges are 15-30, 17-25, 19-23 and 19-21 a pair of nucleotides.

The agents shown in table 1 have a chain length of 21 nucleotides. iRNA agents contain a double-stranded region of 19 nucleotides in size and a protrusion at each 3′-end of 2 nucleotides in size. These agents can be modified as described herein to obtain equivalent agents containing at least a portion of these sequences and / or modifications of the bases and bonds of oligonucleotides.

In general, the iRNA agents of the present invention contain a region of sufficient complementarity with the virus gene, for example, Ρ RSV or PIV protein, and of sufficient length based on the number of nucleotides, so that the iRNA agent or its fragment can mediate reverse regulation of a specific viral gene. The antisense strands of the iRNA agents of the present invention are preferably fully complementary to the viral gene mRNA sequences, and Ρ RSV and PIV protein herein. However, absolute complementarity of the iRNA agent and the target is not required, but matching should be sufficient to allow the iRNA agent or its cleavage product to control sequence-specific silencing, for example, by means of RNAi cleavage of RSV mRNA.

Thus, iRNA agents of the present invention encompass agents comprising a sense strand and an antisense strand, each of which contains a sequence of at least 16, 17 or 18 nucleotides that is substantially identical, as defined below, to one of the viral gene sequences, in particular, protein P RSV or PIV, except that no more than 1, 2 or 3 nucleotides in the chain are respectively substituted by other nucleotides (for example, adenosine is replaced by uracil), while the ability to inhibit ex RSV compression in cultured human cells, as defined below. These agents will thus contain at least 15 nucleotides that are identical to one of the viral gene sequences, especially the RSV or PIV gene P gene, with the exception of 1, 2 or 3 bases that do not match the sequence of the viral target mRNA or differ between the sense and antisense chain. Mismatches with the sequence of the viral target mRNA, in particular in the antisense strand, are more permissible in the terminal regions, and if present, they are preferably in the terminal region or regions, for example, within 6, 5, 4 or 3 nucleotides with 5'- and / or 3'-end, most preferably within 6, 5, 4 or 3 nucleotides from the 5'-end of the sense strand or the 3'-end of the antisense strand. It is necessary that the sense strand be only sufficiently complementary to the antisense strand to support the general double-stranded nature of the molecule.

Preferably, the sense and antisense chains are chosen such that at one or both ends of the molecule of the iRNA agent there is a single-chain or unpaired region, such as those presented in Table 1. Thus, the iRNA agent contains a sense and antisense chain, preferably paired so to contain a protrusion, for example one or two 5'- or 3'-protrusions, but preferably a 3'-protrusion of 2-3 nucleotides. Most embodiments will have a 3'-overhang. Preferred siRNA agents will have single-stranded protrusions, preferably a 3'-protrusion of 1-4 length, or preferably 2 or 3 nucleotides, at one or both ends of the iRNA agent. The protrusions can be formed as a result of the fact that one chain is longer than the other, or as a result of the fact that both chains of the same length are located by a ledge. Preferably, the 5'-ends are phosphorylated.

The preferred length of the duplex region is from 15 to 30, most preferably 18, 19, 20, 21, 22, and 23 nucleotides, for example, in the range of siRNA agents, as discussed above. Also included are embodiments in which two siRNA agent chains are linked, for example, covalently linked. Also included in the scope of the invention is a hairpin or other single-stranded structures that create the desired double-stranded region and preferably a 3 ′ protrusion.

Evaluation of a candidate for iRNA tools

A candidate for an iRNA agent can be assessed by its ability to reverse regulate target gene expression. For example, an iRNA candidate candidate can be obtained and brought into contact with a cell, for example, a human cell that has been or will be infected with a virus of interest, for example, a virus containing a target gene. Alternatively, the cell can be transfected with a construct on which the viral target gene is expressed, thereby eliminating the need for a model of virus infectivity. The level of expression of the target gene before and after contact with the candidate for iRNA agents can be compared, for example, by mRNA, protein level or virus titer. If it is determined that the amount of RNA, protein, or virus expressed by the target gene after contact with the iRNA agent is lower, then it can be concluded that the iRNA agent inversely regulates the expression of the target gene. The level of the viral target RNA or viral protein in the cell or the titer of the virus in the cell or tissue can be determined by any desired method. For example, the level of the target RNA can be determined by Northern blotting, reverse transcription polymerase chain reaction (RT-PCR), branched DNA method or RNase protection method. Protein levels can be determined, for example, by Western blotting or immunofluorescence. The titer of the virus can be determined by the formation of plaques.

Stability testing, modification and retesting of iRNA tools

A candidate for an iRNA agent can be evaluated for stability, for example, susceptibility to cleavage with an endonuclease or exonuclease, such as is observed when an iRNA agent is introduced into an individual. Methods can be used to identify sites that are susceptible to modification, especially cleavage, for example cleavage by a component found in an individual.

If cleavage sites are identified, an additional iRNA agent can be developed and / or synthesized in which the potential cleavage site is rendered resistant to cleavage, for example by introducing 2'-modifications to the cleavage site, for example, the 2'-O-methyl group. This additional iRNA agent can be re-tested for stability, and this process can be repeated until an iRNA agent exhibiting the desired stability is found.

In vivo testing

In an iRNA agent identified as capable of inhibiting RSV gene expression, in vivo functionality can be tested in an animal model (e.g., a mammal such as a mouse or rat), as shown in the examples. For example, an iRNA agent can be administered to an animal, and an iRNA agent is evaluated for its biodistribution, stability, and ability to inhibit the expression of viral genes, such as RSV or PIV, or to lower the titer of the virus.

The iRNA agent can be introduced directly to the target tissue, for example by injection, or the iRNA agent can be introduced to the animal model in the same way that it would be administered to humans. As shown herein, the agent may preferably be administered by inhalation as a treatment for viral infection.

An iRNA agent can also be evaluated for its intracellular distribution. Evaluation may involve determining whether an iRNA agent has entered the cell. The evaluation may also include determining the stability (e.g., half-life) of the iRNA agent. In vivo evaluation of an iRNA agent can be facilitated by using an iRNA agent conjugated to a traceable marker (e.g., a fluorescent marker such as fluorescein; a radioactive label such as ³⁵ S, ³² P, ³³ P or ³ N; gold particles; or antigen particles for immunohistochemistry).

An iRNA agent useful for tracking biodistribution may not actively induce gene silencing in vivo. For example, an iRNA agent can be targeted to a gene that is not in the animal (for example, the target of an iRNA agent introduced into a mouse may be luciferase), or an iRNA agent can have a nonsense sequence that is not targeted to any gene, for example any endogenous gene.

The localization / biodistribution of iRNA can be monitored, for example, using a traceable label attached to an iRNA agent, such as the traceable substance described above.

An iRNA agent can be evaluated for its ability to reverse regulate viral gene expression. In vivo viral gene expression levels can be measured, for example, by in situ hybridization or RNA isolation from tissue before and after exposure to an iRNA agent. Where the animal must be killed to obtain tissue, a control animal that has not received treatment will be used for comparison. The viral target mRNA can be detected by any desired method, including but not limited to RT-PCR, Northern blotting, branched DNA, or RNase protection. Alternatively or in addition, viral gene expression can be monitored by performing Western blotting with tissue extracts treated with an iRNA agent. Virus titer can be measured by counting PFU (plaque forming units).

Chemistry iRNA

The individual iRNA agents described herein are, for example, RNA molecules (double-stranded; single-stranded) that mediate RNAi to inhibit the expression of a viral gene, for example, RSV or PIV protein P.

The RNA agents discussed herein include otherwise unmodified RNAs, as well as RNAs that have been modified, for example, in order to improve efficiency, and polymers of nucleoside substituents. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases and phosphate residues, are the same or essentially the same as those found in nature, preferably those that naturally occur in the human body. The prior art mentions rare or unusual, but naturally occurring RNAs as modified RNAs, see, for example, Limbach et ah, (1994) Nucleic Acids Res. 22: 2183-2196. Such rare or unusual RNAs, which are often called modified RNAs (obviously because they are usually the result of post-transcriptional modification), remain within the term unmodified RNA, which is used in this document. Modified RNA, as used herein, refers to a molecule in which one or more components of a nucleic acid, namely sugars, bases and phosphate residues, are different from natural ones, preferably different from those found in the human body. Although they are called modified “RNAs,” as a result of the modification they will, of course, contain molecules that are not RNAs. Nucleoside substituents are molecules in which the ribophosphate backbone is replaced by a non-ribophosphate construct that allows the bases to be in the correct spatial relationship, such that hybridization is essentially similar to that observed with the ribophosphate backbone, for example, uncharged ribophosphate backbone simulators. Examples of each of the above are discussed herein.

Modifications described herein can be incorporated into any double-stranded RNA and RNA-like molecule described herein, for example, an iRNA agent. It may be desirable to change one or both of the antisense and sense chains of the iRNA agent. Since nucleic acids are polymers of subunits or monomers, many of the modifications described below occur at a position that repeats within the nucleic acid, for example, modifications of the base or phosphate residue or non-Ο phosphate residue. In some cases, the modification will occur in all dependent positions in the nucleic acid, but in many and in most cases this will not happen. For example, modification can occur only at the 3 'or 5'-terminal position, can occur only in the terminal region, for example, at the position of the terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of the chain. Modification can occur in the double chain region, the single chain region, or both. For example, phosphorothioate modification in the unbound position Ο can occur only at one or both ends, can occur only in terminal regions, for example, in the position of a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a chain, or it can occur in regions double or single chain, especially at the ends. Similarly, modification can occur in the sense chain, antisense chain, or both. In some cases, the sense and antisense chains will have the same modifications or the same class of modifications, but in other cases the sense and antisense chains will have different modifications, for example, in some cases it may be desirable to change only one chain, for example, the sense chain.

The two main goals for introducing modifications to iRNA agents are their stabilization against degradation in biological media and the improvement of pharmacological properties, for example, pharmacodynamic properties, which are further discussed below. Other suitable modifications to the sugar, base or scaffold of an iRNA agent are described in co-owned PCT Application No. PCT / US2004 / 01193, filed January 16, 2004. An iRNA agent may contain a non-naturally occurring base, such as those described in co-owned PCT Application No. PCT / US2004 / 011822, filed April 16, 2004. An iRNA agent may contain an unnatural sugar, such as a non-carbohydrate ring carrier molecule. Typical features of non-naturally occurring sugar for use in iRNAs are described in co-owned PCT Application No. PCT / US2004 / 011829, filed April 16, 2003.

