WO2021255262A1 - Arnsi et compositions pour le traitement prophylactique et thérapeutique des maladies virales - Google Patents

Arnsi et compositions pour le traitement prophylactique et thérapeutique des maladies virales Download PDF

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WO2021255262A1
WO2021255262A1 PCT/EP2021/066665 EP2021066665W WO2021255262A1 WO 2021255262 A1 WO2021255262 A1 WO 2021255262A1 EP 2021066665 W EP2021066665 W EP 2021066665W WO 2021255262 A1 WO2021255262 A1 WO 2021255262A1
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antisense strand
sirna
sense strand
strand
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Ana Isabel JIMÉNEZ
Tamara MARTÍNEZ
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Sylentis Sau
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/17—Metallocarboxypeptidases (3.4.17)
    • C12Y304/17023—Angiotensin-converting enzyme 2 (3.4.17.23)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification

Definitions

  • the present invention provides compositions and methods for modulating the expression of angiotensin converting enzyme 2 (ACE2).
  • ACE2 angiotensin converting enzyme 2
  • this invention relates to oligonucleotide compounds, such as for example small interfering RNA (siRNA) compounds, which in some embodiments hybridize with nucleic acid molecules encoding ACE2, a host protein which is used by coronaviruses, including SARS-CoV-2, for entering into the cell.
  • siRNA small interfering RNA
  • Such compounds are shown herein to modulate the expression of ACE2 and to reduce the virus replication and are useful for reducing preventing and treating such viral infections or their related diseases.
  • Coronaviridae Several members of the family Coronaviridae usually cause mild respiratory disease in humans (Corman et al., 2019). However, other family members such as the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV) are transmitted from animals to humans and cause severe respiratory diseases in afflicted individuals, SARS and MERS, respectively (Fehr et al., 2017).
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • coronavirus disease 2019 2019 (COVID-19) and is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus closely related to SARS- CoV identified at the end of the year 2019 (Hoffmann et al., 2020), previously known as “2019 novel coronavirus” or 2019-nCoV.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • 2019-nCoV 2019-nCoV
  • Vaccination is still the most efficacious method of preventing viral infections (other than avoiding exposure); however, this is still a challenge for many viruses, especially in highly mutating viruses, such as RNA viruses (a group which includes the Coronaviruses). Among them we can find the influenza virus, that require annual vaccine re-formulation and prediction of likely prevalent strains each year.
  • viruses such as respiratory syncytial virus (RSV)
  • RSV respiratory syncytial virus
  • the vaccine development has major challenges such as the inadequate response to vaccination during immunization of very young infants and the circulation of two antigenically distinct RSV groups (A and B).
  • a and B antigenically distinct RSV groups
  • current drug research and development can also offer specific biologically active molecules designed against targets that are responsible for virus disease infection and progression.
  • nucleic acid-based molecules have shown tremendous potential to block gene expression either during the transcriptional or post-transcriptional phases (Asha et al., 2019).
  • SARS-CoV-2 uses the receptor angiotensin converting enzyme 2 (ACE2) for entry and the serine protease TMPRSS2 (Transmembrane Protease Serine 2) for S protein priming, similarly to the severe acute respiratory syndrome coronavirus SARS-CoV (also now known as SARS-CoV-1) as had been previously described (Hoffmann et al., 2020).
  • ACE2 receptor angiotensin converting enzyme 2
  • TMPRSS2 Transmembrane Protease Serine 2
  • inhibiting or reducing the expression levels of any of these proteins could be a useful approach for managing the prevention or treatment of SARS-CoV-2 infection or COVID-19, as well as of other coronavirus infections or their related diseases where the virus enters through the ACE2 expressed in host cells.
  • the strong binding interactions between human ACE2 and the coronavirus spike protein receptor binding domains (RDB) has been related to the high pathogenicity of SARS- CoV-2 in relation to the less virulent Singapore strain of coronavirus OC43 or the SARS- CoV which caused the 2002 SARS epidemic.
  • SARS-CoV2 RBDs seems to form a more stable complex with ACE2 in comparison to SARS-CoV1 and OC43, while outcompeting the host binding partner of ACE2, the angiotensin 2 receptor type I (ATR1), by occupying the greatest number of residues in the ATR1 binding site, which could explain its increased pathogenicity. (Chowdhury and Maranas, 2020).
  • ACE2 mRNA expression is present in a huge of human tissues, including bronchial, lung, parenchymal, ileum, testis, pancreas, cardiovascular, renal and gastrointestinal tissues (Harmer et al. 2002), and that the surface expression of ACE2 protein is found on lung alveolar epithelial cells, on enterocytes of the small intestine, in vascular endothelium (arterial and venous endothelial cells), but also arterial muscle cells of several human organs, including among others, oral and nasal mucosa, nasopharynx, lung, and skin (Hamming et al. 2004).
  • type I and type II pneumocytes are markedly positive for ACE2, which may suggest that alveolar pneumocytes in the lung could be a possible site of entrance of SARS-CoV, but also other respiratory viruses that use ACE2 as functional receptor for its route of entry, such as SARS-CoV-2.
  • SARS-CoV-2 other respiratory viruses that use ACE2 as functional receptor for its route of entry
  • ACE2 gene is expressed in corneal epithelial cells, suggesting that the eye could be susceptible to SARS-CoV-2 infections (Sun et al. 2020).
  • ACE inhibitors and angiotensin- receptor blockers have prompted concerns about the potential increased susceptibility of patients receiving these drugs to SARS-CoV-2 infection and/or COVID-19 severity (South et al., 2020). Indeed, some media sources and health systems have recently called for the discontinuation of ACE inhibitors and ARBs, both prophylactically and in the context of suspected COVID-19 (Vaduganathan et al., 2020).
  • angiotensin II levels which is known to act at the type 1 angiotensin receptor (AT1R) to increase blood pressure by inducing vasoconstriction, increasing kidney reabsorption of sodium and water, and increasing oxidative stress to promote inflammation and fibrosis.
  • A1R angiotensin receptor
  • This process can drive an angiotensin 11— AT 1 R- mediated inflammatory response in the lungs and potentially induce direct parenchymal injury, as shown in an experimental model of SARS-CoV (South et al. , 2020).
  • RNA interference RNA interference
  • RNAi is a naturally occurring post-transcriptional regulatory mechanism present in most eukaryotic cells that uses small double stranded RNA (dsRNA) molecules to direct homology-dependent gene silencing. Its discovery by Fire and Mello in the worm C. elegans (Fire et al 1998) was awarded the Nobel Prize in 2006. Shortly after its first description, RNAi was also shown to occur in mammalian cells by means of double- stranded small interfering RNAs (siRNAs) 21 nucleotides long (Elbashir et al 2001).
  • siRNAs double- stranded small interfering RNAs
  • RNA interference is thought to be an evolutionarily-conserved cellular defence mechanism used to prevent the expression of foreign genes and is commonly shared by diverse phyla and flora, where it is called post-transcriptional gene silencing. Since the discovery of the RNAi mechanism there has been an explosion of research to uncover new compounds that can selectively alter gene expression as a new way to treat human disease by addressing targets that are otherwise “undruggable” with traditional pharmaceutical approaches involving small molecules or proteins.
  • RNAi is initiated when long double stranded RNAs are processed by an RNase Ill-like protein known as Dicer.
  • the protein Dicer typically contains an N-terminal RNA helicase domain, an RNA-binding so-called Piwi/Argonaute/Zwille (PAZ) domain, two RNase III domains and a double-stranded RNA binding domain (dsRBD) (Collins et al 2005) and its activity leads to the processing of the long double stranded RNAs into 21-24 nucleotide double stranded siRNAs with 2 base 3’ overhangs and a 5’ phosphate and 3’ hydroxyl group.
  • PAZ RNA-binding so-called Piwi/Argonaute/Zwille
  • dsRBD double-stranded RNA binding domain
  • RNA-induced silencing complex RISC
  • the antisense or guide strand of the siRNA guides RISC to recognize and cleave target mRNA sequences (Elbashir et al 2001) upon adenosine- triphosphate (ATP)-dependent unwinding of the double-stranded siRNA molecule through an RNA helicase activity (Nykanen et al 2001).
  • ATP adenosine- triphosphate
  • the catalytic activity of RISC which leads to mRNA degradation, is mediated by the endonuclease Argonaute 2 (AG02) (Liu et al 2004; Song et al 2004).
  • AG02 belongs to the highly conserved Argonaute family of proteins.
  • Argonaute proteins are ⁇ 100 KDa highly basic proteins that contain two common domains, namely PIWI and PAZ domains (Cerutti et al 2000).
  • the PIWI domain is crucial for the interaction with Dicer and contains the nuclease activity responsible for the cleavage of mRNAs.
  • AG02 uses one strand of the siRNA duplex as a guide to find messenger RNAs containing complementary sequences and cleaves the phosphodiester backbone between bases 10 and 11 relative to the guide strand's 5' end (Elbashir et al 2001).
  • RNAi has been applied in biomedical research such as treatment for HIV, viral hepatitis, cardiovascular and cerebrovascular diseases, metabolic disease, neurodegenerative disorders and cancer (Angaji SA et al 2010) and it has also emerged as a viable therapy with two RNAi based therapies being approved in the last two years (Godinho and Khvorova, 2019).
  • the first formulation-based RNAi therapeutic, Patisiran (OnpattroTM) was approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA), and approximately a year later, another breakthrough was achieved with the approval of Givosiran (GIVLAARITM), the first siRNA using the conjugate-mediated approach for targeted delivery to hepatocytes.
  • FDA Food and Drug Administration
  • EMA European Medicines Agency
  • siRNA selection approaches have become more sophisticated as mechanistic details have emerged; in addition, further analysis of existing and new data can provide additional insights into further refinement of these approaches (Walton SP et al 2010).
  • siRNAs unintended genes
  • OTEs off-target effects
  • modified nucleotides such as 2’-0-methyl nucleotides, 2’- amino nucleotides, or nucleotides containing 2’-0 or 4’-C methylene bridges.
  • modification of the ribonucleotide backbone connecting adjacent nucleotides has been described, mainly by the introduction of phosphorothioate modified nucleotides. It seems that enhanced stability and/or reduction of immunogenicity are often inversely proportional to efficacy (Parrish, 2000), and only a certain number, positions and/or combinations of modified nucleotides may result in a stable and non-immunogenic silencing compound.
  • RNAi therapies include the requirement for the siRNA drugs to be delivered to the intended targeted cells and tissues, and to be internalized into the specific cells, where the compound further will need to be further released from the endosomal compartment. These problems are not yet completely resolved. Indeed, many efforts have been also made to find optimal delivery strategies for in vivo applications of RNAi drugs, including formulation and conjugation, two approaches widely used and demonstrated to be useful in the clinic, as demonstrated by the two RNAi therapeutic products currently approved for use. Formulations can be employed with or without modifications to the siRNA molecule itself, whereas the conjugation approach relies heavily on chemical modifications to protect the oligonucleotide from nuclease attack.
  • RNAi compounds examples include cationic lipids (e.g. D-Lin-MC3- DMA), polymers (e.g. cyclodextrin-based polymers and biocollagen), polypeptides, and exosomes.
  • cationic lipids e.g. D-Lin-MC3- DMA
  • polymers e.g. cyclodextrin-based polymers and biocollagen
  • polypeptides e.g. cyclodextrin-based polymers and biocollagen
  • cationic vectors including for example cationic lipids and/or cationic nanoparticles
  • endosomolytic moieties e.g. melittin-like peptides
  • fusogenic lipids e.g. DOPE
  • PEG polyethyleneoglycol
  • conjugation of ligands to improve bioavailability of RNAi drugs ligands have been successfully attached to the 5’- and/or 3’-end of the sense strand without affecting RISC loading.
  • lipids for example, cholesterol, docosahexanoic acid
  • carbohydrates for example, N-acetylgalactosamine (GalNAc)
  • glycoproteins for example, transferrin, tat peptide, GLP1
  • peptides or peptide derivatives for example, transferrin, tat peptide, GLP1
  • folate for example, transferrin, tat peptide, GLP1
  • vitamin E vitamin E
  • lipid bioconjugates such as cholesterol and docosahexaenoic acid (DHA), which preferably are selected depending on their specific distribution profile and the desired tissue intended to be targeted.
  • DHA docosahexaenoic acid
  • conjugation to cholesterol has been suggested to extend the duration, but not the magnitude of siRNA-mediated knockdown on the target mRNA in mouse lung (Moschos et al. 2007).
  • Other ligands enable targeted delivery to specific cell types, such as GalNAc which enables targeted delivery of conjugated siRNAs to the liver, since the conjugates bind to the asialoglycoprotein receptor (ASGPR), which is highly expressed by hepatocytes.
  • ASGPR asialoglycoprotein receptor
  • the lung is a well-suited organ for the uptake of small, hydrophobic drug molecules due to the vast surface area and strong perfusion, although the air-blood barrier is only permeable to a limited extent for large, hydrophilic molecules, such as therapeutic nucleic acids.