An iRNA agent may contain an internucleotide bond ( e.g., a chiral phosphorothioate bond) useful for enhancing resistance to nucleases. In addition, or alternatively, to increase resistance to nucleases, the iRNA agent may contain a ribose mimic. Typical internucleotide bonds and ribose mimetics for increasing resistance to nucleases are described in co-owned PCT Application No. PCT / US2004 / 07070, filed March 8, 2004.

An iRNA agent may contain ligand-conjugated monomer subunits and monomers for oligonucleotide synthesis. Typical monomers are described in co-owned U.S. Patent Application No. 10/916185, filed August 10, 2004.

An iRNA agent may have a ZXY structure, such as described in co-owned PCT application PCT / US2004 / 07070, filed March 8, 2004.

An iRNA agent can complex with an amphiphilic residue. Typical amphiphilic residues for use with iRNA agents are described in co-owned PCT Application No. PCT / US2004 / 07070, filed March 8, 2004.

In another embodiment, the iRNA agent may complex with an agent that is characterized by a modular complex. The complex may comprise a carrier agent bonded to one or more (preferably two or more, more preferably all three) of (a) a condensing agent ( e.g., an agent capable of attracting, e.g., binding, nucleic acid, e.g., by ionic or electrostatic interaction); (b) means of fusion ( for example, means that can merge and / or transported through the cell membrane); and (c) a targeting group, for example, an agent aimed at the cell or tissue, for example, a lectin, glycoprotein, lipid or protein, for example an antibody that binds to cells of a particular type. iRNAs that form a complex with a delivery vehicle are described in co-owned PCT Application No. PCT / US2004 / 07070, filed March 8, 2004.

An iRNA agent can form non-canonical compounds, such as compounds between the sense and antisense sequences of the iRNA duplex. Typical features of noncanonical iRNAs are described in PCT's co-owned PCT Application No. PCT / US2004 / 07070, filed March 8, 2004.

Increased Nuclease Resistance

An iRNA agent, for example an iRNA agent that targets RSV, may have increased resistance to nucleases. One way to increase resistance is to identify cleavage sites and modify these sites in order to inhibit cleavage. For example, 5'-UA-3 ', 5'-UG-3', 5'-CA-3 ', 5'-UU-3 or 5'-CC-3' dinucleotides may serve as cleavage sites.

To increase the resistance to nucleases and / or the binding affinity of the target, an iRNA agent, for example sense and / or antisense chains of an iRNA agent, may contain, for example, 2'-modified ribose units and / or phosphorothioate bonds. For example, 2'-hydroxyl groups (OH) can be modified or replaced by a number of different “hydroxy” or “deoxy” substituents.

Examples of the “oxy” modification of the 2'-hydroxyl group include alkoxy or aryloxy (OR, for example, R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroalkyl or sugar); polyethylene glycols (PEG), O (CH ₂ CH ₂ O) _n CH ₂ CH ₂ OR; “Locked” nucleic acids (LNAs) in which the 2'-hydroxyl is connected, for example, by a methylene bridge to the 4 ′ carbon atom of the same ribose sugar; O-AMINE (AMINE = NH ₂ ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino) and aminoalkoxy, O (CH ₂ ) _n AMINE (e.g. AMINE = NH ₂ ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino). It is noteworthy that oligonucleotides containing only a methoxyethyl group (MOE), (OCH ₂ CH ₂ OCH ₃ , a derivative of PEG), exhibit resistance to nucleases comparable to that modified by robust modification of phosphorothioate.

“Deoxy” modifications include hydrogen (for example, deoxyribose sugars, which are of particular importance for protruding regions of partially ds RNA); halogen (e.g. fluorine); amino (e.g., NH ₂ ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino or amino acid); NH (CH ₂ CH ₂ NH) _n CH ₂ CH ₂ -AMINE (AMINE = NH ₂ ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino), -NHC (O) R (R = alkyl, cycloalkyl, aryl , aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl thioalkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which optionally may be substituted, for example, with an amine functional group. Preferred substituents are 2′-methoxyethyl, 2′-OCH ₃ , 2′-O-allyl, 2′-C-allyl and 2′-fluoro.

To maximize resistance to nucleases, 2'-modifications can be used in combination with one or more modifications of a phosphate linker (e.g., phosphorothioate). The so-called "chimeric" oligonucleotides are oligonucleotides that contain two or more different modifications.

In certain embodiments, all pyrimidines of the iRNA agent carry 2′-modifications and therefore the iRNA agent has increased resistance to endonucleases. Increased resistance to nucleases can also be achieved by modification of 5'-nucleotides, which results, for example, at least one 5'-uridine-adenine-3 '- (5'-UA-3') dinucleotide in which uridine is 2'-modified nucleotide; at least one 5'-uridine-guanine-3 '- (5'-UG-3') dinucleotide in which 5'-uridine is a 2'-modified nucleotide; at least one 5'-cytidine-adenine-3 '- (5'-CA-3') dinucleotide, where 5'-cytidine is a 2'-modified nucleotide; at least one 5'-uridine-uridin-3 '- (5'-UU-3') dinucleotide in which 5'-uridine is a 2'-modified nucleotide; or at least one 5'-cytidine-cytidin-3 '- (5'-CC-3') dinucleotide, where 5'-cytidine is a 2'-modified nucleotide. An iRNA agent may contain at least 2, at least 3, at least 4, or at least 5 such dinucleotides.

The incorporation of furanose sugar into the oligonucleotide framework can also reduce endonuclease cleavage. The iRNA agent can be further modified by the inclusion of a 3'-cationic group or by inverting the nucleoside of the 3'-end with a 3'-3'-bond. According to another alternative, the 3'-end may be blocked by an aminoalkyl group, for example a 3'-C5-aminoalkyl dT. Other 3'-conjugates can inhibit 3'-5'-cleavage by exonucleases. Without being bound by theory, a 3'-conjugate, such as naproxen or ibuprofen, can inhibit exonuclease cleavage by sterically blocking the binding of exonuclease to the 3'-end of the oligonucleotide. Even small alkyl chains, aryl groups or heterocyclic conjugates, or modified sugars (D-ribose, deoxyribose, glucose, etc.) can block 3'-5'-exonucleases.

Similarly, 5'-conjugates are able to inhibit 5-3'-cleavage by exonucleases. Without being bound by theory, a 5'-conjugate, such as naproxen or ibuprofen, can inhibit exonuclease cleavage by sterically blocking the binding of exonuclease to the 5'-end of the oligonucleotide. Even small alkyl chains, aryl groups or heterocyclic conjugates, or modified sugars (D-ribose, deoxyribose, glucose, etc.) can block 3'-5'-exonucleases.

An iRNA agent may have enhanced resistance to nucleases if the duplex iRNA agent at least at one end contains a single stranded nucleotide protrusion. In preferred embodiments, the nucleotide protrusion comprises 1-4, preferably 2-3, unpaired nucleotides. In a preferred embodiment, an unpaired single-stranded protrusion nucleotide that is directly adjacent to a terminal pair of nucleotides contains a purine base and the terminal nucleotide pair is a G-C pair, or at least two of the last four complementary nucleotide pairs are G-C pairs. In further embodiments, the nucleotide protrusion may have 1 or 2 unpaired nucleotides, and in a typical embodiment, the nucleotide protrusion is 5′-GC-3 ′. In preferred embodiments, the nucleotide protrusion is at the 3 ′ end of the antisense strand. In one embodiment, the iRNA agent comprises a 5'-CGC-3 'motif at the 3'-end of the antisense strand, such that a 2-nt protrusion of 5'-GC-3' is formed.

Thus, an iRNA agent may contain monomers that have been modified to inhibit cleavage, for example, by nucleases, for example endonucleases or exonucleases found in an individual. These monomers are referred to herein as NRM or monomers promoting nuclease resistance or modification. In many cases, these modifications will also affect other properties of the iRNA agent, for example, the ability to interact with a protein, such as a transport protein, such as serum albumin or a member of RISC, or the ability of the first and second sequences to duplex with each other or to duplex with another sequence, for example a target molecule.

Modifications that may be applicable to obtain an iRNA agent that meets the preferred criteria for resistance to nucleases outlined above may include one or more of the following chemical and / or stereochemical modifications of the sugar, base and / or phosphate backbone:

(i) chiral (Sp) thioates. Thus, preferred NRMs include nucleotide dimers enriched or pure in a particular chiral form with a modified phosphate group containing a heteroatom in a non-bonding position, e.g., Sp or Rp, at position X, which is a position normally occupied by oxygen. The atom in X may also be S, Se, Nr ₂ or Br ₃ . If X is S, an enriched or chiral pure Sp bond is preferred. "Enriched" means "containing at least 70, 80, 90, 95 or 99% of the preferred form." Such NRMs are discussed in more detail below;

(ii) the addition of one or more cationic groups to a sugar, base and / or phosphorus atom of a phosphate or a modified residue of a phosphate scaffold. Thus, preferred NRMs include derivatives of monomers substituted at the terminal position with a cationic group. Since the 5'-end of the antisense sequence must end with an -OH or phosphate group, the 5'-end of the antisense sequence is preferably not used in this NRM. The group should be attached to the base position, which minimizes interference with the formation of Η-bonds and hybridization, for example, away from the side that interacts with the complementary base of the second chain, for example, at position 5 ′ of pyrimidine or position 7 of purine. They are discussed in more detail below;

(iii) non-phosphate bonds at the ends. Accordingly, preferred NRMs contain non-phosphate bonds, for example, a 4-atom bond, which gives a higher resistance to cleavage than a phosphate bond. Examples include 3 'CH ₂ -NCH ₃ -O-CH ₂ -5'-and 3'-CH ₂ -NH- (O =) - CH ₂ -5';

(iv) 3'-compounds of thiophosphates and 5'-compounds of thiophosphates. Accordingly, preferred NRMs may contain these structures;

(v) L-RNA, 2'-5'-bonds, inverted bonds, a-nucleosides. Accordingly, other preferred NRMs comprise L nucleosides and dimeric nucleotides derived from L nucleosides; 2'-5'-phosphate, non-phosphate and modified phosphate bonds (e.g., thiophosphates, phosphoramidates and boronophosphates); dimers having inverted bonds, for example 3'-3'-or 5'-5'-bonds; monomers having an alpha bond at the 1 ′ position of sugar, for example, the structures described herein and having an alpha bond;

(vi) conjugated groups. Accordingly, preferred NRMs may contain, for example, a targeting moiety or a conjugated ligand described herein conjugated to a monomer, for example via sugar, base or scaffold;

(vi) non-core relationships. Accordingly, preferred NRMs may include a non-basic monomer, for example, a non-basic monomer as described herein (for example, a monomer that does not contain a nucleic base); aromatic or heterocyclic or polyheterocyclic aromatic monomer as described herein; and

(vii) 5′-phosphonates and 5′-phosphate prodrugs. Accordingly, preferred NRMs contain monomers, preferably in the terminal position, for example at the 5'-position, in which one or more phosphate atoms are protected by a protective group, and this protective group or groups are removed as a result of the action of a component in the body of an individual, for example, carboxyesterase or an enzyme present in an individual. For example, a phosphate prodrug, in which carboxy esterase cleaves a protected molecule, leads to the formation of a thioate anion, which attacks a carbon atom adjacent to O phosphate, which leads to the formation of unprotected phosphate.