  • the lung imposes intrinsic hurdles to efficient siRNA delivery, including the reticulate pulmonary architecture ranging from the trachea to the alveoli, active clearance processes, such as mucociliary clearance and cough clearance, and effective immune responses, mainly mediated by macrophages and the influx of polymorphic neutrophils (PM Ns) that prevent the invasion of foreign material into the lung, as well as the presence of respiratory mucus in the upper airways and airway surface liquid (surfactant) in the lower airways that constitute major physical and chemical barriers entrapping substances and slowing down their transport velocities.
  • PM Ns polymorphic neutrophils
  • siRNA can be overcome by inhalation or aerosol delivery, which provides some advantages such as local targeting, immediate availability, decreased systemic side effects, and non-invasive application for the treatment of pulmonary diseases that affect lung epithelial cells such as cystic fibrosis, chronic obstructive pulmonary disease (COPD), asthma, and pulmonary fibrosis, as well as for preventing or treating infection of various viruses that infect the lung epithelium, including respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza, rhinoviruses, and severe acute respiratory syndrome (SARS) coronaviruses SARS-CoV and SARS-CoV-2.
  • RSV respiratory syncytial virus
  • PAV parainfluenza virus
  • influenza influenza
  • rhinoviruses and severe acute respiratory syndrome coronaviruses SARS-CoV and SARS-CoV-2.
  • SARS severe acute respiratory syndrome
  • RNAi respiratory syncytial virus
  • SARS severe acute respiratory syndrome
  • adenoviruses known to cause respiratory and gastrointestinal infections
  • siRNAs designed against viral genes of respiratory syncytial virus (RSV) and parainfluenza virus (PIV) was shown to prevent and inhibit the individual or combined virus infection in mice, both in the presence or the absence of transfection agents (Barik and Lu, 2015).
  • intranasal siRNA complexed with oligofectamine or polyethylenimine (PEI) and targeted to essential viral genes of influenza was also protective against influenza A viruses in mice.
  • PKI polyethylenimine
  • the authors remark the transient activity of these siRNAs, which only lasts a few days, and the continuing goal of enhancing the intra- and extracellular stability of the synthetic siRNAs while their silencing activities are increased or maintained for their therapeutic translation.
  • the presence of pulmonary surfactants in the airway has been suggested as a natural carrier that facilitates the cellular uptake of siRNA lacking any delivery system.
  • Oligonucleotides that modulate ACE2 expression are already known.
  • W02006021817 describes small interfering nucleic acid compounds targeting ACE2 mRNA, which are useful for the treatment of ocular diseases.
  • W02005086804 describes antisense oligonucleotides which hybridize with nucleic acid molecules encoding ACE2 and inhibit human ACE2 mRNA expression levels in cells. These antisense compounds are able to inhibit in vitro SARS-CoV activity.
  • Lu et al. 2008 disclose the role of ACE2 as a cellular receptor for SARS-associated coronavirus (SARS-CoV) and show that two clones of Vero E6 cells stably transfected with plasmids generating, and constitutively expressing, two short hairpin RNAs that target ACE2 mRNA were able to produce a reduction of ACE2 mRNA of about 96% and block modestly the replication of SARS-CoV.
  • SARS-CoV SARS-associated coronavirus
  • the present invention provides improved products, in particular siRNA compounds, which are targeted to a nucleic acid encoding ACE2, and which modulate the expression of ACE2, which are useful for preventing or treating virus infections which require the host ACE2 in any step of the viral cycle, for example for the virus entry as in case of coronavirus SARS-CoV-2 or SARS-CoV, and consequently useful for interfering with or reducing the virus replication.
  • siRNA products for managing such virus infections versus traditional ACE2 inhibitors are highly specific for their target and are optimized for minimum off-target effect. Thus, their side effects are expected to be negligible.
  • treatments based on siRNA will have a longer-lasting effect. This feature is due to the fact that siRNAs block the synthesis of the target protein. When treatment is suspended the cell will have to synthesise new target proteins from scratch; whereas traditional treatments would leave the target protein intact, ready to be active again once the inhibitor is no longer present.
  • Another advantage is that their potency for preventing or treating the virus infection can be increased by using a combination of different siRNAs, which could be achieved by combining siRNAs targeting ACE2 with other modulators or inhibitors of ACE2 and/or other molecular proteins or effectors required for the virus replication, including other host proteins or essential viral proteins for virus recognition and entry in host cells, replication and formation of the virion.
  • Such additional targets may include the host protease TMPRSS2 or the viral structural protein spike (S) essential for entry of SARS-CoV-2; or other known viral structural proteins, such as envelope (E) and membrane (M) proteins that together with S protein create the viral envelope of SARS-CoV-2; or the nucleocapsid (N) protein that holds the RNA genome of virus SARS-CoV-2.
  • siRNA may be combined with other therapeutic agents (for example, antibodies or other immunoglobulins; small molecule therapeutics) which target ACE2 and/or the additional targets referred to herein.
  • Nsps which are involved in RNA transcription, translation, protein synthesis processing and modification, virus replication and infection of the host, such as Nsp3b, Nsp3e, Nsp7_Nsp8 complex, Nsp9, Nsp10, Nsp14, NSp15, Nsp16, 3C-like main protease (3CLpro), papain-like proteinase (PLpro), RNA-dependent RNA polymerase (RdRp) and helicase; virus structural proteins such as spike (S), envelope (E) or E-channel, nucleocapside (N) and membrane (M) proteins; and some coronavirus virulence factors related to interfering with the host’s innate immunity and assisting coronavirus immune escape, such
  • Wu et al. also describes examples of drug molecules that can be potentially used for inhibiting any of these viral target proteins, and also of known molecules which are know that bind host target proteins involved during the viral infection such as ACE2 or TMPRSS2. Equivalent viral genes in other strains of coronavirus may also be potential targets for treatment or prevention of said other coronaviruses.
  • the siRNA compounds comprise two complementary oligonucleotide strands, wherein the antisense strand has from 19 to 21 nucleobases and is complementary to at least 19 consecutive nucleotides of the ACE2 mRNA, and the sense strand has from 19 to 21 nucleobases with at least 15 consecutive nucleotides from the 5’-end being complementary to at least 15 consecutive nucleotides of the antisense strand, preferably from 15 to about 16, 17, 18 or 19 nucleobases.
  • the compound comprises at least one modified nucleobase, modified sugar moiety or modified internucleoside linkage.
  • the compound comprises at least one 2'-0-methoxy modification and/or at least one 2’-fluoro modification and/or at least one phosphorothioate linkage.
  • the siRNA is blunt ended; that is, each strand is the same length, and there are no overhangs.
  • Other embodiments may also include 3’ mononucleotide or dinucleotide overhangs (for example, U, UU, dT or dTdT).
  • the siRNA compounds may be short hairpin oligonucleotides, being a single strand which forms a hairpin such that a double stranded region is formed wherein a first portion of the oligonucleotide has from 19 to 21 nucleobases and is complementary to at least 19 consecutive nucleotides of the ACE2 mRNA, and a second portion of the oligonucleotide has from 19 to 21 nucleobases with at least 15 consecutive nucleotides from the 5’-end being complementary to at least 15 consecutive nucleotides of the first portion.
  • kits, compositions, including pharmaceutical compositions, and medicaments comprising compounds of the invention.
  • siRNA compounds which are targeted to a nucleic acid encoding ACE2, and which modulate the expression of ACE2, for use in preventing or treating virus infections which require the host ACE2 in any step of the viral cycle, for example for the virus entry as in case of coronavirus SARS-CoV-2 or SARS-CoV, and consequently useful for interfering with or reducing the virus replication.
  • an siRNA compound in the manufacture of a medicament for preventing or treating virus infections which require the host ACE2 in any step of the viral cycle, wherein the siRNA compound is targeted to a nucleic acid encoding ACE2 and modulates the expression of ACE2.
  • the virus infection is a coronavirus infection, more preferably a SARS-CoV infection, and most preferably a SARS-CoV-2 infection.
  • an siRNA compound in the manufacture of a medicament for preventing or treating a coronavirus infection wherein the siRNA compound is targeted to a nucleic acid encoding ACE2 and modulates the expression of ACE2.
  • an siRNA compound in the manufacture of a medicament for preventing or treating COVID-19 wherein the siRNA compound is targeted to a nucleic acid encoding ACE2 and modulates the expression of ACE2.
  • Methods of modulating the expression of ACE2 in one or more cells or tissues comprising contacting the cell(s) or tissue(s) with one or more compounds or compositions of the invention.
  • Methods of treating a subject, particularly a human, suspected of having or being prone to a disease or condition associated with expression of ACE2, or in need of treatment therefore, are also set forth herein.
  • Such methods comprise, for example, administering a therapeutically or prophylactically effective amount of one or more of the compounds or compositions of the invention to the subject being treated.
  • the subject to be treated has been diagnosed with having a disease or condition associated with expression of ACE2, such as a SARS coronavirus infection or its related disease, preferably SARS-CoV-2 or COVID-19.
  • the present invention also provides methods of inhibiting a SARS coronavirus, preferably SARS-CoV-2 in one or more cells or tissues comprising, for example, contacting the cell(s) or tissue(s) with one or more compounds of the invention.
  • the present invention also provides methods of treating a subject, particularly a human, having a disease or condition associated with a SARS coronavirus, preferably SARS- CoV-2, or in need of treatment therefore, comprising administering to the subject a therapeutically or prophylactically effective amount of a compound described herein so that expression of ACE2 is inhibited.
  • Such methods comprise administering a therapeutically or prophylactically effective amount of one or more of the compounds or compositions of the invention to the subject being treated.
  • the animal or person being treated has been diagnosed with having a disease or condition associated with a SARS virus.
  • Figure 1 shows the short fragments of the target gene sequence of ACE2 chosen as the target sequences for the siRNAs of the present invention.
  • Figure 2 shows the oligonucleotide sequences for the blunt ended siRNA molecules of the present invention targeting ACE2 encompassed by the present invention.
  • the SEQ ID NOs given in the Figure refer to the oligonucleotide sequence of the antisense and sense (5’ -> 3’) strands; typically, siRNAs will be administered as dsRNAs, so siRNAs will include both the antisense strand and its complementary sense strand.
  • SEQ ID NO. 46 to SEQ ID NO. 90 are antisense strands of siRNAs targeting SEQ ID NO. 1 to SEQ ID NO. 45, respectively.
  • siRNA 135 correspond respectively to the complementary sense strands of siRNAs having antisense strands SEQ ID NO. 46 to SEQ ID NO. 90.
  • a siRNA will include the antisense and sense strand, and may also include 3’ mononucleotide or dinucleotide overhangs (for example, U, UU, dT or dTdT). However, this is not essential.
  • Figure 3 shows the oligonucleotide sequences of the siRNA molecules targeting ACE2 with at least one 3’-overhang in any of their strands encompassed by the present invention.
  • the SEQ ID NOs given in the Figure refer to the oligonucleotide sequence of the antisense and sense (5’ -> 3’) strands; typically, siRNAs will be administered as dsRNAs, so siRNAs will include both the antisense strand and its complementary sense strand.
  • SEQ ID NO. 136 to SEQ ID NO. 556 are antisense strands of siRNAs targeting any of sequences SEQ ID NO. 1 to SEQ ID NO. 45, and SEQ ID NO. 557 to SEQ ID NO. 977 respectively correspond to complementary sense strands of siRNAs having antisense strands SEQ ID NO. 136 to SEQ ID NO. 556.
  • X is dT (deoxythymidine).
  • FIG. 4 modified 19 nucleotides blunt-ended siRNAs targeting ACE2.
  • SEQ ID NO 978 to SEQ ID NO 1007 and SEQ ID NO: 1008 to SEQ ID NO: 1037 refer respectively to the modified antisense (5’ -> 3’) strands and modified sense strands (5’ -> 3’) of different siRNAs which targets sequences SEQ ID NO 1 to SEQ ID NO 45 of the ACE2 gene.
  • Figure 5 shows oligonucleotide sequences comprising short fragments of 15-21 consecutive nucleotides corresponding to the nucleotide sequences of the sense (SEQ ID NO: 1048 - SEQ ID NO: 1056) and antisense (SEQ ID NO: 1038 - SEQ ID NO: 1047) strands for the siRNAs of the present invention.
  • Figure 6 shows ACE2 expression levels after transfection of different siRNAs targeting the ACE2 gene in human lung BEAS-2B cells.
  • Figure 7 shows ACE2 expression levels after transfection of different siRNAs targeting ACE2 gene in African Green Monkey Vero E6 cells.
  • Figure 8 shows ACE2 expression levels after transfection of different siRNAs targeting ACE2 gene in human huh-7 cells.
  • the present invention relates to the provision of siRNA molecules targeting ACE2 mRNA, the biological receptor of the spike (S) protein of SARS coronavirus SARS-CoV- 2 or SARS-CoV which is involved in the virus entry into the cell.
  • siRNA molecule for use as a medicament or in the prophylactic or therapeutic treatment of a virus infection involving the host angiotensin I converting enzyme 2 (ACE2) in any step of the virus cycle of replication, preferably a SARS coronavirus including SARS-CoV-2 and/or SARS-CoV-1.
  • Said siRNA molecule specifically targets a sequence selected from the group consisting or comprising of: SEQ ID NO. 1 to SEQ ID NO. 45 and reduces expression of the ACE2 gene when introduced in a cell.