One or more different NRM modifications may be introduced into an iRNA agent or sequence of an iRNA agent. In an iRNA sequence or tool, an NRM modification may be used more than once. Since some NRMs interfere with hybridization, the total number of NRMs included in the composition must be such as to maintain acceptable levels of duplex formation of the iRNA agent.

In some embodiments, NRM modifications are introduced at a terminal cleavage site or at a cleavage region of a sequence (sense strand or sequence) that is not targeted to the desired sequence or gene of an individual. This reduces silencing for non-target sequences.

Nuclease-resistant modifications include some that can only be placed at the end, and others that can be in any position. Typically, modifications that can inhibit hybridization are used only in terminal regions and preferably not in the cleavage site or in the cleavage region of a sequence that targets a sequence or individual gene. They can be used in any part of the sense sequence, provided that a sufficient degree of hybridization between the two sequences of the iRNA agent is maintained. In some embodiments, it is desirable to place the NRM at the cleavage site or at the cleavage site of a sequence that is not targeted to the sequence or the individual gene, as this can minimize silencing outside the target sequence.

In most cases, modifications that promote resistance to nucleases will be distributed differently depending on whether the sequence is aimed at the sequence in the individual (often called the antisense sequence) or will not be aimed at the sequence in the individual (often called the sense sequence). If a sequence is to be targeted at sequences in an individual, modifications that interfere with cleavage or inhibit cleavage by endonucleases should not be inserted into a region that is an individual of cleavage mediated by RISC, for example, a cleavage site or cleavage region (as described in Elbashir et al., 2001, Genes and Dev. 15: 188, hereby incorporated by reference). Target cleavage occurs approximately in the middle of the 20 or 21 nt “leading” RNA, or approximately 10 or 11 nucleotides above the first nucleotide, which is complementary to the leading sequence. As used herein, a cleavage site refers to a nucleotide located on either side of the cleavage site, on a target, or on an iRNA agent chain that is hybridized with it. The cleavage region means a nucleotide with 1, 2 or 3 nucleotides of the cleavage site, in any direction.

Such modifications may be embedded in terminal regions, for example, in a terminal position or in the terminal positions 2, 3, 4 or 5 of a sequence that is targeted, or a sequence that is not aimed at a sequence in an individual.

Related Ligands

The properties of the iRNA agent, including its pharmacological properties, can be modified and adapted for a specific application by, for example, the introduction of ligands, for example, bound ligands.

A variety of objects can be associated with an iRNA agent, for example, ligands, for example, those associated with a ligand conjugated monomeric subunit carrier. Examples are described below in the context of a ligand conjugated monomeric subunit, but it is only preferred that the objects can be attached to the iRNA agent at other points.

Preferred residues are ligands, which are preferably connected covalently, either directly or indirectly via an intermediate bond, to a carrier. In preferred embodiments, the ligand is coupled to the carrier via an intermediate bond. The ligand or bound ligand may be present on the ligand conjugated monomer when the ligand conjugated monomer is included in the growing chain. In some embodiments, the ligand may be included in the “precursor” of the ligand-conjugated monomer subunit after the “precursor” of the ligand-conjugated monomer subunit is incorporated into the growing chain. For example, a monomer having, for example, a bond terminating in an amino group, for example TAP- (CH2) _n NH ₂ , can be integrated into a growing sense or antisense chain. In a subsequent step, that is, after incorporation of the monomer unit of the precursor into a chain, a ligand having an electrophilic group, for example a pentafluorophenyl ester group or an aldehyde group, can be attached to the precursor of the ligand-conjugated monomer by connecting the electrophilic group of the ligand to the terminal nucleophilic group of the conjugated precursor bond of the precursor with a ligand of a monomeric subunit.

In preferred embodiments, the ligand affects the distribution, targeting, or duration of the iRNA agent in which it is included. In preferred embodiments, the implementation of the ligand provides increased affinity for the selected target, for example a molecule, cell or cell type, compartment, for example a cell compartment or compartment of an organ, tissue, organ or part of the body, for example, compared with a species that does not have such a ligand.

Preferred ligands can improve transport, hybridization, and specificity characteristics and can also increase the resistance to nucleases of the resulting natural or modified oligonucleotide or polymer molecule containing any combination of monomers described herein and / or natural or modified ribonucleotides.

Ligands, in General, may contain therapeutic modifiers, for example, in order to enhance absorption; diagnostic compounds or reporter groups, for example, to control distribution; crosslinking agents; residues conferring resistance to nucleases; and natural or unusual nucleic bases. Common examples include lipophilic substances, lipids, steroids (e.g. uvol, gecigenin, diosgenin), terpenes (e.g. triterpenes, e.g. sarsasapogenin, Fridelin, epiphyridanol derivative of lithocholic acid), vitamins (e.g., folic acid, Vitamin A, biotin, p ), carbohydrates, proteins; protein binding agents; molecules targeted by integrin; polycationic substances, peptides, polyamines and mimetic peptides.

Ligands may contain a naturally occurring substance (for example, human serum albumin (HSA), low density lipoprotein (LDL) or globulin); a carbohydrate (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); amino acid or lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, for example a synthetic polyamino acid. Examples of polyamino acids include polyamino acid, polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, a copolymer of styrene and maleic anhydride, a copolymer of poly (L-lactide-co-glycolide), a copolymer of divinyl ether and maleic anhydride N- (2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly (2-ethyl acrylic acid), N-isopropyl acrylamide polymers or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic residues, for example cationic lipid, cationic porphyrin, quaternary polyamine salt or alpha-spiral peptide .

Ligands can also contain targeting groups, for example, means for targeting a cell or tissue, for example a lectin, glycoprotein, lipid or protein, for example an antibody that binds to a cell of a given type, such as a liver cell or a jejunum cell. The targeting group may be thyrotropin, melanotropin, lectin, glycoprotein, surfactant A protein, mycine carbohydrate, polyvalent lactose, polyvalent galactose, N-acetylgalactosamine, N-acetylglucosylamine, multivalent multivalent, multivalent, multivalent, multivalent, multivalent, multivalent, multivalent, multivalent, multivalent, multivalent, multivalent, multivalent, multivalent, multivalent, multivalent, polyvalent , polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B ₁₂ , biotin or RGD peptide, or RGD peptide mimetic.

Ligands can be proteins, for example glycoproteins, or peptides, for example molecules having a specific affinity for a coligand, or antibodies, for example, an antibody that binds to a cell of a given type, such as a cancer cell, endothelial cell or bone cell. Ligands can also contain hormone receptors and hormones. They may also include non-peptide species such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetylgalactosamine, N-acetylglucosamine, polyvalent mannose or polyvalent fucose. The ligand may be, for example, a lipopolysaccharide, p38 MAP kinase activator, or NF-κB activator.

The ligand can be a substance, for example, a medicine, which can increase the flow of an iRNA drug into a cell, for example, by breaking down the cytoskeleton of a cell, for example, by breaking down microtubules, microfilaments, and / or intermediate cell filaments. The medicament may be, for example, a taxon, vincristine, vinblastine, cytochalazine, nocodazole, japlacinolide, latrunculin A, phalloidin, swine solid A, indanocin or myoservin.

In one aspect, the ligand is a lipid or lipid based molecule. Such a lipid or lipid-based molecule preferably binds whey protein A, for example human serum albumin (HSA). The HAS binding ligand enables the distribution of the conjugate to the target tissue, for example, liver tissue, including parenchymal liver cells. Other molecules that can bind to HSA can also be used as ligands. For example, neproxin or aspirin may be used. A lipid-based lipid or ligand can (a) increase the degradability of the conjugate, (b) improve targeting or transport to the target cell or cell membrane, and / or (c) can be used to regulate binding to a serum protein, such as HSA.

A lipid-based ligand can be used to modulate, for example control, the binding of the conjugate to the target tissue. For example, a lipid-based lipid or ligand that binds more strongly to HSA will be less likely to target the kidneys and therefore will be less likely to be excreted. A lipid-based lipid or ligand that binds less strongly to HSA can be used to target the conjugate to the kidneys.

In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, it binds to HSA with an affinity sufficient so that the conjugate is preferably distributed into non-renal tissues. However, it is preferred that the affinity is not so strong that the binding of HAS to the ligand becomes irreversible.

In another aspect, the ligand is a residue, for example a vitamin, that is captured by a target cell, for example a proliferating cell. They are particularly useful for treating disorders characterized by undesired cell proliferation, for example, of a malignant or benign type, for example, cancer cells. Exemplary vitamins include Vitamin A, E, and K. Other exemplary vitamins include Β vitamins, such as folic acid, B ₁₂ , riboflavin, biotin, pyridoxal, or other vitamins or nutrients captured by cancer cells. HSA and low density lipoprotein (LDL) are also included.

In another aspect, the ligand is a cell penetration enhancer, preferably a spiral penetration enhancer. Preferably, the agent is amphipathic. A typical remedy is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including peptidyl mimetics, inverters, non-peptide or pseudopeptide bonds and the use of D-amino acids. The helical agent is preferably an alpha-helical agent that preferably has lipophilic and lipophobic phases.