  • the target sequence comprises or consists of any of the nucleotide sequences SEQ ID NO. 1 to SEQ ID NO. 14
  • the target sequence comprises or consists of SEQ ID NO. 3.
  • the target sequence comprises or consists of SEQ ID NO. 9.
  • a gene is “targeted” by a siRNA according to the present invention when, for example, the siRNA molecule selectively decreases or inhibits the expression of the gene.
  • the phrase “selectively decrease or inhibit” as used herein encompasses siRNAs that affect expression of one gene, in this case ACE2.
  • a siRNA targets a gene when (one strand of) the siRNA hybridizes under stringent conditions to the gene transcript, i.e. its mRNA.
  • Hybridizing “under stringent conditions” means annealing to the target mRNA region under standard conditions, e.g., high temperature and/or low salt content which tend to disfavour hybridization.
  • a suitable protocol (involving O.lxSSC, 68 °C for 2 hours) is described in Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982, on pages 387-389.
  • nucleic acid sequences cited herein are written in a 5’ to 3’ direction unless otherwise indicated.
  • the term “nucleic acid” refers to either DNA or RNA or a modified form thereof comprising the purine or pyrimidine bases present in DNA (adenine “A”, cytosine “C”, guanine “G”, thymine “T”) or in RNA (adenine “A”, cytosine “C”, guanine “G”, uracil “U”).
  • Interfering RNAs provided herein may comprise “T” bases, for example at 3’ ends, even though “T” bases do not naturally occur in RNA. In some cases, these bases may appear as “dT” to differentiate deoxy ribonucleotides present in a chain of ribonucleotides.
  • target sequence as defined above is described as a target DNA sequence as used for definition of transcript variants in databases used for the purposes of designing siRNAs, whereas the specific compounds to be used will be RNA sequences defined as such.
  • GenBank Accession Number corresponding to two human transcripts of ACE2 mRNA are NM_001371415.1 (transcript variant 1) and NM_021804.3 (transcript variant 2).
  • ENSEMBL MBL-EBI/Wellcome Trust Sanger Institute
  • ACE2 human Accession Numbers ENST00000252519, ENST00000427411 , ENST00000471548, ENST00000473851 and ENST00000484756. All this information is in the free-access Ensembl data base.
  • Said target regions identified by the present invention comprise or consist of at least one sequence selected from SEQ ID NO. 1 to SEQ ID NO. 45. These sequences present 100% homology between the following species: Homo sapiens, Macaca mulatta, Macaca fascicularis, Canis lupus familiaris, and Mus musculus, including Mus musculus C57BU6NJ.
  • said target regions comprise or consist of a sequence selected from SEQ ID NO. 3, SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 , SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO: 14.
  • the target regions are those which are targeted by any or some of the siRNA molecules of the present invention having a high gene silencing efficiency in cells of different species, preferably of 60% and above, or 70% and above, or 80% and above, or 90% and above or 95% and above, in cells of at least two of the species above mentioned having 100% homology.
  • the target regions are those that are targeted by any or some of the siRNA molecules having gene silencing efficiency higher than 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% in cells of at least two different species, or in cells of at least three different species, or in cells of four different species having 100% homology in the target region of the ACE2 gene, and even more preferably in cells of all different species above mentioned having 100% homology.
  • RNAi field when in vitro studies demonstrate that a human siRNA is not able to induce knock-down of the animal model gene, a surrogate compound (animal-active analogue) is synthetized in order to analyze the efficacy of the siRNA in the relevant animal model.
  • This surrogate is designed against the same region as the human siRNA, thus the two siRNAs have the same sequence except for a few nucleotides, depending on the homology between the human and the animal target gene.
  • This approach has been widely used for development of other oligonucleotides, specifically for toxicology and efficacy studies (Kornbrust et al 2013).
  • a siRNA according to the aspects of the present invention will preferably comprise a double stranded RNA molecule, whose antisense strand will comprise an RNA sequence substantially complementary to at least one sequence consisting of SEQ ID NO. 1 to SEQ ID NO. 45, and whose sense strand will comprise an RNA sequence having at least the first 15 nucleotides from the 5’-end complementary to the antisense strand, wherein both strands are hybridised by standard base pairing between nucleotides. More preferably, a siRNA according to aspects of the present invention will preferably comprise a double stranded RNA molecule, whose antisense strand will comprise an RNA sequence substantially complementary to SEQ ID NO. 1 to SEQ ID NO.
  • the antisense strand comprises or consists of an RNA sequence substantially complementary to at least one of the sequences from the group consisting of SEQ ID NO. 1 to SEQ ID NO. 14.
  • the antisense strand comprises or consists of an RNA sequence substantially complementary to SEQ ID NO. 3.
  • the antisense strand comprises or consists of an RNA sequence substantially complementary to SEQ ID NO. 9.
  • substantially complementary to a target mRNA sequence, may also be understood as “substantially identical” to said target sequence. “Identity” as is known by one of ordinary skill in the art, is the degree of sequence relatedness between nucleotide sequences as determined by matching the order and identity of nucleotides between sequences. In one embodiment the antisense strand of an siRNA having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementarity to the target mRNA sequence are considered substantially complementary and may be used in the present invention.
  • the percentage of complementarity describes the percentage of contiguous nucleotides in a first nucleic acid molecule that can base pair in the Watson-Crick sense with a set of contiguous nucleotides in a second nucleic acid molecule.
  • the antisense siRNA strand is 100% complementary to the target mRNA sequence
  • the sense strand is 100% complementary to the antisense strand over the double stranded portion of the siRNA.
  • the siRNA may also include unpaired overhangs, for example, 3’ mononucleotide overhangs, preferably U or dT, 3’ dinucleotide overhangs, preferably UU or dTdT.
  • Preferred siRNA antisense strands that target sequences SEQ ID NO. 1 to SEQ ID NO. 45 of ACE2 gene or mRNA comprise or consist of oligonucleotide sequences having 19, 20 or 21 consecutive nucleotides from one of the nucleotide sequences defined by SEQ ID NO: 1038 (5’-
  • siRNA sense strands which are complementary to these antisense strands comprise or consist of oligonucleotide sequences having 19, 20 or 21 consecutive nucleotides from one of the nucleotide sequences defined by SEQ ID NO: 1048 (5’-
  • SEQ ID NO: 1049 5’-CAGAAACCAAACAUAGAUGUUACUG-3'
  • SEQ ID NO: 1050 5’- AAAUCAUGUCACUUUCUGCAGCC-3'
  • SEQ ID NO: 1051 5’-
  • siRNA sense strands which are complementary to these antisense strands consist of oligonucleotide sequences of 19 nucleotides in length that comprises at least 15, 16, 17 or 18, and preferably only 15, consecutive nucleotides from one of the nucleotide sequences defined by SEQ ID NO: 1048, SEQ ID NO: 1049, SEQ ID NO: 1050, SEQ ID NO: 1051, SEQ ID NO: 1052 or SEQ ID NO: 1053, respectively.
  • siRNA antisense strands that target sequences SEQ ID NO. 1 to SEQ ID NO. 45 of ACE2 gene or mRNA consist of oligonucleotide sequences which are selected from one or more of SEQ ID NO. 48, SEQ ID NO. 46 to SEQ ID NO. 47, SEQ ID NO. 49 to SEQ ID NO: 90, SEQ ID NO: 152 to SEQ ID NO: 159, SEQ ID NO: 136 to SEQ ID NO: 151 , SEQ ID NO: 160 to SEQ ID NO: 556 and SEQ ID NO: 978 to SEQ ID NO: 1007.
  • Preferred siRNA sense strands which are complementary to these antisense strands consist of oligonucleotide sequences which are selected from one or more of SEQ ID NO. 93, SEQ ID NO. 91 to SEQ ID NO. 92, SEQ ID NO. 94 to SEQ ID NO: 135, SEQ ID NO: 573 to SEQ ID NO: 580, SEQ ID NO: 557 to SEQ ID NO: 572, SEQ ID NO: 581 to SEQ ID NO: 977 and SEQ ID NO: 1008 to SEQ ID NO: 1037, respectively.
  • Preferred siRNA antisense strands that target sequences SEQ ID NO. 1 to SEQ ID NO. 14 of ACE2 gene or mRNA comprise or consist of oligonucleotide sequences having 19, 20 or 21 consecutive nucleotides from one of the nucleotide sequences defined by SEQ ID NO: 1044 (5’-AGUACAGAUUUGUCCAAAAUCUAC-3'), SEQ ID NO: 1045 (5’- CACAUAUCACCAAGCAAAU-3'), SEQ ID NO: 1046 (5’-
  • siRNA sense strands which are complementary to these antisense strands comprise or consist of oligonucleotide sequences having 19, 20 or 21 consecutive nucleotides from one of the nucleotide sequences defined by SEQ ID NO: 1054 (5’-AGAUUUUGGACAAAUCUGUACU-3'), SEQ ID NO: 1055 (5’-AUUUGCUUGGUGAUAUGUG-3'), SEQ ID NO: 1056 (5’- GGUAGAUUUUGGACAAAUCUGU-3'), or SEQ ID NO: 1049 (5’-
  • siRNA sense strands which are complementary to these antisense strands consist of oligonucleotide sequences of 19 nucleotides in length that comprises at least 15, 16, 17 or 18, and preferably only 15, consecutive nucleotides from one of the nucleotide sequences defined by SEQ ID NO: 1054, SEQ ID NO: 1055, SEQ ID NO: 1056, or SEQ ID NO: 1057, respectively.
  • siRNA antisense strands that target sequences SEQ ID NO. 1 to SEQ ID NO. 14 of ACE2 gene or mRNA consist of oligonucleotide sequences which are selected from one or more of SEQ ID NO. 48, SEQ ID NO: 152 to SEQ ID NO: 159, SEQ ID NO: 409 to SEQ ID NO: 411 , SEQ ID NO: 497 to SEQ ID NO: 498, SEQ ID NO. 46 to SEQ ID NO. 47, SEQ ID NO.
  • siRNA sense strands which are complementary to these antisense strands consist of oligonucleotide sequences which are selected from one or more of SEQ ID NO.
  • one aspect of the invention refers to a small interfering RNA or siRNA molecule which targets the angiotensin I converting enzyme 2 (ACE2) gene, preferably against a target sequence of at least 19 nucleotides of the ACE2 mRNA, comprising an antisense strand and a sense strand complementary to the antisense strand for use in the prophylactic or therapeutic treatment in a subject of a virus infection involving host ACE2 in the virus cycle, preferably SARS-CoV-2, wherein the antisense strand comprises or consists of at least 19 consecutive nucleotides of any of the nucleotide sequences selected from the group SEQ ID NO: 1038, SEQ ID NO: 1039, SEQ ID NO: 1040, SEQ ID NO: 1041 , SEQ ID NO: 1042 and SEQ ID NO: 1043, and both strands are base paired to form a double stranded RNA (dsRNA) structure comprising at least 15 base pairs (bp) wherein the 3’
  • the sense strand is an oligonucleotide consisting of a sequence of 19 nucleotides complementary to the antisense strand which form a blunt ended dsRNA structure, preferably consisting of any of the nucleotide sequences SEQ ID NO. 93, SEQ ID NO. 91 to SEQ ID NO. 92 and SEQ ID NO. 94 to SEQ ID NO. 135; or ii.
  • the antisense strand when the antisense strand consists of 19 consecutive nucleotides of any of the nucleotide sequences and further comprises an additional nucleotide or dinucleotide of U ordT at the 3’-end, preferably consisting of any of the nucleotide sequences SEQ ID NO. 136 to SEQ ID NO. 495, the antisense strand has a 3’- overhang consisting of said additional nucleotide or dinucleotide, and the sense strand is one selected from: a.
  • nucleotide sequence consisting of a sequence of 19 nucleotides in which at least the first 15 consecutive nucleotides from the 5’-end are complementary to the antisense strand, preferably consisting of any of the nucleotide sequences SEQ ID NO. 573 to SEQ ID NO. 580, SEQ ID NO. 557 to SEQ ID NO. 572 and SEQ ID NO. 581 to SEQ ID NO. 828, or b.
  • nucleotide sequence consisting of a sequence of 20 or 21 nucleotides in which the first 19 consecutive nucleotides from the 5’-end are complementary to the antisense strand, and are followed by an additional nucleotide or dinucleotide of U or dT at the 3’-end, preferably consisting of any of the nucleotide sequences SEQ ID NO. 830 to SEQ ID NO. 832, SEQ ID NO. 829 and SEQ ID NO. 833 to SEQ ID NO. 916, to form a dsRNA structure of 19 bp with 3’-overhangs of U, dT, UU or dTdT on both strands; or iii.
  • the antisense strand when the antisense strand consists of 20 or 21 consecutive nucleotides of any of the nucleotide sequences SEQ ID NO. 1038 to SEQ ID NO. 1043, preferably consisting of any of the nucleotide sequences SEQ ID NO. 497 to SEQ ID NO. 498, SEQ ID NO. 496, and SEQ ID NO. 499 to SEQ ID NO. 556, the sense strand is an oligonucleotide consisting of a sequence of 20 or 21 consecutive nucleotides of any of the nucleotide sequences SEQ ID NO: 1048 to SEQ ID NO: 1053, respectively, preferably consisting of any of the nucleotide sequences SEQ ID NO. 918 to SEQ ID NO. 919, SEQ ID NO. 917 and SEQ ID NO. 920 to SEQ ID NO. 977, to form a dsRNA structure of 19 bp with 3’- overhangs on both strands.
  • the antisense strand of the siRNA molecule comprises or consists of at least 19 consecutive nucleotides of any of the nucleotide sequences SEQ ID NO: 1038 or SEQ ID NO: 1039, more preferably of any of nucleotide sequences SEQ ID NO: 1044, SEQ ID NO: 1045, SEQ ID NO: 1046 or SEQ ID NO: 1047, and: i. when the antisense strand consists of 19 consecutive nucleotides, preferably consisting of any of the nucleotide sequences SEQ ID NO. 48, SEQ ID NO. 46 to SEQ ID NO. 47 and SEQ ID NO. 49 to SEQ ID NO.
  • the sense strand is an oligonucleotide consisting of a sequence of 19 nucleotides complementary to the antisense strand which form a blunt ended dsRNA structure, preferably consisting of any of the nucleotide sequences SEQ ID NO. 93, SEQ ID NO. 91 to SEQ ID NO. 92 and SEQ ID NO. 94 to SEQ ID NO. 104; or ii. when the antisense strand consists of 19 consecutive nucleotides of any of the nucleotide sequences and further comprises an additional nucleotide or dinucleotide of U ordT at the 3’-end, preferably consisting of any of the nucleotide sequences SEQ ID NO.
  • the antisense strand has a 3’-overhang consisting of said additional nucleotide or dinucleotide, and the sense strand is one selected from: a.
  • nucleotide sequence consisting of a sequence of 19 nucleotides in which at least the first 15 consecutive nucleotides from the 5’-end are complementary to the antisense strand, preferably consisting of any of the nucleotide sequences SEQ ID NO. 573 to SEQ ID NO. 580, SEQ ID NO. 557 to SEQ ID NO. 572 and SEQ ID NO. 581 to SEQ ID NO. 644, or b.
  • nucleotide sequence consisting of a sequence of 20 or 21 nucleotides in which the first 19 consecutive nucleotides from the 5’-end are complementary to the antisense strand, and are followed by an additional nucleotide or dinucleotide of U or dT at the 3’-end, preferably consisting of any of the nucleotide sequences SEQ ID NO. 830 to SEQ ID NO. 832, SEQ ID NO. 829 and SEQ ID NO. 833 to SEQ ID NO. 852, to form a dsRNA structure of 19 bp with 3’-overhangs of U, dT, UU or dTdT on both strands; or iii.
  • the antisense strand when the antisense strand consists of 20 or 21 consecutive nucleotides of any of the nucleotide sequences SEQ ID NO. 1038 or SEQ ID NO. 1039, preferably consisting of any of the nucleotide sequences SEQ ID NO. 497 to SEQ ID NO. 498, SEQ ID NO. 496 and SEQ ID NO. 499 to SEQ ID NO. 514, the sense strand is an oligonucleotide consisting of a sequence of 20 or 21 consecutive nucleotides of any of the nucleotide sequences SEQ ID NO: 1048 or SEQ ID NO: 1049, respectively, preferably consisting of any of the nucleotide sequences SEQ ID NO. 918 to SEQ ID NO. 919, SEQ ID NO. 917 and SEQ ID NO. 920 to SEQ ID NO. 935, to form a dsRNA structure of 19 bp with 3’-overhangs on both strands.
  • the antisense strand of the siRNA molecule consists of at least 19 consecutive nucleotides of any of the nucleotide sequences SEQ ID NO: 1038 or SEQ ID NO: 1039, and preferably consisting of any of the nucleotide sequences SEQ ID NO. 48, SEQ ID NO. 46 to SEQ ID NO. 47 and SEQ ID NO. 49 to SEQ ID NO. 59, and the sense strand is an oligonucleotide consisting of a sequence of 19 nucleotides complementary to the antisense strand which form a blunt ended dsRNA structure, preferably consisting of any of the nucleotide sequences SEQ ID NO. 93, SEQ ID NO. 91 to SEQ ID NO.
  • the siRNA molecule is one selected from the siRNA duplexes defined in Figure 2 selected from the following group: siRNA ID 335: antisense strand SEQ ID NO: 48, sense strand SEQ ID NO: 93; siRNA ID 389: antisense strand SEQ ID NO: 54, sense strand SEQ ID NO: 99; siRNA ID 361 : antisense strand SEQ ID NO: 46, sense strand SEQ ID NO: 91 ; siRNA ID 350: antisense strand SEQ ID NO: 47, sense strand SEQ ID NO: 92; siRNA ID 320: antisense strand SEQ ID NO: 49, sense strand SEQ ID NO: 94; siRNA ID 371 : antisense strand SEQ ID NO: 50, sense strand SEQ ID NO: 95; siRNA ID 419: antisense strand SEQ ID NO: 51 , sense strand SEQ ID NO: 96;
  • the antisense strand of the siRNA molecule consists of at least 19 consecutive nucleotides of any of the nucleotide sequences SEQ ID NO: 1038 or SEQ ID NO: 1039, preferably consisting of at least 19 consecutive nucleotides of any of the nucleotide sequences SEQ ID NO: 1044, SEQ ID NO: 1045, SEQ ID NO: 1046 and SEQ ID NO: 1047, and more preferably consisting of any of the nucleotide sequences SEQ ID NO. 152 to SEQ ID NO. 159, SEQ ID NO. 136 to SEQ ID NO. 151 and SEQ ID NO. 160 to SEQ ID NO.
  • the sense strand is an oligonucleotide consisting of a sequence of 19 nucleotides complementary to the antisense strand which form a blunt ended dsRNA structure, preferably at least 19 consecutive nucleotides of any of the nucleotide sequences SEQ ID NO: 1048 or SEQ ID NO: 1049, preferably consisting of at least 19 consecutive nucleotides respectively of any of the nucleotide sequences SEQ ID NO: 1054, SEQ ID NO: 1055, SEQ ID NO: 1056 and SEQ ID NO: 1057, and more preferably consisting of any of the nucleotide sequences SEQ ID NO. 573 to SEQ ID NO. 580, SEQ ID NO. 557 to SEQ ID NO.
  • the siRNA molecule is one selected from the siRNA duplexes defined in Figure 3 selected from the following group: siRNA ID 341 : antisense strand SEQ ID NO: 152, sense strand SEQ ID NO: 573; siRNA ID 342: antisense strand SEQ ID NO: 153, sense strand SEQ ID NO: 574; siRNA ID 343: antisense strand SEQ ID NO: 154 sense strand SEQ ID NO: 575; siRNA ID 344: antisense strand SEQ ID NO: 155, sense strand SEQ ID NO: 576; siRNA ID 346: antisense strand SEQ ID NO: 156, sense strand SEQ ID NO: 577; siRNA ID 347: antisense strand SEQ ID NO: 157, sense strand SEQ ID NO: 578; siRNA ID 348: antisense strand SEQ ID NO: 158, sense strand SEQ ID
  • the siRNA molecule is one of the siRNA duplexes defined in Figure 3 selected from the following group: siRNA ID 344: antisense strand SEQ ID NO: 155, sense strand SEQ ID NO: 576; siRNA ID 342: antisense strand SEQ ID NO: 153, sense strand SEQ ID NO: 574; siRNA ID 346: antisense strand SEQ ID NO: 156, sense strand SEQ ID NO: 577; siRNA ID 348: antisense strand SEQ ID NO: 158, sense strand SEQ ID NO: 579; siRNA ID 349: antisense strand SEQ ID NO: 159, sense strand SEQ ID NO: 580; siRNA ID 365: antisense strand SEQ ID NO: 139, sense strand SEQ ID NO: 560; siRNA ID 369: antisense strand SEQ ID NO: 143, sense strand SEQ ID NO: 564; siRNA ID 356: antisense strand SEQ
  • Another aspect of the invention refers to a small interfering RNA or siRNA molecule against a target sequence of at least 19 nucleotides of the angiotensin I converting enzyme 2 (ACE2) mRNA comprising an antisense strand and a sense strand complementary to the antisense strand, wherein the antisense strand comprises or consists of at least 19 consecutive nucleotides of any of the nucleotide sequences selected from the group SEQ ID NO: 1038 and SEQ ID NO: 1039 (more preferably of one any of the nucleotide sequences selected from the group SEQ ID NO: 1044, SEQ ID NO: 1045, SEQ ID NO: 1046 and SEQ ID NO: 1047), and both strands are base paired to form a double stranded RNA (dsRNA) structure comprising at least 15 base pairs (bp) wherein the 3’ end of the antisense strand is a blunt end or has a one nucleotide or dinucleotide 3’
  • the sense strand is an oligonucleotide consisting of a sequence of 19 nucleotides complementary to the antisense strand which form a blunt ended dsRNA structure, preferably consisting of any of the nucleotide sequences SEQ ID NO. 93, SEQ ID NO. 91 to SEQ ID NO. 92 and SEQ ID NO. 94 to SEQ ID NO. 101 , SEQ ID NO.
  • the antisense strand consists of 19 consecutive nucleotides of any of the nucleotide sequences and further comprises an additional nucleotide or dinucleotide of U ordT at the 3’-end, preferably consisting of any of the nucleotide sequences SEQ ID NO. 152 to SEQ ID NO. 159, SEQ ID NO. 136 to SEQ ID NO. 151 , SEQ ID NO. 160 to SEQ ID NO. 223, SEQ ID NO. 409 to SEQ ID NO. 411, SEQ ID NO. 408 and SEQ ID NO. 412 to SEQ ID NO.
  • the antisense strand has a 3’-overhang consisting of said additional nucleotide or dinucleotide
  • the sense strand is one selected from: a. a nucleotide sequence consisting of a sequence of 19 nucleotides in which at least the first 15 consecutive nucleotides from the 5’-end are complementary to the antisense strand, preferably consisting of any of the nucleotide sequences SEQ ID NO. 573 to SEQ ID NO. 580, SEQ ID NO. 557 to SEQ ID NO. 572, SEQ ID NO. 581 to SEQ ID NO. 644, or b.
  • nucleotide sequence consisting of a sequence of 20 or 21 nucleotides in which the first 19 consecutive nucleotides from the 5’-end are complementary to the antisense strand, and are followed by an additional nucleotide or dinucleotide of U or dT at the 3’-end, preferably consisting of any of the nucleotide sequences SEQ ID NO. 830 to SEQ ID NO. 832, SEQ ID NO. 829 and SEQ ID NO. 833 to SEQ ID NO. 854, to form a dsRNA structure of 19 bp with 3’-overhangs of U, dT, UU or dTdT on both strands; or iii.
  • the antisense strand consists of 20 or 21 consecutive nucleotides of any of the nucleotide sequences SEQ ID NO: 1044, SEQ ID NO: 1045, SEQ ID NO: 1046 or SEQ ID NO: 1047, preferably consisting of any of the nucleotide sequences SEQ ID NO. 497 to SEQ ID NO. 498, SEQ ID NO. 496 and SEQ ID NO. 499 to SEQ ID NO.
  • the sense strand is an oligonucleotide consisting of a sequence of 20 or 21 consecutive nucleotides of any of the nucleotide sequences SEQ ID NO: 1054, SEQ ID NO: 1055, SEQ ID NO: 1056 or SEQ ID NO: 1057, respectively, preferably consisting of any of the nucleotide sequences EQ ID NO. 918 to SEQ ID NO. 919, SEQ ID NO. 917 and SEQ ID NO. 920 to SEQ ID NO. 935, to form a dsRNA structure of 19 bp with 3’-overhangs on both strands.
  • the antisense strand of the siRNA molecule comprises or consists of at least 19 consecutive nucleotides of any of the nucleotide sequences selected from the group SEQ ID NO: 1044, SEQ ID NO: 1045, SEQ ID NO: 1046 and SEQ ID NO: 1047, and: i. when the antisense strand consists of any of the nucleotide sequences SEQ ID NO. 48, SEQ ID NO. 46 to SEQ ID NO. 47 and SEQ ID NO. 49 to SEQ ID NO. 56, SEQ ID NO. 58 and SEQ ID NO.
  • the sense strand is an oligonucleotide consisting of a sequence of 19 nucleotides complementary to the antisense strand which form a blunt ended dsRNA structure, preferably consisting of any of the nucleotide sequences SEQ ID NO. 93, SEQ ID NO. 91 to SEQ ID NO. 92 and SEQ ID NO. 94 to SEQ ID NO. 101 , SEQ ID NO. 103 and SEQ ID NO. 104; or ii.
  • the antisense strand consists of 19 consecutive nucleotides of any of the nucleotide sequences and further comprises an additional nucleotide or dinucleotide of U or dT at the 3’-end, and it consists of any of the nucleotide sequences SEQ ID NO. 152 to SEQ ID NO. 159, SEQ ID NO. 136 to SEQ ID NO. 151 and SEQ ID NO. 160 to SEQ ID NO. 223, SEQ ID NO. 409 to SEQ ID NO. 411 , SEQ ID NO. 408 and SEQ ID NO. 412 to SEQ ID NO.