5′-phosphate modifications

In preferred embodiments, the iRNA agents are 5'-phosphorylated or contain an analogue of a phosphorus group at the 5'-primary end. Modifications of the 5′-antisense strand phosphate include those that are compatible with RISC mediated gene silencing. Suitable modifications include: 5′-monophosphate ((HO) 2 (O) P-O-5 ′); 5′-diphosphate ((ΉΟ) 2 (O) Ρ-O-Ρ (ΗΟ) (O) -O-5 '); 5′-triphosphate ((HO) 2 (O) P-O- (HO) (O) P-O-P (HO) (O) -O-5 ′); 5′-guanosine cap (methylated or unmethylated at position 7) (7m-G-O-5 '- (HO) (O) P-O- (HO) (O) P-O-P (HO) (O) -O-5'); 5′-adenosine cap (Arrr) and any modified or unmodified nucleotide cap structure (N-O-5 ′ - (HO) (O) P-O- (HO) (O) P-O-P (HO) (O) -O-5 ′); 5'-monothiophosphate (phosphorothioate; (HO) 2 (S) P-O-5 '); 5'-monodithiophosphate (phosphorodithioate; (HO) (HS) (S) P-O-5 '), 5'-phosphorothiolate ((HO) 2 (O) P-S-5'); any additional combination of substituted oxygen / sulfur monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO) 2 (O) P- NH-5 ', (HO) (NH2) (O) PO-5'), 5'-alkylphosphonates (R = alkyl = methyl, ethyl, isopropyl, propyl, etc., e.g. RP (OH) (O) -O-5'-, (OH) 2 (O) P-5'-CH2-), 5′-alkyl terphosphonates (R = alkyl ether = methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP (OH) (O) -O-5'-).

The sense strand can be modified to inactivate the sense strand and prevent the formation of an active RISC, thereby potentially reducing effects outside the target. This can be achieved with a modification that prevents 5'-phosphorylation of the sense strand, for example with 5'-O-methyl modification with ribonucleotides (see Nykanen et al., (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309-321). Other modifications that prevent phosphorylation can also be used, such as simply replacing 5'-OH with Η rather than O-Me. Alternatively, a bulky group can be added to 5′-phosphate, turning it into a phosphodiester bond.

Delivery of iRNA products to tissues and cells

A drug

Preparations for administration to an individual, preferably for local administration into the lungs and nasal passages (respiratory tissues) by inhalation or intranasal administration, or parenterally, for example by injection, can be prepared from iRNA agents described herein.

For ease of description, the preparations, compositions, and methods discussed in this section relate primarily to unmodified iRNA agents. However, it should be understood that these preparations, compositions and methods may be carried out with other iRNA agents, for example modified iRNA agents, and such an implementation is included in the scope of the invention.

A formulated iRNA composition may take many conditions. In some examples, the composition is at least partially crystalline, uniformly crystalline and / or anhydrous (for example, contains less than 80, 50, 30, 20, or 10% water). In another example, iRNA is in the aqueous phase, for example in a solution that includes water, this form is the preferred form for inhalation.

The aqueous phase or crystalline compositions may be included in a delivery vehicle, for example, liposomes (especially for the aqueous phase) or particles (for example, microparticles, which may be suitable for the crystalline composition). Typically, an iRNA composition is prepared in a manner that is consistent with the proposed route of administration.

An iRNA preparation can be prepared in combination with another agent, for example, another therapeutic agent or agent that stabilizes iRNA, for example, a protein that complexes with iRNA to form iRNP. Other agents include chelating agents, e.g. EDTA (e.g., to remove divalent cations, such as Mg ²⁺ ), salts, RNase inhibitors (e.g., a broad specificity RNase inhibitor, such as RNAsin), etc.

In one embodiment, the iRNA preparation comprises another iRNA agent, for example, a second iRNA agent that can mediate RNAi in relation to the second gene. Other drugs may include at least three, five, ten, twenty, fifty, or one hundred or more different types of iRNA. In some embodiments, the agents are directed against the same virus, but against different target sequences. In another embodiment, each of the iRNA agents is directed against a different virus. As shown in the example, it is possible to inhibit more than one virus by administering two iRNA agents simultaneously or at short time intervals, each agent directed against one of the viruses.

Methods of treatment and route of administration

A composition that contains an iRNA agent of the present invention, for example an iRNA agent that targets RSV or PIV, can be administered to an individual in a variety of ways. Typical routes include inhalation, intrathecal, parenchymal, intravenous, nasal, oral and ocular administration. The preferred route of administration of the iRNA agents of the present invention is by direct administration into the lungs and nasal passages, or systemic via parenteral administration.

An iRNA agent may be formulated into pharmaceutical compositions suitable for administration. For example, the compositions may contain one or more iRNA agents and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion medium, coatings, antibacterial and antifungal agents, isotonic agents and anti-absorption agents and the like compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional medium or agent is incompatible with the active compound, their use in the compositions is contemplated. Additional active compounds may also be included in the composition.

The pharmaceutical compositions of the present invention can be administered in many ways, depending on whether topical or systemic treatment is desired, as well as on the area to be treated. Administration may be topical (including ophthalmic, intranasal, transdermal, intrapulmonary), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

In general, the iRNA agents of the present invention are administered in order to achieve delivery to the individual site of infection. The preferred way to achieve this is either by local administration into the lungs or nasal passages, for example into respiratory tissues via inhalation or intranasal administration, or from systemic administration, for example parenteral administration.

Preparations for inhalation or parenteral administration are known in the art. Such a preparation may contain sterile aqueous solutions, which may also contain buffers, diluents and other suitable additives. For intravenous administration, it is necessary to control the total concentration of dissolved substances in order to make the drug isotonic.

The active compounds disclosed herein are preferably administered to the lung (s) or nasal passages of an individual by any suitable means. Active compounds can be administered by applying an aerosol suspension of particles suitable for inhalation and consisting of the active compound or active compounds that the individual inhales. The active compound may be aerosolized in a variety of forms, such as, but not limited to, dry powder inhalation agents, metered doses of the inhalation agent, or a liquid / liquid suspension. Particles suitable for inhalation may be a liquid or a solid. Optionally, the particles may contain other therapeutic ingredients, such as amiloride, benzamyl or phenamyl, where the selected compound is contained in an amount capable of effectively inhibiting the reabsorption of water from the mucous membranes of the respiratory tract, as described in US Pat. 4,501,729.

A pharmaceutical composition consisting of particles may optionally be combined with a carrier to facilitate dispersion or transport. A suitable carrier, such as sugar (i.e., lactose, sucrose, trehalose, mannitol) can be mixed with the active compound or compounds in any suitable ratio (e.g., 1: 1 by weight).

Particles consisting of an active compound for carrying out the present invention should contain particles suitable for inhalation size, that is, particles small enough to penetrate through the mouth or nose and larynx into the bronchi and alveoli of the lungs. In general, particles whose size is about 1-10 microns (more specifically, less than about 5 microns) are suitable for inhalation. Particles of a size not suitable for inhalation that are contained in the aerosol tend to settle in the throat and be swallowed, and the amount of particles unsuitable for inhalation in the aerosol is preferably minimized. For nasal administration, a particle size of 10-500 microns is preferred to provide a delay in the nasal cavity.

Liquid pharmaceutical compositions of the active compound for the preparation of an aerosol can be prepared by combining the active compound with a suitable solvent, such as sterile, pyrogen-free water. The hypertonic saline solutions used to practice the present invention are preferably sterile, pyrogen-free solutions containing one to fifteen percent (by weight) of a physiologically acceptable salt, and more preferably three to seven percent by weight of a physiologically acceptable salt.

Aerosols of liquid particles containing the active compound can be obtained by any suitable means, for example, by means of a jet propellant driven by pressure, or an ultrasonic atomizer. See, for example, US Pat. No. 4,501,729. Nebulizers are commercially available devices that convert solutions or suspensions of the active ingredient into therapeutic spray mist, either by accelerating compressed gas, typically air or oxygen passing through a narrow venturi, or by ultrasonic shaking.

Suitable formulations for use in nebulizers consist of the active ingredient in a liquid carrier, where the active ingredient is 40% w / w. drug, but preferably less than 20% wt./wt. The carrier is typically water (and most preferably sterile, pyrogen-free water) or a dilute aqueous alcoholic solution, preferably made isotonic, but it can also be made hypertonic to body fluids by adding, for example, sodium chloride. Optional additives include preservatives if the drug is not sterile, such as methyl hydroxybenzoate, antioxidants, aromatics, essential oils, buffers, and surfactants.

Similarly, aerosols of solid particles containing the active compound can be obtained using any generator of therapeutic aerosols composed of particles. Aerosol generators for administering therapeutic agents consisting of particulate matter to an individual produce particles that are suitable for inhalation and create an aerosol volume containing a specific measured dose of the therapeutic agent at a speed suitable for administration to humans. One illustrative type of particulate aerosol generator is an insufflator. Suitable preparations for administration by injection include finely ground powders, which can be administered by means of an insufflator or placed into the nasal cavity by drawing in by the nose. In the insufflator, the powder (for example, a metered dose of powder effective to perform the treatment described herein) is contained in capsules or cartridges, typically made of gelatin or plastic, which are either punctured or open in situ and the powder is delivered by air, pulled through the device during inhalation, or through a hand pump. The powder used in the insufflator consists either solely of the active ingredient or of a powder mixture containing the active ingredient, a suitable powder diluent, such as lactose, and optionally a surfactant. The active ingredient typically ranges from 0.1 to 100 wt./wt. composition.

A second type of illustrative aerosol generator includes a metered dose inhaler. Dosage inhalers are pressurized aerosol nebulizers that typically contain a suspension or solution of the active ingredient in a liquefied propellant. During use, these devices dispense the drug through a valve adapted to discharge a metered volume, typically from 10 to 200 μl, to obtain an aerosol of fine particles containing the active ingredient. Suitable propellants include certain chlorofluorocarbon compounds, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof. The preparation may additionally contain one or more additional solvents, for example ethanol, surfactants such as oleic acid or sorbitan trioleate, an antioxidant and suitable flavorings.

Administration may be by an individual or another person, such as a carer. A caregiver can be any object related to providing care for a person: for example, a hospital, hospice, doctor’s office, outpatient clinic; a healthcare professional, such as a doctor, nurse, or other practitioner; or a spouse or guardian, such as a parent. The drug can be provided in metered doses or in a nebulizer that administers the measured dose.