  • the antisense strand has a 3’-overhang consisting of said additional nucleotide or dinucleotide
  • the sense strand is one selected from: a. a nucleotide sequence consisting of a sequence of 19 nucleotides in which at least the first 15 consecutive nucleotides from the 5’-end are complementary to the antisense strand, preferably consisting of any of the nucleotide sequences SEQ ID NO. 573 to SEQ ID NO. 580, SEQ ID NO. 557 to SEQ ID NO. 572 and SEQ ID NO. 581 to SEQ ID NO. 644, or b.
  • nucleotide sequence consisting of a sequence of 20 or 21 nucleotides in which the first 19 consecutive nucleotides from the 5’-end are complementary to the antisense strand, and are followed by an additional nucleotide or dinucleotide of U or dT at the 3’-end, preferably consisting of any of the nucleotide sequences SEQ ID NO. 830 to SEQ ID NO. 832, SEQ ID NO. 829 and SEQ ID NO. 833 to SEQ ID NO. 854, to form a dsRNA structure of 19 bp with 3’-overhangs of U, dT, UU or dTdT on both strands; or iii.
  • the antisense strand when the antisense strand consists of 20 or 21 consecutive nucleotides, it consists of any of the nucleotide sequences SEQ ID NO. 497 to SEQ ID NO. 498, SEQ ID NO. 496 and SEQ ID NO. 499 to SEQ ID NO. 514, the sense strand is an oligonucleotide consisting of any of the nucleotide sequences SEQ ID NO. 918 to SEQ ID NO. 919, SEQ ID NO. 917 and SEQ ID NO. 920 to SEQ ID NO. 935, to form a dsRNA structure of 19 bp with 3’-overhangs on both strands.
  • the antisense strand of the siRNA molecule consists of at least 19 consecutive nucleotides of any of the nucleotide sequences SEQ ID NO: 1038 or SEQ ID NO: 1039, and preferably consisting of any of the nucleotide sequences SEQ ID NO. 48, SEQ ID NO. 46 to SEQ ID NO. 47 and SEQ ID NO. 49 to SEQ ID NO. 59, and the sense strand is an oligonucleotide consisting of a sequence of 19 nucleotides complementary to the antisense strand which form a blunt ended dsRNA structure, preferably consisting of any of the nucleotide sequences SEQ ID NO. 93, SEQ ID NO. 91 to SEQ ID NO.
  • the siRNA molecule is one selected from the siRNA duplexes defined in Figure 2 selected from the following group: siRNA ID 335: antisense strand SEQ ID NO: 48, sense strand SEQ ID NO: 93; siRNA ID 361 : antisense strand SEQ ID NO: 46, sense strand SEQ ID NO: 91 ; siRNA ID 350: antisense strand SEQ ID NO: 47, sense strand SEQ ID NO: 92; siRNA ID 320: antisense strand SEQ ID NO: 49, sense strand SEQ ID NO: 94; siRNA ID 371 : antisense strand SEQ ID NO: 50, sense strand SEQ ID NO: 95; siRNA ID 419: antisense strand SEQ ID NO: 51 , sense strand SEQ ID NO: 96; siRNA ID 404: antisense strand S
  • the antisense strand of the siRNA molecule consists of at least 19 consecutive nucleotides of any of the nucleotide sequences SEQ ID NO: 1044, SEQ ID NO: 1045, SEQ ID NO: 1046 and SEQ ID NO: 1047, and preferably consisting of any of the nucleotide sequences SEQ ID NO. 152 to SEQ ID NO. 159, SEQ ID NO. 136 to SEQ ID NO. 151 and SEQ ID NO. 160 to SEQ ID NO.
  • the sense strand is an oligonucleotide consisting of a sequence of 19 nucleotides complementary to the antisense strand which form a blunt ended dsRNA structure, preferably consisting of at least 19 consecutive nucleotides respectively of any of the nucleotide sequences SEQ ID NO: 1054, SEQ ID NO: 1055, SEQ ID NO: 1056 and SEQ ID NO: 1057, and more preferably consisting of any of the nucleotide sequences SEQ ID NO. 573 to SEQ ID NO. 580, SEQ ID NO. 557 to SEQ ID NO. 572 and SEQ ID NO. 581 to SEQ ID NO. 644.
  • the siRNA molecule is one selected from the siRNA duplexes defined in Figure 3 selected from the following group: siRNA ID 344: antisense strand SEQ ID NO: 155, sense strand SEQ ID NO: 576; siRNA ID 342: antisense strand SEQ ID NO: 153, sense strand SEQ ID NO: 574; siRNA ID 346: antisense strand SEQ ID NO: 156, sense strand SEQ ID NO: 577; siRNA ID 348: antisense strand SEQ ID NO: 158 sense strand SEQ ID NO: 579; siRNA ID 349: antisense strand SEQ ID NO: 159, sense strand SEQ ID NO: 580; siRNA ID 341 : antisense strand SEQ ID NO: 152, sense strand SEQ ID NO: 573; siRNA ID 343: antisense strand SEQ ID NO: 154, sense strand SEQ ID NO: 575; siRNA ID 347: antisense strand SEQ
  • siRNA ID 288 antisense strand SEQ ID NO: 214, sense strand SEQ ID NO: 635; siRNA ID 289: antisense strand SEQ ID NO: 215, sense strand SEQ ID NO: 636; siRNA ID 296: antisense strand SEQ ID NO: 216, sense strand SEQ ID NO: 637;
  • siRNA ID 297 antisense strand SEQ ID NO: 217, sense strand SEQ ID NO: 638; siRNA ID 298: antisense strand SEQ ID NO: 218, sense strand SEQ ID NO: 639;
  • siRNA ID 299 antisense strand SEQ ID NO: 219, sense strand SEQ ID NO: 640;
  • siRNA ID 301 antisense strand SEQ ID NO: 220, sense strand SEQ ID NO: 641;
  • siRNA ID 302 antisense strand SEQ ID NO: 221, sense strand SEQ ID NO: 642;
  • - siRNA ID 303 antisense strand SEQ ID NO: 222, sense strand SEQ ID NO: 643;
  • - siRNA ID 304 antisense strand SEQ ID NO: 223, sense strand SEQ ID NO: 644.
  • the siRNA molecule is one of the siRNA duplexes defined in Figure 3 selected from the following group:
  • siRNA ID 344 antisense strand SEQ ID NO: 155, sense strand SEQ ID NO: 576;
  • siRNA ID 342 antisense strand SEQ ID NO: 153, sense strand SEQ ID NO: 574;
  • siRNA ID 346 antisense strand SEQ ID NO: 156, sense strand SEQ ID NO: 577
  • siRNA ID 348 antisense strand SEQ ID NO: 158, sense strand SEQ ID NO: 579;
  • siRNA ID 349 antisense strand SEQ ID NO: 159, sense strand SEQ ID NO: 580;
  • - siRNA ID 365 antisense strand SEQ ID NO: 139, sense strand SEQ ID NO: 560;
  • siRNA ID 369 antisense strand SEQ ID NO: 143, sense strand SEQ ID NO: 564;
  • siRNA ID 356 antisense strand SEQ ID NO: 147, sense strand SEQ ID NO: 568;
  • - siRNA ID 360 antisense strand SEQ ID NO: 151, sense strand SEQ ID NO: 572;
  • siRNA ID 329 antisense strand SEQ ID NO: 163, sense strand SEQ ID NO: 584; siRNA ID 334: antisense strand SEQ ID NO: 167, sense strand SEQ ID NO: 588;
  • siRNA ID 425 antisense strand SEQ ID NO: 171, sense strand SEQ ID NO: 592;
  • siRNA ID 429 antisense strand SEQ ID NO: 175, sense strand SEQ ID NO: 596;
  • siRNA ID 413 antisense strand SEQ ID NO: 179, sense strand SEQ ID NO: 600;
  • siRNA ID 418 antisense strand SEQ ID NO: 183, sense strand SEQ ID NO: 604; siRNA ID 398: antisense strand SEQ ID NO: 187, sense strand SEQ ID NO: 608; siRNA ID 403: antisense strand SEQ ID NO: 191 , sense strand SEQ ID NO: 612;
  • siRNA ID 383 antisense strand SEQ ID NO: 195, sense strand SEQ ID NO: 616;
  • siRNA ID 388 antisense strand SEQ ID NO: 199, sense strand SEQ ID NO: 620; siRNA ID 434: antisense strand SEQ ID NO: 203, sense strand SEQ ID NO: 624; siRNA ID 438: antisense strand SEQ ID NO: 207, sense strand SEQ ID NO: 628;
  • siRNA ID 284 antisense strand SEQ ID NO: 211, sense strand SEQ ID NO: 632; siRNA ID 289: antisense strand SEQ ID NO: 215, sense strand SEQ ID NO: 636; siRNA ID 299: antisense strand SEQ ID NO: 219, sense strand SEQ ID NO: 640; siRNA ID 304: antisense strand SEQ ID NO: 223, sense strand SEQ ID NO: 644.
  • siRNA molecules instability in biological fluids due to the ubiquitous nature of Ribonucleases (RNases). Consequently, the use of many different chemical modifications to nucleotides has been described with the purpose of enhancing compound stability.
  • RNases Ribonucleases
  • siRNAs have been found to induce nonspecific activation of the innate immune system, including up-regulation of certain cytokines, e.g. type I and/or type II interferon as well as IL-12, IL-6 and/or TNF-alpha production.
  • cytokines e.g. type I and/or type II interferon as well as IL-12, IL-6 and/or TNF-alpha production.
  • the origin of these effects is thought to be activation of Toll-like receptors such as TLR7, TLR8 and/or TLR3 by siRNA.
  • the siRNA further comprises at least one nucleotide with a chemical modification and/or at least one chemical modification in the ribonucleotide backbone.
  • Preferred chemical modifications which enhance stability and reduce immunogenic effects include 2’-0-methyl nucleotides, 2’-fluoro nucleotides, 2’-amino nucleotides, 2’- deoxy nucleotides, or nucleotides containing 2’-0 or 4’-C methylene bridges.
  • Other preferred chemical modifications for exonuclease protection include the ExoEndoLight pattern of modification (EEL): modification of all pyrimidines in the sense strand to 2’-0- methyl residues, and modification of all pyrimidines in a 5’-UA-3’ or 5’-CA-3’ motif in the antisense strand to 2’-0-methyl residues.
  • EEL ExoEndoLight pattern of modification
  • position 1 of the sense strand can also be changed to 2’-0-methyl to prevent 5’-phosphorylation of the sense strand and thus increase strand-specificity of the siRNA.
  • the sense strand can also include a 2’-0-methyl modification in position 14, because 2’-0-Me residues at this position inactivate the sense strand and therefore increase strand-specificity of the siRNAs.
  • other preferred chemical modifications for nuclease protection include Methyl-Fluoro modification pattern (MEF): alternating 2’-fluoro and 2’-0-methyl modifications starting (5’-end) with a 2’-F on the sense strand and starting with 2’-0-Me on the antisense strand.
  • MEF Methyl-Fluoro modification pattern
  • position 1 of the sense strand can also be changed to 2’-0-Me and position 1 of the antisense strand to 2’-F (as 2’-F residues are compatible with 5’-phosphorylation whereas 2’0-Me residues are bulky and generally impair phosphorylation).
  • This modification pattern not only stabilizes the molecule but also disables the ability of the RISC to use the sense strand thus promoting strand-specificity.
  • modification of the ribonucleotide backbone can be performed by binding the nucleotides by using phosphorothioate bonds instead of phosphodiester links.
  • a further preferred chemical modification within the meaning of the present invention relates to: 4’-thioribose, 5-propynyluracil 3’, 5'-methyluridine or the substitution of uracyl ribonucleotides with deoxythymidine (deoxy ribonucleotides).
  • the at least one chemically modified nucleotide and/or the at least one chemical modification in the ribonucleotide backbone is on the sense strand, on the antisense strand or on both strands of the siRNA.
  • the siRNA comprises or consists of at least one sequence with a sense strand and/or an antisense strand selected from the group consisting of SEQ ID NO. 982 to SEQ ID NO. 983, SEQ ID NO. 978 to SEQ ID NO. 981 , and SEQ ID NO. 984 to SEQ ID NO. 1037.
  • the siRNA of the different aspects of the invention comprises or consists of an antisense strand which comprises or consists of at least one sequence selected from the group consisting of SEQ ID NO. 982 to SEQ ID NO. 983, SEQ ID NO. 978 to SEQ ID NO. 981, and SEQ ID NO. 984 to SEQ ID NO. 1007, and a sense strand which is complementary to the antisense strand and which is selected from the group consisting of SEQ ID NO. 1012 to SEQ ID NO. 1013, SEQ ID NO. 1008 to SEQ ID NO. 1011 and SEQ ID NO. 1014 to SEQ ID NO. 1037, respectively.
  • compositions that includes or comprises an siRNA compound of the invention, e.g., a double-stranded siRNA compound as any of the described above.
  • the pharmaceutical composition can be a solution, an emulsion, microemulsion, cream, jelly, or liposome.
  • siRNA compounds of the invention described herein can be formulated for administration to a subject, preferably for administration locally to the lungs and nasal passage (respiratory tissues) via inhalation or intranasal administration, or simultaneous administration to the mouth and nose or to the mouth, nose and eyes, for example as an aerosol to be administered using a face mask covering mouth and nose and optionally also covering the eye, or topically, or systemically, such as parenterally, e.g., via injection.
  • formulations, compositions, and methods in this section are discussed with regard to unmodified siRNA compounds. It should be understood, however, that these formulations, compositions, and methods can be practiced with other siRNA compounds, e.g., modified siRNA compounds, and such practice is also described herein.
  • a formulated siRNA composition can assume a variety of states.
  • the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (for example, less than 90, 80, 70, 60, 50, 40, 30, 20, or 10% water).
  • the siRNA compound is in an aqueous phase, for example, in a solution that includes water, this form being the preferred form for administration via inhalation.
  • the aqueous phase or the crystalline compositions can be incorporated into a delivery vehicle, for example, a liposome (particularly for the aqueous phase), or a particle (for example, a microparticle as can be appropriate for a crystalline composition).
  • a delivery vehicle for example, a liposome (particularly for the aqueous phase), or a particle (for example, a microparticle as can be appropriate for a crystalline composition).
  • the siRNA composition is formulated in a manner that is compatible with the intended method of administration.
  • siRNA compound preparation can be formulated in combination with another agent, for example, another therapeutic agent or an agent that stabilizes an siRNA compound, for example, a protein that complexes with the siRNA compound to form an RNA-induced silencing complex.
  • agents include chelators, for example, EDTA (for example, to remove divalent cations such as Mg 2+ ), salts, RNase inhibitors and so forth.
  • therapeutic agent include therapies which are being experimentally tested for treatment of COVID-19, including: antibodies, such as bevacizumab, nivolumab, leronlimab (PRO 140), camrelizumab, sarilumab, tocilizumab, gimsilumab, TJM2 (TJ003234, an anti- granulocyte-macrophage colony stimulating factor antibody), lenzilumab, siltuximab, eculizumab, canakinumab, emapalumab, meplazumab, LY3127804 (an anti- Angiopoietin 2 (Ang2) antibody), IFX-1 (an anti-C5a antibody), otilimab or antibodies from recovered SARS patients or COVID-19 patients (for example, VIR-7831 or VI R- 7832); antivirals, such as lopinavir/ritonavir, remdesivir, galidesivir
  • the siRNA compound preparation may include another siRNA compound, e.g., a second siRNA compound or RNAi agent that can mediate RNAi with respect to a second gene or with respect to a different target region of the same gene.
  • the siRNA compounds or RNAi agents may be directed to the same virus but different target sequences, or each siRNA compound or RNAi agent may be directed to a different virus; or one or more siRNA compounds may be directed to a target sequence in a host (e.g., patient, e.g., human patient) gene, and one or more siRNA compounds may be directed to a target sequence in a virus (e.g., coronavirus, e.g. SARS-CoV or SARS CoV-2); or each siRNA compound may be directed to a different gene in a host.
  • a virus e.g., coronavirus, e.g. SARS-CoV or SARS CoV-2
  • each siRNA compound may be directed to a different gene in a host
  • the pharmaceutical composition includes or comprises an siRNA compound of the invention mixed with a topical delivery agent.
  • the topical delivery agent can be a plurality of microscopic vesicles.
  • the microscopic vesicles can be liposomes.
  • the liposomes are cationic liposomes. Cationic lipids or liposomes can spontaneously form complexes with negatively charged siRNA or oligonucleotides through electrostatic interactions with the positively charged lipids.
  • cationic lipids examples include 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium (DDAB), 1 ,2-di-0-octadecenyl-3-trimethylammonium- propane (DOTMA) , 3p-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC- CHOL), 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP), cetyl trimethylammonium bromide (CTAB), 1,3-dioleoyloxy-2-(6-carboxy-spermyl)- propylamide (DOSPER), or 1 ,2-dioleoyl-sn-glycero-3-[(N- (5-amino-l-carboxypentyl) iminodiacetic acid
  • Cationic lipids with a hydrophilic head and at least one hydrophobic tail are disclosed in any of the WO 1995/026356 A1 , WO 2000/027795 A1 , WO 2002/066012 A2, WO 2004/002454 A1 , WO 2004/002468 A1 , WO 2007/107304 A2, WO 2007/130073 A2, WO 2010/008582 A2, WO 2011/119901 A1 and WO 2012/068176 A1.
  • hydrophilic polymers such as polyethylene glycol (PEG) can be also used to shield the positive charge of the cationic lipids and reduce inflammatory response.
  • PEG polyethylene glycol
  • the pharmaceutical composition comprising the siRNA compound of the invention, e.g., a double-stranded siRNA compound, is admixed with a topical penetration enhancer.
  • a topical penetration enhancer is a fatty acid.
  • the fatty acid can be arachidonic acid, oleic acid, lauric acid, caprylic acid, capricacid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monolein, dilaurin, glyceryl 1-monocaprate, 1 -dodecylazacycloheptan-2-one, an acyl carnitine, an acyl choline, or a C1-10 alkyl ester, monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.
  • the topical penetration enhancer is a bile salt.
  • the bile salt can be cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, sodium tauro- 24,25-dihydro-fusidate, sodium glycodihydrofusidate, polyoxyethylene-9-lauryl ether or a pharmaceutically acceptable salt thereof.
  • the penetration enhancer is a chelating agent.
  • the chelating agent can be EDTA, citric acid, a salicyclate, a N-acyl derivative of collagen, laureth-9, an N-amino acyl derivative of a beta-diketone or a mixture thereof.
  • the penetration enhancer is a surfactant, e.g., an ionic or nonionic surfactant.
  • the surfactant can be sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether, a perfluorchemical emulsion or mixture thereof.
  • the penetration enhancer can be selected from a group consisting of unsaturated cyclic ureas, 1-alkyl- alkones, 1-alkenylazacyclo-alakanones, steroidal anti-inflammatory agents and mixtures thereof.
  • the penetration enhancer can be a glycol, a pyrrol, an azone, or a terpene.
  • the pharmaceutical composition including or comprising the siRNA compound is in a form suitable for oral delivery, and/or is in an oral dosage form.
  • oral delivery can be used to deliver an siRNA compound composition to a cell or a region of the throat, the gastro-intestinal tract, e.g., small intestine, colon, and so forth.
  • the oral delivery form can be tablets, capsules or gel capsules.
  • the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach. In some embodiments the enteric material is a coating.
  • the coating can be acetate phthalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methylcellulose phthalate or cellulose acetate phthalate.
  • the oral dosage form of the pharmaceutical composition includes a penetration enhancer.
  • the penetration enhancer can be a bile salt or a fatty acid.
  • the bile salt can be ursodeoxycholic acid, chenodeoxycholic acid, and salts thereof.
  • the fatty acid can be capric acid, lauric acid, and salts thereof.
  • the oral dosage form of the pharmaceutical composition includes an excipient, which can be for example polyethyleneglycol or precirol.
  • the oral dosage form of the pharmaceutical composition includes a plasticizer.
  • the plasticizer can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethyl citrate.
  • the pharmaceutical composition includes or comprises the siRNA compound of the invention and a delivery vehicle.
  • the delivery vehicle can deliver the siRNA compound to a cell by a topical route of administration.
  • the delivery vehicle can be microscopic vesicles.
  • the microscopic vesicles are liposomes.
  • the liposomes are cationic liposomes. Examples of cationic lipids that form cationic liposomes with negatively charged siRNA or oligonucleotides are described above.
  • the microscopic vesicles are micelles.
  • micelle structures are of particular interest because of their small size, good biocompatibility, high stability both in vitro and in vivo, and the ability to transport pharmaceuticals.
  • Amphiphilic compounds spontaneously self associate into micelles when dispersed in water at a concentration above their critical micelle concentration (CMC).
  • CMC critical micelle concentration
  • polymeric micelles for siRNA delivery can be designed by direct conjugation of hydrophilic (Polyethylene glycol -PEG-) or hydrophobic (lipid) moieties to siRNA via degradable (e.g., disulfide) or non-degradable linkages, followed by their condensation with polycationic ions to form micellar structures called polyion complex micelles (PICs) or polyelectrolyte complex micelles (PECs).
  • PICs polyion complex micelles
  • PECs polyelectrolyte complex micelles
  • the polyion segments are usually made of poly(amino acids) like poly(aspartic acid) or poly(L-lysine) or polyethylenimine (PEI).
  • siRNA can be complexed with an amphiphilic block copolymer containing polycation (or lipid) segment followed by micellization of the block copolymer-siRNA complex.
  • Useful block copolymers can be PEG-b-poly(L-lysine) (PEG- b-PLL) containing lysine amines modified with 2-iminothiolane (2IT), which can be further modified with cyclo-Arg-Gly-Asp (cRGD) peptide at the PEG terminus.
  • PEG-b-poly(L-lysine) PEG- b-PLL
  • 2IT 2-iminothiolane
  • cRGD cyclo-Arg-Gly-Asp
  • Another useful copolymer can be the triblock copolymer poly(BMA-co-PAA-co-DMAEMA)-b- poly(DMAEMA)-b-poly(AzEMA) resulting of micelle blocks consisting of a core-forming terpolymer of butyl methacrylate-co-2-propyl acrylic acid-co-2-dimethylaminoethyl methacrylate) (BMA-co-PAA-co-DMAEMA), a cationic block for condensing siRNA (DMAEMA) and an azide-presenting corona-forming block for the attachment of alkyne- functionalized mannose (2-azidoethyl methacrylate (AzEMA), which can be further mannosylated by reaction with alkyne functionalized mannose.
  • BMA-co-PAA-co-DMAEMA butyl methacrylate-co-2-propyl acrylic acid-co-2-dimethylaminoethyl methacrylate
  • Cationic micelles can be also formed from diblock copolymers of dimethylaminoethyl methacrylate (pDMAEMA) and butyl methacrylate (BMA), from diblock copolymers of linear PEI and PCL (PEI-PCL) (Jhaveri et al. 2014).
  • pDMAEMA dimethylaminoethyl methacrylate
  • BMA butyl methacrylate
  • PEI-PCL linear PEI and PCL
  • the siRNA compounds of the invention can be formulated in a pharmaceutical composition with a pharmaceutically acceptable vehicle or carrier that comprises a polymeric nanoparticle.
  • the polymeric nanoparticle is a nanoparticle comprising poly(beta-amino ester) s (PBAEs), preferably modified with at least one oligopeptide.
  • PBAEs poly(beta-amino ester) s
  • End-modified PBAEs and related nanoparticles useful for the delivery of polynucleotides in medical applications, as well as methods for their preparation have been described for example in W02014136100 A1, Dosta et al. 2018 and Segovia et al. 2014.
  • Oligopeptide-modified pBAEs can be products obtained by end- modification of an acrylate-terminated polymer (for example, a C32, C6 or C16 polymers resulting from polymeric addition reaction of primary amines to 1 ,4-butanediol diacrylate) with thiol-terminated oligopeptides (for example, H-CysArgArgArg-Nhh, H- CysLysLysLys-NH2, H-CysHisHisHis-NH2, H-CysGluGluGlu-Nhh, or H-CysAspAspAsp- NH2).
  • an acrylate-terminated polymer for example, a C32, C6 or C16 polymers resulting from polymeric addition reaction of primary amines to 1 ,4-butanediol diacrylate
  • thiol-terminated oligopeptides for example, H-CysArgArgArg-Nhh, H
  • C32 polymers can be obtained by polymerisation of 5-amino-pentanol and 1 ,4- butanediol diacrylate.
  • C6 polymers may be synthesized by conjugate addition of different ratios of hexylamine/5-amino-1 -pentanol (for example, 1 :0, 1 :1 or 1:3 for C6-100, C6-50 or C6-25 polymers, respectively) to 1,4-butanediol.
  • C16 polymers may be synthesized by conjugate addition of different ratios of hexadecylamine/5-amino-1- pentanol (for example, 1 :1 or 1 :3 for C16-50, C6-50 or C 16-25 polymers, respectively) to 1,4-butanediol.
  • C32, C6 or C16 polymers may be also optionally modified with cholesterol by different degrees of esterification (for example, 50%, 25%, 12.5%) of the polymer hydroxyl groups with carboxylic acid-modified cholesterol (cholesterol-COOH).
  • the pharmaceutical composition comprising a siRNA compound of the invention is in a pulmonary or nasal dosage form.
  • the siRNA compound is incorporated into a particle, e.g., a macro particle, e.g., a microsphere.
  • the particle can be produced by spray drying, lyophilisation, evaporation, fluid bed drying, vacuum drying, or a combination thereof.
  • the microsphere can be formulated as a suspension, a powder, or an implantable solid.
  • the pharmaceutical composition comprising the siRNA compound is in an injectable dosage form.
  • the injectable dosage form of the pharmaceutical composition includes sterile aqueous solutions or dispersions and sterile powders.
  • the sterile solution can include a diluent such as water; saline solution; fixed oils, polyethylene glycols, glycerine, or propylene glycol.
  • T reatment Methods and Routes of Delivery Another aspect of the invention relates to a method of reducing the expression of ACE2 gene in a cell, comprising contacting said cell with a siRNA compound of the invention.