The term “therapeutically effective amount” refers to the amount present in a composition that is necessary to provide the desired level of drug for the individual who is being treated to give the expected physiological response. In one embodiment, a therapeutically effective amount of two or more iRNA agents, each directed against a different respiratory virus, such as RSV and PIV, is administered to the individual simultaneously.

The term “physiologically effective amount” means the amount administered to an individual to obtain the desired palliative or therapeutic effect.

The term "pharmaceutically acceptable carrier" means that the carrier can enter the lungs without significant adverse toxicological effects on the lungs.

Types of pharmaceutical excipients that are useful as carriers include stabilizers, such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; etc. These carriers may be in crystalline or amorphous form, or may be a mixture of both.

Particularly valuable bulking agents include compatible carbohydrates, polypeptides, amino acids, or combinations thereof. Suitable carbohydrates include monosaccharides such as galactose, D-mannose, sorbose and the like; disaccharides such as lactose, trehalose and the like; cyclodextrins such as 2-hydroxypropyl-beta. cyclodextrin; and polysaccharides such as raffinose, maltodextrins, dextrans and the like; aldites, such as mannitol, xylitol, etc. A preferred carbohydrate group includes lactose, trehalose, raffinose, maltodextrins and mannitol. Suitable polypeptides include aspartame. Amino acids include alanine and glycine, glycine being preferred.

Suitable pH adjusters or buffers include organic salts derived from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred.

Dosage. An iRNA agent can be administered in a single dose of less than about 75 mg per kg of body weight or less than about 70, 60, 50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0, 05, 0.01, 0.005, 0.001 or 0.0005 mg per 1 kg of body weight and less than 200 nmol of RNA agent (for example, approximately 4.4 × 10 ¹⁶ copies) per 1 kg of body weight or less than 1500, 750, 300 , 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmol of RNA agent per 1 kg of mass body. A single dose, for example, may be administered by injection (e.g., intravenous or intramuscular, intrathecal or directly into the organ), inhalation of the dose or topical application.

Administration of an iRNA agent directly to an organ (e.g., directly to the liver) can be carried out at a dosage of from about 0.00001 mg to about 3 mg per organ, or preferably about 0.0001-0.001 mg per organ, about 0.03-3.0 mg per organ, approximately 0.1-3.0 mg per eye, or approximately 0.3-3.0 mg per organ.

The dosage may be an amount effective to treat or prevent a disease or disorder.

In one embodiment, a unit dose is administered less than once a day, for example less than every 2, 4, 8, or 30 days. In another embodiment, the unit dose is not administered at a certain frequency ( for example, not at a regular frequency). For example, a unit dose may be administered once.

In one embodiment, an effective dose is administered along with other conventional therapeutic effects.

In one embodiment, the individual is administered an initial dose and one or more maintenance doses of an iRNA agent, for example a double helix iRNA agent or siRNA agent (for example, a precursor, for example, a larger iRNA agent that can be converted into a siRNA agent, or DNA, which encodes an iRNA agent, for example a double helix iRNA agent or siRNA agent or its precursor). The maintenance dose or doses are typically lower than the initial dose, for example, half the initial dose. The maintenance regimen may include treating an individual with a dose or doses of 0.01 μg to 75 mg / kg body weight per day, for example 70, 60, 50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0 , 1, 0.05, 0.01, 0.005, 0.001 or 0.0005 mg per 1 kg of body weight per day. Maintenance doses are preferably administered no more than every 5, 10, or 30 days. In addition, the treatment regimen may last for a period of time that will vary depending on the nature of a particular disease, its severity and general condition of the patient. In preferred embodiments, the dose is administered no more than once a day, for example no more than once in 24, 36, 48 or more hours, for example no more than once every 5 or 8 days. After treatment, the patient can be examined for changes in his condition and alleviation of the symptoms of the disease state. The dosage of the compound may be increased when the patient does not give a significant reaction to the current dosage, or the dose may be reduced if there is a relief of symptoms of the disease state, if the disease state has disappeared or if undesirable side effects are observed.

An effective dose may be administered as a single dose or two or more doses, which is desirable or considered appropriate in certain circumstances. If it is desired to facilitate repeated or frequent infusions, it may be appropriate to implant a device for administering, for example, a pump, a semi-permanent stent (e.g., intravenous, intraperitoneal, intracysteal or intracapsular) or reservoir.

In one embodiment, the pharmaceutical composition of an iRNA agent comprises a number of varieties of iRNA agent. Varieties of iRNA agents can have sequences that do not overlap and are not contiguous with respect to the natural target sequence, for example, the target sequence of the RSV gene. In another embodiment, a number of varieties of the iRNA agent are specific for various natural target genes. For example, an iRNA agent that targets the Ρ RSV protein gene may be present in the same pharmaceutical composition as an iRNA agent targeting another gene, such as the Ν protein gene. In another embodiment, iRNA agents are specific for various viruses, for example RSV and PIV.

After successful treatment, it may be desirable to subject the patient to maintenance therapy in order to prevent a relapse of the disease state where the compound of the invention is administered in maintenance doses ranging from 0.01 μg to 100 g per 1 kg of body weight (see US 6107094).

The concentration of the composition of the iRNA agent is an amount sufficient to effectively treat or prevent disorders or to regulate the physiological state in humans. The concentration or amount of the iRNA agent administered will depend on the parameters established for the agent and route of administration, for example, nasal, buccal or pulmonary. For example, in nasal preparations, the concentration of some ingredients is usually much lower to avoid irritation or burning of the nasal passages. Sometimes, an oral preparation is preferably diluted 10-100 times to obtain a suitable nasal preparation.

Certain factors may affect the dosage required for effective treatment of an individual, including but not limited to the severity of the disease or disorder, previous treatment, general condition and / or age of the individual and other existing diseases. In addition, treating an individual with a therapeutically effective amount of an iRNA agent, for example a double helix iRNA agent or siRNA agent (for example, a precursor, for example a larger iRNA agent that can be converted into a siRNA agent, or DNA that encodes an iRNA agent, for example a double-stranded iRNA agent or siRNA agent or its precursor) may comprise a single course of treatment or, preferably, may include a number of courses of treatment. In addition, it will be appreciated that the effective dosage of an iRNA agent, such as a siRNA agent used for treatment, during a particular course of treatment may increase or decrease. Dosage changes can result in and become apparent from the results of diagnostic tests, as described herein. For example, after applying the composition of an iRNA agent, an individual may be observed. Based on the information obtained by observation, an additional amount of the composition of the iRNA agent may be administered.

Dosing depends on the severity and severity of the response of the disease state that is being treated, the course of treatment will last from several days to several months, or until a cure occurs or the disease condition is weakened. Optimum dosage regimens can be calculated from measurements of drug accumulation in the patient’s body. Professionals can easily determine the optimal dosage, dosing methodology and repetition rate. Optimal dosages can vary depending on the relative activity of the individual compounds and can usually be estimated based on EC50, which were found to be effective in vitro and in an animal model in vivo . In some embodiments, the animal model includes transgenic animals that express a human gene, for example, a gene that produces an RSV target RNA. A transgenic animal may have a lack of appropriate endogenous RNA. In another embodiment, the test composition comprises an iRNA agent that is complementary to at least the inner region, a sequence that is conserved for the RSV target RNA in the animal model and RSV target RNA in the human.

The invention is further illustrated by the following examples, which should not be construed as an additional limitation.

EXAMPLES

Construction of antiviral siRNAs against RSV mRNA and HPIV3 phosphoprotein

siRNA against RSV Ρ and siRNA against HPIV3 м mRNA were chemically synthesized (Bitko, V. & Bank, S. BMC Microbiol. 1, 34 (2001)) and their IC50 (siRNA concentration causing a 50% decrease in the target) was determined ex vivo (Fig. . 1a). The siRNA sequences and IC50 values are listed (Table 1). Two anti-RSV-P siRNAs (# 1, # 2) and one against HPIV3 (# 4) showed significant inhibitory activity and were selected for further study. The correlation of the target mRNA with protein, as illustrated for siRNA # 1 (Fig. 1a), is consistent with the RNAi mechanism, and as shown below, the inhibitory activity of siRNA ex vivo is also consistent with its activity in an animal (in vivo). Thus, ex vivo analysis provides a reliable, inexpensive, fast and convenient method for initial testing of an antiviral siRNA agent.

Intranasal (IN) siRNAs inhibit RSV and HPIV3 replication in mouse lung

An animal model was used to determine whether ex vivo active siRNAs would be effective in a true infection. The BALB / c mouse is a traditional laboratory model for RSV infection to study disease progression, pathology, and immunology (Graham, BS, et al., J. Med. Virol. 26,153-162 (1988), van Schaik, SM, et al., J. Infect. Dis. 177,269-276 (1998), Haeberle, HA et al. J. Virol. 75, 878-890 (2001)). Mice were intranasally injected with siRNA in combination with TransIT-TKO® reagent, and after 4 hours each animal was infected with 10 ⁷ PFU RSV or HPIV3, also intranasally. The maximum multiplication of RSV in the lung of the mouse was observed after approximately 5-6 days pi and this point in time was used in further studies. siRNAs that were effective ex vivo (Fig. 1a) showed a potent antiviral effect in animals (Fig. 1b, c). At a dose of 5 nmol IN siRNA (average ~ 70 μg double-stranded siRNA) per mouse, siRNA # 1 and siRNA # 4, respectively, reduced the RSV titer and the HPIV3 titer in the lung by about 5000 and 100 times with separate infection (Fig. 1b, c) It is important that siRNA free of transfection reagents also significantly inhibited lung titer virus titers (Figure 1d). This proves that inhalation-based antiviral treatment is possible using simple pharmaceutical compositions containing an iRNA agent. It should be noted that HPIV3 does not infect mice as easily as RSV (Durbin, AR, Elkins, WR & Murphy, BR Vaccine 18,2462-2469 (2000)), which causes relatively lower replication of HPIV3 in the lung of the mouse (Fig. 1c). The use of sucrose purified high titer HPIV3 as an inoculum made it possible to obtain a measurable lung mouse infection on the model.