  • the cell is a cell from the higher or lower respiratory tract.
  • Another aspect of the invention relates to a method of reducing the expression of ACE2 gene in a subject, comprising administering to the subject siRNA compound of the invention.
  • Another aspect of the invention relates to a method for prophylactic and/or therapeutic treatment of a virus infection or its related diseases in a subject, comprising administering to the subject a therapeutically effective amount of the siRNA compound of the invention, thereby treating the subject.
  • the virus infection is caused by a virus that involves host ACE2 protein during the viral cycle, such as several types of known coronavirus, including SARS-CoV-2 or SARS-CoV, or their related diseases.
  • Exemplary related diseases that can be prevented or treated by the method of the invention include COVID-19, SARS or MERS.
  • the siRNA or compositions of the invention can be delivered to a subject by a variety of routes, being preferred the topical or the pulmonary routes.
  • the siRNA and pharmaceutical compositions described herein may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including intranasal, intratracheal, intrapulmonary or onto the corneal surface of the eye), oral or parenteral. Exemplary routes include inhalation, nasal, oral, ophthalmic or intravenous delivery.
  • the delivery of the siRNA compounds described herein is done to achieve delivery into the subject to the site of infection.
  • This objective can be achieved through local (i.e. , topical) administration to the lungs, nasopharynx, pharynx or nasal passage, e.g., into the respiratory tissues via inhalation, nebulization or intranasal administration, local (i.e., topical) administration to the eye, e.g., administration to the corneal surface of the eye through eye drops, ocular spray or nebulization, or via systemic administration, e.g., parental administration.
  • Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.
  • the preferred means of administering the siRNA compounds described herein is through direct topical administration to the lungs, nasopharynx, pharynx and/or nasal passage by inhalation of an aerosolized liquid such as a nebulized mist or a nasal spray.
  • compositions can include one or more siRNA compounds and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying 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. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • Formulations for inhalation, intranasal, or parenteral administration are well known in the art.
  • Such formulations may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives, an example being PBS or Dextrose 5% in water.
  • buffers, diluents and other suitable additives an example being PBS or Dextrose 5% in water.
  • the total concentration of solutes should be controlled to render the preparation isotonic.
  • the active compounds disclosed herein are preferably administered to the lung(s), nasopharynx, pharynx or nasal passage of a subject by any suitable means.
  • Active compounds may be administered by administering an aerosol suspension of respirable particles comprised of the active compound or active compounds, which the subject inhales.
  • the active compound can be aerosolized in a variety of forms, such as, but not limited to, dry powder inhalants, metered dose inhalants (including pressured metered dose inhalants), or liquid/liquid suspensions.
  • the respirable particles may be liquid or solid.
  • the particles may optionally contain other therapeutic ingredients such as amiloride, benzamil or phenamil, with the selected compound included in an amount effective to inhibit the reabsorption of water from airway mucous secretions (for example, as described in U.S. Pat. No. 4,501,729).
  • other therapeutic ingredients such as amiloride, benzamil or phenamil
  • the particulate pharmaceutical composition may optionally be combined with a carrier to aid in dispersion or transport.
  • a suitable carrier such as a sugar (e.g., dextrose, lactose, sucrose, trehalose, mannitol) may be blended with the active compound or compounds in any suitable ratio (e.g., a 1 to 1 ratio by weight).
  • An active compound may be topically administered by inhalation.
  • administration by "inhalation” generally refers to the inspiration of particles comprised of the active compound that are of respirable size, that is, particles of a size sufficiently small to pass through the mouth or nose and larynx upon inhalation and into the bronchi and alveoli of the lungs. In general, particles ranging from about 1 to 10 microns in size (more particularly, less than about 5 microns in size) are respirable and suitable for administration by inhalation.
  • Topical administration refers to the delivery to a subject by contacting the formulation directly to a surface of the subject. The most common form of topical delivery is to the skin, but a composition disclosed herein can also be directly applied to other surfaces of the body, e.g., to the eye, a mucous membrane, to surfaces of a body cavity or to an internal surface.
  • An active compound may be topically delivered by intranasal administration.
  • intranasal administration refers to administration of a dosage form formulated and delivered to topically treat the nasal epithelium.
  • Particles or droplets used for intranasal administration generally have a diameter that is larger than those used for administration by inhalation.
  • a particle size in the range of 10-500 microns is preferred to ensure retention in the nasal cavity.
  • Particles of non- respirable size which are included in the aerosol tend to deposit in the throat and be swallowed, and the quantity of non-respirable particles in the aerosol is preferably minimized.
  • Liquid pharmaceutical compositions of active compound for producing an aerosol can be prepared by combining the active compound with a suitable vehicle, such as sterile pyrogen free water.
  • a suitable vehicle such as sterile pyrogen free water.
  • hypertonic saline solutions are used to carry out the present invention.
  • sterile, pyrogenfree solutions comprising from one to fifteen percent (by weight) of a physiologically acceptable salt, and more preferably from three to seven percent by weight of the physiologically acceptable salt.
  • Aerosols of liquid particles comprising the active compound may be produced by any suitable means, such as with a pressure-driven jet nebulizer or an ultrasonic nebulizer. See, e.g., U.S. Pat. No. 4,501,729.
  • Nebulizers are commercially available devices which transform solutions or suspensions of the active ingredient into a therapeutic aerosol mist either by means of acceleration of compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation.
  • Suitable formulations for use in nebulizers may consist of the active ingredient in a liquid carrier, the active ingredient comprising up to 40% w/w of the formulation, but preferably less than 20% w/w.
  • the carrier is typically water (and most preferably sterile, pyrogen free water) or a dilute aqueous-alcoholic solution, preferably made isotonic but may be hypertonic with body fluids by the addition of, for example, sodium chloride.
  • Optional additives include preservatives if the formulation is not made sterile, for example, methyl hydroxybenzoate, antioxidants, flavouring agents, volatile oils, buffering agents and surfactants. Aerosols of solid particles comprising the active compound may likewise be produced with any solid particulate therapeutic aerosol generator.
  • Aerosol generators for administering solid particulate therapeutics to a subject produce particles which are respirable and generate a volume of aerosol containing a predetermined metered dose of a therapeutic at a rate suitable for human administration.
  • One illustrative type of solid particulate aerosol generator is an insufflator.
  • Suitable formulations for administration by insufflation include finely comminuted powders which may be delivered using an insufflator or taken into the nasal cavity in the manner of a snuff.
  • the powder e.g., a metered dose thereof effective to carry out the treatments described herein
  • the powder is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in situ and the powder delivered by air drawn through the device upon inhalation or employing a manually-operated pump.
  • the powder employed in the insufflator consists either solely of the active ingredient or of a powder blend comprising the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant.
  • the active ingredient typically comprises from 0.1 to 100 w/w of the formulation.
  • a second type of illustrative aerosol generator comprises a metered dose inhaler.
  • Metered dose inhalers are pressurized aerosol dispensers, typically containing a suspension or solution formulation of the active ingredient in a liquefied propellant. During use these devices discharge the formulation through a valve adapted to deliver a metered volume, typically from 10 mI to 200 pi, to produce a fine particle spray containing the active ingredient.
  • Suitable propellants include certain chlorofluorocarbon compounds, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof.
  • the formulation may additionally contain one or more co-solvents, for example, ethanol, surfactants, such as oleic acid or sorbitan trioleate, antioxidant and suitable flavouring agents.
  • An active siRNA compound may also be administered to the buccal cavity of a human being by spraying into the cavity, without inhalation, from a metered dose spray dispenser, a mixed micellar pharmaceutical formulation as described above and a propellant.
  • the dispenser is first shaken prior to spraying the pharmaceutical formulation and propellant into the buccal cavity.
  • the medication can be sprayed into the buccal cavity or applied directly, e.g., in a liquid, solid, or gel form to a surface in the buccal cavity.
  • the buccal administration is by spraying into the cavity, e.g., without inhalation, from a dispenser, e.g., a metered dose spray dispenser that dispenses the pharmaceutical composition and a propellant.
  • a dispenser e.g., a metered dose spray dispenser that dispenses the pharmaceutical composition and a propellant.
  • Administration can be provided by the subject or by another person, e.g., a caregiver.
  • a caregiver can be any entity involved with providing care to the human: for example, a hospital, hospice, doctor's office, outpatient clinic; a healthcare worker such as a doctor, nurse, or other practitioner; or a spouse or guardian, such as a parent.
  • the medication can be provided in measured doses or in a dispenser which delivers a metered dose.
  • the prophylactic and therapeutic treatments are used in human subjects, although the treatments can be also applied to other mammals, including ACE2 humanized mice, dogs, monkeys and macaques, or others expressing surface protein ACE2 in tissues accessible to the virus which can be bound to the viral spike (S) protein and being susceptible of a virus infection with SARS-CoV-2 or the viral entry into their cells.
  • mammals including ACE2 humanized mice, dogs, monkeys and macaques, or others expressing surface protein ACE2 in tissues accessible to the virus which can be bound to the viral spike (S) protein and being susceptible of a virus infection with SARS-CoV-2 or the viral entry into their cells.
  • S viral spike
  • Coronavirus disease 2019 or COVID-19 refers to an infectious disease caused by the new coronavirus discovered at the end of year 2019, SARS-CoV-2.
  • COVID-19 affects different people in different ways. While understanding of the disease is still developing, most people infected with the COVID-19 virus experience mild to moderate respiratory illness and recover without requiring special treatment. Older people, and those with underlying medical problems like cardiovascular disease, diabetes, chronic respiratory disease, and cancer are more likely to develop serious illness.
  • Most common symptoms are fever or chills, dry cough and tiredness, although less common symptoms include aches and pains, sore throat, diarrhoea, conjunctivitis, headache, loss of taste or smell, congestion or runny nose, nausea or vomiting, and a rash on skin, or discolouration of fingers or toes.
  • Serious symptoms of COVID-19 are difficulty breathing or shortness of breath, chest pain or pressure, and loss of speech or movement, which normally require to seek immediate medical attention. People with mild symptoms who are otherwise healthy should manage their symptoms at home. On average it takes 5-6 days from when someone is infected with the virus for symptoms to show, however it can take up to 14 days.
  • COVID-19 virus spreads primarily through droplets of saliva or discharge from the nose when an infected person coughs or sneezes, so it’s important to practice respiratory etiquette (for example, by coughing into a flexed elbow).
  • a “suspected case” would include: (a) a patient or subject with acute respiratory illness (fever and at least one sign/symptom of respiratory disease, e.g., cough, shortness of breath), and a history of travel to or residence in a location reporting community transmission of COVID-19 disease during the 14 days prior to symptom onset; or (b) a patient or subject with any acute respiratory illness and having been in contact with a confirmed or probable COVID-19 case (see definition of contact below) in the last 14 days prior to symptom onset; or (c) a patient or subject with severe acute respiratory illness (fever and at least one sign/symptom of respiratory disease, e.g., cough, shortness of breath; and requiring hospitalization) and in the absence of an alternative diagnosis that fully explains the clinical presentation.
  • acute respiratory illness fever and at least one sign/symptom of respiratory disease, e.g., cough, shortness of breath
  • a suspect case for whom testing for the COVID-19 virus is inconclusive (“inconclusive” being the result of the test reported by the laboratory) or a suspect case for whom testing could not be performed for any reason can be considered as a “probable case”, for which a confirmation of infection with SARS-CoV-2 or COVID-19 virus could not be determined.
  • a “contact” is a person or a subject who experienced any one of the following exposures during the 2 days before and the 14 days after the onset of symptoms of a probable or confirmed case: (i) face-to-face contact with a probable or confirmed case within 2 meter and for more than 15 minutes; (ii) direct physical contact with a probable or confirmed case; (iii) direct care for a patient or a subject with probable or confirmed COVID-19 disease without using proper personal protective equipment; or (iv) other situations as indicated by local risk assessments.
  • the period of contact is measured as the 2 days before through the 14 days after the date on which the sample was taken which led to confirmation.
  • a contact who experienced any one of the above exposures can be also understood as a person who has been exposed to a close contact with a confirmed case, probable case or suspect case of COVID-19 or SARS-CoV-2 infection.
  • subject diagnosed of an infection with SARS-CoV-2 refers to a person or subject with laboratory confirmation of COVID-19 infection, irrespective of clinical signs and symptoms, similarly as for the definition of “confirmed case” established by the WHO.
  • the current diagnostic strategy recommended to identify or diagnose patients with COVID-19 or with a SARS-CoV-2 infection is to test samples taken from the respiratory tract of the subject to assess for the presence of one or several nucleic acid targets specific to SARS-CoV-2.
  • serum samples in the acute or the convalescent phase could support diagnosis based on validated serologic tests available for SARS-CoV-2, and especially by diagnosing from paired serum samples in the acute and convalescent phase, the initial sample having been collected preferably in the first week of illness, and the second ideally collected 2-4 weeks later.
  • WHO World Health Organization
  • upper respiratory specimens such as nasopharyngeal and oropharyngeal swab or wash in ambulatory patients
  • lower respiratory specimens such as sputum (if produced) and/or endotracheal aspirate or bronchoalveolar lavage in patients with more severe respiratory
  • additional clinical specimens may be collected as COVID-19 virus has been detected in blood and stool.