Several additional features of the example are applicable to various embodiments of the present invention. First, the results demonstrate a virus-specific effect of siRNA (FIG. 1). Even the most powerful anti-RSV siRNA (# 1) inhibits only RSV, but not HPIV3, and vice versa, which in itself argues against the non-specific antiviral effect of IΝ siRNA. Second, anti-luciferase siRNA (Elbashir, SM et al. Nature 411, 494-498 (2001)) did not inhibit any virus even at the highest dose tested (50 nmol or 700 μg / mouse) (Fig. 1b- d) Finally, IN siRNA alone or together with the Transit-TKO reagent in uninfected mice did not cause obvious discomfort (which was determined by normal coat, activity, appetite and weight gain, and the absence of respiratory disorders), which indicates a favorable pharmacology for the potential development of a drug.

It should be noted that in all the experiments described in this document, the results of viral protein immunoblotting always coincided with the titer of the virus, and therefore, for this experiment, each can serve as a backup marker of the other, and all complementary data are not given.

Specific antiviral effects of IN siRNA prevent pulmonary infection

Although the results presented above confirm inhibition of viral replication, they are not direct evidence of cessation of infection in the lung tissue. Therefore, the authors investigated various sections of both lungs after 5 days pi of the use of antibodies specific for the corresponding virus. On instillation of 10 ⁷ PFU of mouse, both viruses caused severe pulmonary infection. In the characteristic results (Fig. 2a), the infection was significantly attenuated in mice that were previously injected with 5 nmol (70 μg) of anti-RSV siRNA # 1 in combination with TransIT-TKO. A similar attenuation of HPIV3 infection was also observed after 5 nmol of anti-HPIV3 siRNA # 4. As with virus titer, siRNA without a transfection reagent showed a significant attenuation of infection, which is presented for RSV. For the same amount of siRNA, the authors calculated that siRNA without reagents would be approximately 70-80% as effective as siRNA in combination with TransIT-TKO. Although the authors only presented data for TransIT-TKO in combination with siRNA in the rest of the document, these results point to an exciting prospect: the intranasal administration of naked siRNA, free of other chemicals, can provide significant protection against respiratory infections. This is especially important since transfection reagents themselves can have side effects. The authors noted here that polyethyleneimine (PEI) was successfully used as a carrier for intravenous (IV) as well as endotracheal (IT) administration of siRNA and DNA against influenza virus (Ge, Q., et al., Proc. Natl. Acad. Sci USA 101, 8676-8681 (2004)). However, according to the authors' experience, direct IN administration of PEI, individually or together with siRNA, often resulted in overt illness and / or death of the mouse.

IN introduced siRNA is localized in the lungs and does not activate interferon

In order to obtain new evidence that the inhibition of the virus observed in the lung was a direct and specific effect of siRNA administered through the nose, two types of experiments were performed. First, the authors were able to detect the siRNA antisense chain in lung tissue (Fig. 2b) using specific Northern blotting. Secondly, the possibility that the antiviral effect of siRNA is due to the activation of interferons (IFN) was excluded as follows. Paramyxoviruses, in general, encode various mechanisms to counteract IFN; RSV, in particular, is highly resistant to type I IFN (IFN-α / β), although it is sensitive to type II IFN (IFN-γ) (Schlender, J., et al., J. Virol. 74, 8234- 8242 (2000), Ramaswamy, M., et al., Am. J. Respir. Cell Mol. Biol. 30, 893-900 (2004)). Previous studies by the authors showed that siRNAs are active against RSV and HPIV3 in Vero cells that contain deletions of type I IFN genes (data not shown). However, the authors measured the levels of IFN-α and IFN-γ in the lung tissue of the mouse on different days after treatment with different siRNAs and did not detect activation of any type of IFN (Fig. 2c).

siRNA selectively protect against RSV and HPIV3 in mixed infection

There is always the possibility of co-infection of the respiratory tract caused by several pathogens, and in some studies a mixed infection caused by RSV and HPIV3 has been diagnosed (Coiras, MT, et al, J. Med. Virol. 72, 484-495 (2004)). In fact, chimeric viruses and recombinant vaccines including RSV, as well as HPIV3 antigens, were designed in the hope that they would provide simultaneous protection against both viruses (Schmidt, AC, et al., J. Virol. 75,4594-4603 (2001), Bernhard, W. et al. Am. J. Respir. Cell Mol. Biol. 25, 725-731 (2001)). The specific antiviral effect of siRNA # 1 and siRNA # 4 against RSV and HPIV3, respectively, led the authors to test them together (5 nmol or 70 μg each) in mixed infection of mice caused by both viruses. Control mice were treated with one type of siRNA (either # 1 or # 4). Since there is no easy way to determine the amount of PFU of each virus in a mixture of two viruses, the authors turned to immunofluorescence microscopy as described above and double-stained sections of lung tissue using a mixture of antibodies to RSV and HPIV3. Mixed infection of lung tissue according to this criterion actually occurred (fig.2d). In mice pretreated with either siRNA # 1 or siRNA # 4, infection caused by RSV and HPIV3, respectively, was prevented, which was observed by the disappearance of either green or red fluorescence, but not both (Fig. 2d). Using a combination of two siRNAs (5 nmol, i.e. 70 μg each), both types of fluorescence disappeared, which confirmed the inhibition of both viruses (Figure 2d). As before, the same siRNA mixture without any transfection reagent was also highly active (data not shown).

Interestingly, if an excessively high amount of one siRNA was used, the effect of another siRNA in the double infection assay was inhibited (FIG. 3). By real-time quantitative RT-PCR, the IC50 siRNA # 4 (versus HPIV3 P) ex vivo increased to 15.35 and 100 nM, since the concentration of siRNA # 1 (versus RSV P) was raised from 0 to 20-200 nM (FIG. 3a ) These results were validated by measuring HPIV3 Protein P in an immunoblot (Fig. 3b). In double infection of mice, quantification by immunoblotting of the protein of each virus yielded a substantially identical conclusion: although 5 nmol (70 μg) of each siRNA effectively inhibited both viruses, 50 nmol of one siRNA attenuated the effect of 5 nmol mutually different (Fig. 3c).

IN administered siRNAs prevent pulmonary pathology

Since siRNAs prevent infection, a logical question arises as to whether they also prevent the development of pathological signs. On visual examination, mice treated with siRNA and exposed to RSV infection behaved and looked essentially the same as uninfected mice with normal activity, shiny coat and good general condition. The authors then measured respiratory rate, leukotriene induction, and pulmonary inflammation. The respiratory rate of BALB / c mice is known to increase in response to an infection caused by RSV (Haeberle, HA et al J. Virol. 75, 878-890 (2001), Volovitz, B., et al., Pediatr. Res. 24 504-507 (1988)). Leukotrienes, a product of the lipoxygenase pathway, bind to leukotriene receptors present in the smooth muscles of the bronchi, and the content of leukotrienes increases in the secretion of the respiratory tract of patients with asthma, infants with RSV infection and mice infected with RSV (Volovitz, B., et al., Pediatr Res. 24, 504-507 (1988), Welliver, RC, 2nd, et al., J. Infect. Dis. 187, 1773-1779 (2003)). These compounds stimulate mucus secretion in the airways, bronchospasm and airway infiltration by inflammatory cells, which are important signs of a serious illness caused by RSV. If the authors administered siRNA # 1 against RSV simultaneously with (or before) RSV, there was a significant decrease in respiratory rate, attenuation of pulmonary histopathology and accumulation of leukotriene in bronchoalveolar fluid (BALF) (Fig. 4). These parameters remained close to the initial ones and were essentially comparable with those in mice subjected to simulated infection. All parameters remained low for at least 14 days after infection - this proves that siRNA really prevents the disease, and does not make its onset later. In fact, mice treated with siRNA did not show visible respiratory distress during the 6 weeks of observation. A negative control of RNA or luc-siRNA (table 1) did not give relief in all experiments (data not shown).

IN administered siRNA has an effective antiviral effect after infection

Having shown that siRNAs introduced before infection can prevent respiratory viral disease, the authors wondered if they could have a therapeutic effect after the infection developed, as this is an important goal in pediatric medicine. In this series of experiments, the authors introduced siRNA # 1 on various days after RSV infection and mice were weighed daily. As is known, the body weight of mice decreases within approximately 8-10 days after RSV infection, after which they either die or slowly restore body weight depending on the size of the initial inoculum - too high or moderate-low (Haeberle, HA et at. J Virol. 75, 878-890 (2001)). In a similar cohort of mice, lungs were taken on certain days for analysis of an infectious virus. As expected, in untreated siRNA mice, body weight remained for approximately 4 days pi, and then gradually decreased, the decrease lasted at least 9 days (Fig. 5a). Mice that received siRNA before (data not shown) or with RSV (day 0) essentially looked uninfected and continued to continuously gain body weight. Most mice that received siRNA on day 1 were also difficult to distinguish from control animals that simulated infection. In mice that received siRNA in the following days (day 1-4), protection was increasingly weaker, although a significant increase in weight was observed all days with all treatment options.

Determination of pulmonary RSV titer in these mice days 2, 4, 6, 8, 10, and 16 gave a similar picture (Fig. 5b). In untreated siRNA mice, the titer increased to day 4-5 and then slowly dropped to an undetectable level by day 16. Administration of siRNA before or simultaneously with RSV infection kept the titer 5,000 times lower on all days on which the study was performed. The effectiveness of administering siRNA at a later time of infection progressively decreased, but the titer of the virus was always lower than in untreated control animals on any day tested. It seemed that siRNA, regardless of the time of administration, slowed down the rate of viral replication, which led to a lower peak titer. Subsequently, the titer fell below a definable level the faster the earlier siRNA was introduced. For example, although pulmonary RSV was detected in infected mice until approximately the 16th day of p.i., in mice that received siRNA on day 1, the virus was not detected after the 10th day of p.i. As before, the negative control siRNA or siRNA of luciferase (table 1) had no effect in all experiments (data not shown). Together, these results showed that anti-Ρ RSV siRNA had a therapeutic effect, even when administered after infection, and that the mice were always less diseased and faster to recover than cohorts of untreated mice.

DISCUSSION

The main conclusion of this document is that appropriately engineered siRNAs administered intranasally protect against respiratory infections and also provide effective treatment if used after infection. siRNAs, administered in a particulate aerosol from a simple pocket inhaler, can be used to prevent or treat lung infections. While the authors' manuscript was in preparation, two reports appeared in which siRNAs inhibited the influenza virus, another major respiratory pathogen in mice (Ge, Q., et al., Proc. Natl. Acad. Sci. USA 101, 8676-8681 (2004), Tompkins, SM, et al., Proc. Natl. Acad. Sci. USA 101, 8682-8686 (2004)). In one study (Ge, Q., et al., Proc. Natl. Acad. Sci. USA 101, 8676-8681 (2004)), synthetic siRNA or plasmid DNA expressing siRNA was introduced using a combination of IV and IT pathways. In another study (Tompkins, SM, et al., Proc. Natl. Acad. Sci. USA 101, 8682-8686 (2004)) siRNA was first administered by hydrodynamic intravenous administration, after 16-24 hours the mice were infected with the influenza virus and a second dose was administered siRNA in a lipid carrier, both intranasally. The presence of the virus in the lungs was determined after 2 days, and inhibition of various strains of the influenza virus was observed 10-50 times. The authors' studies regarding RSV and PIV suggested the following: improvement and simplification of the previously mentioned: (i) administration is only intranasal, therefore, relatively non-invasive and painless, can be carried out using an inhaler or therapeutic fog, (ii) siRNA is highly effective without any carrier , which reduces the potential risk of side effects caused by the carrier, (iii) A single dose of approximately 5 nmol siRNA (70 μg double-stranded RNA), apparently provides a beneficial effect throughout the long STI infection. A more thorough verification of the target sequence (i.e., RSV P) and the use of new chemistry can produce siRNAs with much lower IC50s and better pharmacokinetics, resulting in a lower dosage. siRNAs to varying degrees exhibit nonspecific effects due to non-target binding, especially at high concentrations (Jackson, AL et al. Nat. Biotechnol. 21, 635-637 (2003), Sledz, CA, et al., Nat. Cell Biol . 5, 834-839 (2003), Persengiev, SP, et al ., RNA 10,12-18 ( 2004), Bridge, AJ, et al ., Nat. Genet. 34,263-264 ( 2003)). This is an obvious source of concern for treatment and intravenous siRNA can lead to systemic side effects. With intranasal administration, siRNA, on the contrary, is most likely to be concentrated or exclusively localized in respiratory tissues, thereby weakening side effects. The lack of activation of IFN by intranasally introduced synthetic siRNA supports and extends previous evidence that chemically synthesized siRNAs lacking 5 ′ phosphates do not activate the IFN pathway in cell culture (Kim, DH et al. Nat. Biotechnol. 22, 321-325 (2004 )). Correlation of antiviral activity with specific mRNA suppression (Fig. 1), detection of siRNA in target tissue (lungs) (Fig. 2b), lack of IFN activation (Fig. 2c) and virus-specific effect of siRNA (Fig. 1-3) - collectively, they prove that the antiviral effect is specific, targeted and mediated by RNAi. (iii) Respiratory viruses, such as RSV and PIV, exhibit high selectivity in tissue tropism by infecting respiratory tissues. Thus, intranasal administration ensures that siRNA will be directed to the location of the infection - an ideal condition for pharmacology.

Although RSV and HPIV sometimes cause mixed infections, their interactions are largely ignored. The exact cause of the observed inhibition of one siRNA by another needs further investigation. The fact that this occurs ex vivo (in cell culture) is an objection to, but does not exclude, in animals, the role of humoral factors and cytokines, such as interferon. RSV actually inhibits the activation of interferon (Schlender, J., et al., J. Virol 74, 8234-8242 (2000), Ramaswamy, M., et al., Am. J. Respir. Cell Mol. Biol. 30 , 893-900 (2004)) and therefore should facilitate rather than inhibit the reproduction of PIV. Another possibility is that the reproduction of one virus inhibits the reproduction of another through other mechanisms, such as competition for intracellular stores. On the other hand, it is known that the RNAi apparatus in a cell is capable of saturation; therefore, two siRNAs can potentially compete for a limited reserve of this apparatus (Barik, S. Virus Res. 102, 27-35 (2004), (Hutvagner, G, et al ., PLoS Biol. 2, E98 (2004). It should be noted that such competition can only be assessed with relatively high doses of siRNA, amounting to tens or hundreds of nanomoles (Fig. 3). On the contrary, only a few nanomoles of siRNA obtained by the authors provide almost complete protection of mice. Thus, the observed competition is not practical when siRNA with IC50 in anomolar range.

When siRNA is used as a prophylactic, it not only prevents infections, but also inhibits the appearance of various aspects of the disease process, as measured by body weight, pulmonary pathology, respiratory parameters, and allergy markers (Fig. 4). The kinetics of the disease process in mice and humans is relatively similar, and in both species, immunopathological changes occur shortly after RSV infection. If siRNA is used as a medicine after the infection has developed, it is expected that they do not correct an already existing pathology. However, even in this case, inhibition of further virus propagation accelerates cure and recovery (Fig. 5). Thus, it seems that the “window of opportunity” for treatment exists all the time in patients with RSV infection, although treatment for any disease leads to a better prognosis. The effectiveness of naked siRNA needs to be explained. It is possible that respiratory tissues, especially in the lungs, are naturally susceptible to the exchange of small molecules or become so after infection.

Finally, depending on the severity of binding of the siRNA to the target, exposure to siRNA can lead to the selection of siRNA resistant viruses, as has been demonstrated for HIV (Das, AT et al. J. Virol. 78, 2601-2605 (2004)). The authors have not yet encountered this problem using siRNAs tested in this document. Viruses that were isolated from the lungs of mice treated with siRNA were grown in A549 cell culture, and it was found that they have the same IC50 for siRNA as the original inoculum (data not shown). In addition, sequencing of the region of the Ρ siRNA gene in six such independent strains purified by plaque formation revealed the wild-type parental sequence (data not shown). Even if resistance is accidentally encountered in the future, in a multi-drug regimen, a second siRNA with a lower IC50 can be used, targeting another Ρ mRNA region or another viral mRNA, thereby reducing the likelihood of virus resistance.

Methods

Virus, siRNA and other reagents. RSV of Long strain and human type 3 PIV (HPIV3) of JS strain were grown on the HEp-2 monolayer as described for RSV (Burke, E., et al., Virology 252, 137-148 (1998), Burke, E., et al., J. Virol. 74, 669-675 (2000), Gupta, S., et al., J. Virol. 72, 2655-2662 (1998)). Extracellular medium containing the released progeny of the virus was collected after about 70 hours for RSV and 50 hours for HPIV3. Viruses were purified and concentrated by precipitation with polyethylene glycol (molecular weight 8000) and sucrose gradient centrifugation as described for RSV (Ueba, O. Acta. Med. Okayama 32, 265-272 (1978)). The final preparations had an infectious titer in the range of 10 ⁸ -10 ⁹ PFU / ml and were stored in small packaging frozen at -80 ° C. All infectious virus titers (PFU) were determined by plaque formation on agarose with a HEp-2 monolayer and staining with neutral red (Burke, E., et al., Virology 252, 137-148 (1998), Burke, E., et al J. Virol. 74,669-675 (2000), Gupta, S., et al., J. Virol 72,2655-2662 (1998).

siRNAs were purchased from Dharmacon and processed as recommended by the manufacturer (Bitko, V. & Barik, S. BMC Microbiol 1, 34 (2001). TransIT-TKO ^® reagent was obtained from Mirnis Bio Corp (Madison, Wisconsin). immunohistochemical reactions used rabbit RSV-P antibodies (Bitko, V. & Barik, S. BMC Microbiol. 1, 34 (2001). Polyclonal antibodies to RSV and HPIV3 were produced against purified goat virions and were purchased from Chemicon (Temecula , California) and BiosPacific (Emeryville, California), respectively; the nucleocapsid protein (N) was the main viral band detected by these antibodies in immunoblotting. Antibodies to profilin are also described (Burke, E., et al., J. Virol 74,669-675 (2000), Gupta, S., et al., J. Virol. 72, 2655-2662 (1998)).

Viral infection and siRNA treatment. Infection and treatment of siRNA of A549 cells grown in a monolayer was performed as described (Bitko, V. & Barik, S. BMC Microbiol. 1, 34 (2001)). Intranasal administration of RSV to mice is an established procedure and causes bronchiolitis. Pathogen-free female BALB / c mice aged 8-10 weeks, weighing 16-20 g, were purchased from Charles River Laboratories. Anesthesia for infection or administration of siRNA was carried out by intraperitoneal injection of 0.2 ml of nembutal (5 mg / ml). Euthanasia was performed by displacing the neck after anesthesia with 0.3 ml of nembutal. siRNA was suitably diluted in the dilution buffer provided by the manufacturer to the desired content of 1 μl. It was mixed with 5 μl TransIT-TKO ^® reagent and 35 μl Opti-MEM (Gibco Life Technologies, Invitrogen, Carlsbad, CA) immediately before the experiment to obtain a total volume of 41 μl. If siRNA was used without a carrier, 5 μl of transfection reagent was replaced with 5 μl of Opti-MEM. The sucrose-purified virus was suitably diluted in cold phosphate-saline (PBS) immediately before infection, so 10 ⁷ PFU of the virus was contained in 30 μl. The same volume of virus-free PBS was used to simulate infection. Both the siRNA mixture and the virus were evenly distributed between the nostrils and injected with a micropipette (i.e., 35 μg siRNA in 20.5 μl and 0.5 × 10 ⁷ PFU of the virus in 15 μl were injected into each nostril). No special equipment was required, since the mice inhaled all the liquid during natural breathing. For mixed infection, the stock solutions of RSV and HPIV3 were diluted, so that each mouse received a mixture of 10 ⁷ PFU of each virus and a mixture of 5 nmol (70 μg) of each siRNA # 1 and siRNA # 4 in the previously indicated volumes. Animal experiments were carried out in accordance with all prescribed standards and were approved by IACUC.

Definition of the virus in the lungs and clinical examination . Animals were checked daily and their weight was measured. Standard RSV symptoms were noted, including nasal mucus, increased respiratory rate due to a rush of blood and bronchiolitis, dull hair, tousled fur and / or hair loss, general lethargy and malaise. Respiratory rate (number of respiratory movements per minute) was determined using video recording (Volovitz, B., et al., Pediatr. Res. 24, 504-507 (1988)). Sneezing, sniffing and sighing were excluded from the count. After a different number of days after infection (pi), the lungs were removed to detect RSV by determining the titer of the infectious virus, immunoblotting or immunohistochemistry, as described below.

To determine the titer of the virus, the lungs were homogenized in DMEM supplemented with 2% FBS (2 ml DMEM per 100 mg of tissue) in the cold. The extract was centrifuged at 2000 g for 10 minutes and serial dilutions of the supernatant were examined for PFU. For immunoblotting of viral proteins (Burke, E., et al., Virology 252,137-148 (1998)) 10 μl of a homogenized sample (before centrifugation) was added to 10 μl of 2 × SDS-PAGE buffer, the mixture was heated at 98 ° C for 5 minutes, was clarified by centrifugation in a microcentrifuge at room temperature, and 10 μl of the clear supernatant was analyzed by immunoblotting using goat anti-RSV and HPIV3 antibodies. To measure IFN (Durbin, JE et al. J. Immunol. 168, 2944-2952 (2002)), the lungs were homogenized in PBS, processed as described above, and serial dilutions were examined using ELISA kits (R&D Systems, Minneapolis, MN), the limit of determination of which was 10 pg / ml.

For histopathology studies, the lungs were washed and fixed in 10% buffered formalin and embedded in paraffin. Multiple sections with a thickness of 4 μm were stained with hematoxylin and eosin, and two independent researchers evaluated inflammatory cells under a light microscope. Inflammatory infiltrates were evaluated by counting the number of layers of inflammatory cells surrounding the vessels and bronchioles. From zero to three layers of inflammatory cells was considered the norm, more than three layers of inflammatory cells, surrounding 50% or more of the circumference of the vessel or bronchioles, were considered pathology. The number of pathological perivascular and peribronchial spaces, divided by the total number of such spaces, was reported as a pathological indicator. For each animal, all of approximately 20 spaces per lung were counted. With 10 ⁷ RSV (and the absence of siRNA) (Haeberle, HA et al. J. Virol. 75, 878-890 (2001)), approximately 30-35% of perivascular and peribronchial spaces could be pathological on day 1, and their number peaked around the 5th day.

For immunohistological studies (Haeberle, HA et al. J. Virol. 75,878-890 (2001)), lung tissue was immersed in a 100% OCT formulation and frozen at -80 ° C. Sections were separated into microscopic preparations, dried in air, fixed in acetone, washed in PBS and increased permeability in 0.2% Triton X-100 in PBS, blocked for 20 minutes with 10% goat serum in PBS at room temperature. After repeated washes with PBS, the tissue was incubated for 2 hours at room temperature with antibodies to either RSV-P or HPIV3 diluted with PBS containing 1.5% goat serum. The preparations were again washed in PBS and two antibodies were detected conjugated with FITC antibodies to rabbit antibodies and conjugated to TRITC antibodies to goat antibodies of the IgG class. After 1 hour of incubation at room temperature, the preparations were washed with PBS, poured with DABCO-DAPI filling medium and examined by fluorescence microscopy (Bitko, V. & Barik, S. BMC Microbiol. 1, 34 (2001)).

Bronchoalveolar lavage (BALF) was obtained by washing the bronchi and lungs with 5 × 1.0 ml of physiological saline (containing 10 μg indomethacin per ml) (Bernhard, W. et al. Am. J. Respir. Cell Mol. Biol. 25,725-731 ( 2001)); the total volume of bronchoalveolar lavage per mouse was 4.2–4.4 ml. Samples containing visible signs of blood contamination were discarded. Cells were removed from BALF by centrifugation at 5000 g for 15 minutes at 4 ° C, and samples were stored at -80 ° C until further analysis. The concentration of cysteinyl-leukotrienes in BALF was determined using an ELISA kit following the manufacturer's protocol (R&D Systems, Minneapolis, Minnesota). According to the introduced product, the cross-reactivity of the kit with respect to various leukotrienes was LTC4 100%, LTD4 115%, LTE4 63% and LTB4 1.2%.

For real-time PCR experiments, RNA was isolated from infected HPIV3 cells, and the first cDNA strand was synthesized using the GeneAmp RNA PCR Core kit (Perkin-Elmer Applied Biosystems, Foster City, California). The primers were designed by Beacon Designer 2.13 from Premier Biosoft. The following primers were used to amplify HPIV3 mRNA: 5'-GGTCATCACACGAATGTACAAC-3 '(SEQ ID NO: 1) and 5'-CTTGGAACATCTGCAGATTGTC-3' (SEQ ID NO: 2). Real-time PCR was performed on an iCycler iQ Quantitative PCR system from BioRad Laboratories (Hercules, California) using iQ Sybr Green SuperMix. Gene expression measurements were calculated using the manufacturer's software and GAPDH as an internal control.

The siRNA antisense strand was extracted from the lungs and determined by Northern hybridization, essentially as described (Reinhart, BJ, et al., Genes & Dev. 16,1616-1626 (2002)), using complementary synthetic oligonucleotides whose ends were labeled ³² P.

Statistical analysis. Differences between treatment groups or between sets of in vitro experiments were analyzed using one-way ANOVA and then Student's t test with Bonferroni correction. The increase in leukotriene concentrations was evaluated by the Mann-Whitney test. All numerical data were obtained in at least 3 separate experiments. The results were expressed as mean ± SEM (error value in the graphs). Differences were considered significant at Ρ < 0.05.

Table 1

SiRNA sequences

GenBank access numbers for the RSV-P, HPIV3-P, and luciferase sequences are M22644, Z11575, and X65324, respectively. Note that siRNA sequences were based on the actual sequencing of viral strains in our laboratory; thus, siRNA # 2 differs from the GenBank sequence in one nucleotide (underlined C represents U in M22644). A negative siRNA control sequence was obtained from Qiagen (Valencia, California).

Claims (16)

1. A method of reducing protein, mRNA, or titer of respiratory syncytial virus (RSV) in an individual's respiratory tract cell, comprising
the stage of intranasal administration of siRNA funds to the specified individual,
where siRNA agent contains a sense strand of 15-23 consecutive nucleotides and an antisense strand consisting of 15-23 consecutive nucleotides,
wherein said antisense strand is complementary to the target RNA encoding RSV virus P protein, and is also complementary to said sense strand, said sense strand containing at least 15 consecutive nucleotides of SEQ ID NO: 5 or SEQ ID NO: 7. 2. The method according to claim 1, wherein said sense strand is selected from the group consisting of SEQ ID NO: 5 and 7, and wherein said antisense strand is selected from the group consisting of SEQ ID NO: 6 and 8. 3. The method according to claim 1, wherein said siRNA agent is administered to an individual by inhalation or nebulization. 4. The method according to claim 1, in which prior to the introduction of the specified siRNA funds in an individual diagnosed with a viral infection. 5. The method according to claim 1, wherein said siRNA agent is administered prophylactically to an individual. 6. The method according to claim 2, where the specified sense chain consists of SEQ ID NO: 5, and the specified antisense chain consists of SEQ ID NO: 6. 7. The method according to claim 2, wherein said sense chain consists of SEQ ID NO: 7, and said antisense chain consists of SEQ ID NO: 8. 8. A method of reducing titer levels of respiratory syncytial virus (RSV) and parainfluenza virus (PIV) in an individual's respiratory tract cell, comprising
the stage of joint intranasal administration of the first and second siRNA funds to the specified individual,
wherein said first siRNA agent contains a sense strand of 15-23 consecutive nucleotides and an antisense strand of 15-23 consecutive nucleotides, wherein said antisense strand is complementary to the target RNA encoding RSV virus protein P, and is also complementary to said sense strand, wherein said sense strand the chain of said first siRNA agent contains at least 15 consecutive nucleotides of the sequence SEQ ID NO: 3, 5 or 7; and
said second siRNA agent contains a sense strand of 15-23 consecutive nucleotides and an antisense strand of 15-23 successive nucleotides, wherein said antisense strand is complementary to target PIV virus RNA and also complementary to said sense strand, said sense strand of said second siRNA means at least 15 consecutive nucleotides of the sequence SEQ ID NO: 9 or 11. 9. The method of claim 8, wherein said siRNA agents are administered by inhalation or nebulization. 10. The method of claim 8, wherein said sense chain of said first siRNA agent is selected from the group consisting of SEQ ID NO: 3, 5, and 7, and where said antisense chain of the first siRNA is selected from the group consisting of SEQ ID NO: 4 , 6 and 8, and said sense chain of said second siRNA agent is selected from the group consisting of SEQ ID NO: 9 and 11, and where said antisense chain of the second siRNA agent is selected from the group consisting of SEQ ID NO: 10 and 12. 11. The method of claim 10, wherein said sense strand of said first siRNA agent consists of SEQ ID NO: 3, and where said antisense chain of a first siRNA agent consists of SEQ ID NO: 4, and said sense chain of said second siRNA agent consists of SEQ ID NO: 9, and said antisense chain of said second siRNA agent consists of SEQ ID NO: 10. 12. The method of claim 10, wherein said sense chain of said first siRNA agent consists of SEQ ID NO: 3, and where said antisense chain of said first siRNA agent consists of SEQ ID NO: 4, and said sense chain of said second siRNA agent consists of SEQ ID NO: 11, and said antisense chain of said second siRNA agent consists of SEQ ID NO: 12. 13. The method of claim 10, wherein said sense strand of said first siRNA agent consists of SEQ ID NO: 5, and where said antisense chain of a first siRNA agent consists of SEQ ID NO: 6, and said sense chain of said second siRNA agent consists of SEQ ID NO: 9, and said antisense chain of said second siRNA agent consists of SEQ ID NO: 10. 14. The method of claim 10, wherein said sense chain of said first siRNA agent consists of SEQ ID NO: 5, and where said antisense chain of said first siRNA agent consists of SEQ ID NO: 6, and said sense chain of said second siRNA agent consists of SEQ ID NO: 11, and said antisense chain of said second siRNA agent consists of SEQ ID NO: 12. 15. The method of claim 10, wherein said sense strand of said first siRNA agent consists of SEQ ID NO: 7, and where said antisense chain of a first siRNA agent consists of SEQ ID NO: 8, and said sense chain of said second siRNA agent consists of SEQ ID NO: 9, and said antisense chain of said second siRNA agent consists of SEQ ID NO: 10. 16. The method of claim 10, wherein said sense chain of said first siRNA agent consists of SEQ ID NO: 7, and where said antisense chain of a first siRNA agent consists of SEQ ID NO: 8, and said sense chain of said second siRNA agent consists of SEQ ID NO: 11, and said antisense chain of said second siRNA agent consists of SEQ ID NO: 12.

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