  • serum samples acute and/or convalescent
  • samples undergo RNA extraction followed by qualitative RT-PCR for target detection.
  • Routine confirmation of cases of SARS-CoV-2 or COVID-19 is based on detection of unique sequences of virus RNA by NAAT such as real-time reverse-transcription polymerase chain reaction (rRT-PCR) with confirmation by nucleic acid sequencing when necessary.
  • NAAT real-time reverse-transcription polymerase chain reaction
  • the viral genes targeted so far include the N, E, S and RdRP genes.
  • a positive NAAT result of a single discriminatory target can be enough in areas with established SARS-CoV-2 or COVID-19 virus circulation, although in some situations, a positive NAAT result for at least two different targets on the COVID-19 virus genome, of which at least one target is preferably specific for COVID-19 virus using a validated assay; or one positive NAAT result for the presence of betacoronavirus and COVID-19 virus further identified by sequencing partial or whole genome of the virus could be more reliable and convenient, such in case of confirmation in areas with no known COVID-19 virus circulation.
  • One or more negative results by NAAT do not rule out the possibility of SARS-CoV-2 or COVID-19 virus infection.
  • subject at risk of an infection with SARS- CoV-2 or “subject at risk of exposure to SARS-CoV-2” refer to a subject potentially exposed to the SARS-CoV-2 virus or COVID-19, for example, a subject who is in contact with, has been in contact with, cares for, or has cared for a subject/patient diagnosed (i.e. confirmed case) or suspected (i.e.
  • Subjects at a higher risk of being exposed to SARS-CoV-2 virus, being infected with SARS-CoV-2 virus or developing a SARS-CoV-2 infection or COVID-19 include healthcare workers (including practitioners, nurses, emergency services, assistants and non-clinical staff), pharmacists and subjects or people working at healthcare facilities; subjects or people living in the same household (for example, relatives, flatmates, roommates, domestic workers and caregivers) as a person who has lab-confirmed COVID-19 or who was diagnosed with COVID-19; subjects or people providing care in a household (for example, caregivers) for a person who has lab-confirmed COVID-19 or who was diagnosed with COVID-19; subjects or people being within 2 meters (or 6 feet) of a person who has lab-confirmed COVID-19 or who was diagnosed with COVID-19 for at least 15 minutes; subjects or people in direct contact with secretions from a person who has lab-confirmed COVID-19 orwho was diagnosed with COVID-19 (e.g
  • terapéuticaally effective amount is the amount present in the composition that is needed to provide the desired level of drug in the subject to be treated to give the anticipated physiological response.
  • physiologically effective amount is that amount delivered to a subject to give the desired palliative or curative effect.
  • pharmaceutically acceptable carrier means that the carrier can be taken into the lungs, the upper or lower airways, or the eye with no significant adverse toxicological effects on the lungs, the upper or lower airways, or the eye, respectively.
  • co-administration refers to administering to a subject two or more agents (e.g. a siRNA compound of the invention with another active principle), and in particular two or more siRNA compounds.
  • agents e.g. a siRNA compound of the invention with another active principle
  • the agents can be contained in a single pharmaceutical composition and be administered at the same time, or the agents can be contained in separate formulations and administered serially to a subject. So long as the two agents can be detected in the subject at the same time, the two agents are said to be co administered.
  • the types of pharmaceutical excipients that are useful as carrier 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; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two.
  • HSA human serum albumin
  • bulking agents such as carbohydrates, amino acids and polypeptides
  • pH adjusters or buffers such as sodium chloride
  • salts such as sodium chloride
  • 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; alditols, such as mannitol, xylitol, and the like.
  • a preferred group of carbohydrates includes lactose, threhalose, raffinose, maltodextrins, and mannitol.
  • Suitable polypeptides include aspartame.
  • Amino acids include alanine and glycine, with glycine being preferred.
  • Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred. Kits
  • kits that include a suitable container containing a pharmaceutical formulation of an siRNA compound.
  • the individual components of the pharmaceutical formulation may be provided in one container.
  • the kit may be packaged in a number of different configurations such as one or more containers in a single box.
  • the different components can be combined, e.g., according to instructions provided with the kit.
  • the components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition.
  • the kit can also include a delivery device.
  • RNAs are preferably chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Substitution of one or both strands of a siRNA duplex by 2'-deoxy or 2'-0-methyl oligoribonucleotides abolished silencing in fly extract (Elbashir et al. 2001). In mammalian cells, however, it seems possible to substitute the sense siRNA by a 2'-0- methyl oligoribonucleotide (Ge et al. 2003).
  • siRNAs were obtained from commercial RNA oligo synthesis suppliers, which sell RNA-synthesis products of different quality; for example, intended for target validation. In general, 19, 20 and 21-nt RNAs are not too difficult to synthesize and are readily provided in a quality suitable for RNAi. A number of commercial custom RNA synthesis companies exist; in the present examples, siRNA may be obtained from Biospring (Germany), unless otherwise specified.
  • siRNAs targeting ACE2 are preferably delivered as a part of a bioconjugate and/or delivered formulated alone/or in conjugation in nanoparticles (NPs) directed to the lung.
  • NPs nanoparticles
  • poly- ⁇ -aminoester polymer already used in previous studies (Dosta et al. 2018). Based on these polymers, new ones will be generated. New polymers will be synthesized and, optionally will include an antiviral molecule, such as hydroxychloroquine and/or plitidepsin, or an anti-inflammatory molecule, such as a corticosteroid or interferon g (IFN-g). These polymeric particles can also be coated with hyaluronic acid that would guarantee their adhesion in the lung and they have a hydrophobic nucleus that could house other drugs of interest.
  • an antiviral molecule such as hydroxychloroquine and/or plitidepsin
  • an anti-inflammatory molecule such as a corticosteroid or interferon g (IFN-g).
  • IFN-g interferon g
  • the newly synthesized polymers will be analyzed through 1 H-NMR, IR and HPLC.
  • Said polymers may be vectorized with ligands and/or small peptides and/or molecules directed to surface proteins of lung epithelial cells and alveolar macrophages such as antibodies or aptamers (e.g.
  • Nanoparticles will be synthesized by electrostatic interaction between positive charges of polymer with negative charges of RNA.
  • NPs Different polymer/RNA ratios will be used to obtain the NPs with the characteristics that ensure successful delivery in the target tissues (for example, size less than 250 nm, slightly positive charge and stability to lyophilization).
  • the NPs will be characterized by size and surface charge (by dynamic light scattering), both in the formulation medium and in contact with blood serum to determine their stability under physiological conditions. Also, NPs will be lyophilized and re-characterized to see their stability.
  • siRNA targeting ACE2 mRNA To validate the efficacy in vitro of a siRNA targeting ACE2 mRNA, culture cells are transfected with the selected ACE2 siRNA (i.e. a siRNA which targets any of the nucleotide sequences defined by SEQ ID NO: 1-14) and the silencing capacity of siRNAs in cells that express good levels of ACE2, but also susceptible to SARS-CoV-2 infection, are evaluated. Transfections are performed with different transfection agents at different times (for example, at 24, 48, and 72 hours) and testing different doses of the siRNA compound (for example, 100 nM). Different doses of siRNA will be administered for the calculation of IC50 values (dose at which 50% of the messenger RNA disappears at a certain time-point).
  • IC50 values dose at which 50% of the messenger RNA disappears at a certain time-point.
  • Cells from different species are used in these experiments which include human cells, and at least one or several cell types from different animal species necessary for further development of the selected compounds, which include murine, macaque and/or dog cells.
  • primary lung cells and established lung cell lines are used.
  • the cell lines preferably human cells from the upper (nasopharynx, Detroit 562 ATCC® CCL-138TM) and lower respiratory tract (lung, A549 (ATCC® CCL-185TM) and/or BEAS-2B (ATCC® CRL-9609TM), human broncho-epithelial primary cells (HBEpC), human Primary Bronchial/Tracheal human cells and human Primary Lobar Epithelial Cells are used.
  • siRNAs are also validated in mouse cells from the higher respiratory tract, such as Mouse Primary Tracheal Epithelial Cells, and from the lower respiratory tract, such as IMLg [Mlg 2908] cells and/or MLE 12 (ATCC® CRL-2110).
  • siRNAs are also validated in Rhesus macaque cells such as 4MBr-5 (Lung) (ATCC® CCL-208TM) and/or RF/6A (Retina) ATCC® CRL- 1780, LLC-MK2 and/or FRhK-4 (Kidney) cells (ATCC® CRL-1688TM) and in Crab-eating Macaque cells such as (CYNOM-K1) (ECACC, 90071809).
  • Rhesus macaque cells such as 4MBr-5 (Lung) (ATCC® CCL-208TM) and/or RF/6A (Retina) ATCC® CRL- 1780, LLC-MK2 and/or FRhK-4 (Kidney
  • siRNAs are also validated in CF41.Mg ( ATCC® CRL-6232TM) and/or MDCK dog cells (ATCC ® CCL 34TM). IC50 values are calculated for those siRNA candidates having a better performance in the different selected cell models.
  • siRNA regions of SEQ ID NO: 1048, SEQ ID NO: 1049, SEQ ID NO: 1050 or SEQ ID NO: 1051 have been tested, which are identified by their corresponding siRNA ID.
  • positive control a commercial species-specific ACE2 siRNA depending on the cell line species
  • NAC non-active siRNA
  • comparator siRNA against a common 19-nt long nucleotide sequence of the human ACE2 mRNA targeted by two shRNAs described in Lu et al 2008 doi: 10.1007/s10096- 008-0495-5).
  • siRNA ID 0342 siRNA ID 0348, siRNA ID 0339 and siRNA ID 0338 which reached reductions >70-80% compared to the control. These reductions were sustained over time, and 72 hours after the transfection the basal levels of expression were not recovered.
  • siRNA ID 0335 reduced gene expression levels to more than 90% 24 hours after transfection and gradually recovered levels over time.
  • siRNA ID 0344 reduced ACE2 levels rapidly 24 hours after transfection, and then recovered slightly to 50%, remaining unchanged until 72 hours after transfection. The non-active compound did not produce any reduction in ACE2 gene expression levels.
  • siRNAs targeting the region defined by SEQ ID NO:3 were better than the positive control and the comparator siRNA. The rest of the siRNAs corresponding to other mRNA regions were less or not effective. Only siRNA ID 0389 and siRNA ID 0491 reduced ACE2 levels by 50% and 40%, respectively, and recovered their expression levels rapidly over time.
  • siRNA ID 0404 siRNA ID 0001, siRNA ID 0374, siRNA ID 03700 and siRNA ID 0439 which lowered ACE2 messenger by 40-50% or siRNA ID 0419, siRNA ID 0389, siRNA 0275 and siRNA ID 0005 which produced decreases > 80%. All of these siRNA candidates produced sustained decreases over time.
  • siRNA ID 0344 and siRNA ID 0346 SEQ ID NO. 1
  • SEQ ID NO. 13 siRNA ID 0275
  • SEQ ID NO. 29 siRNA ID 0491
  • siRNA ID 0360 which showed a reduction of about 27%. Indeed, an increase in cell viability levels was seen for the siRNA candidates targeting SEQ ID NO: 5, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12 and SEQ ID NO: 13, but also in the siRNA compounds targeting SEQ ID NO. 3 that had shown reduced viability at 24 h and 48 h post transfection.
  • siRNA ID 0335 siRNA ID 0339, siRNA ID 0340, siRNA ID 0342, siRNA ID 0344, siRNA ID 0346, siRNA ID 0348
  • siRNA compounds targeting SEQ ID NO: 9 siRNA ID 0389
  • SEQ ID NO: 13 siRNA ID 0275
  • siRNA compounds targeting SEQ ID NO. 3 which include siRNA ID 0335, siRNA ID 0338, siRNA ID 0339, siRNA ID 0340, siRNA ID 0342, siRNA ID 0344, siRNA ID 0346 and siRNA ID 0348, have a better performance in reducing ACE2 expression levels in cells of different species, without altering substantially the cell viability, regardless of the structural differences comprised in the siRNA compound, such as the 3’-overhangs in their sense and/or the antisense strands, and/or the number of unpaired nucleotides in the 5’-end of their antisense strands.
  • siRNA ID 0338 siRNA ID 0339 and siRNA ID 0340 (corresponding to the region SEQ ID NO:3) as candidates for further development.
  • siRNA ID 0340 corresponding to the region SEQ ID NO:3
  • CPE cytopathic effect
  • viral titer reduction assays viral titer reduction assays
  • plaque reduction assays according to the protocol described by Herzog et al 2008 and/or Xia et al 2020 , through CKK-8 and/or Luminiscent and/or MTS cytotoxic assays
  • African green monkey kidney VeroE6 cells ATCC-1586
  • human Caco2 and/or Huh7 cells human Caco2 and/or Huh7 cells.
  • the viral titers are incubated for 72-144 hours.
  • the detection reagents ATP and/or MTS
  • Luminescence or formazan are measured and correlated with viral dilutions. Dilutions containing infectious virus demonstrate low cell viability and, therefore, low luminescence values due to generalized CPE. In contrast, dilutions that do not contain or limit virus show higher luminescence values.
  • TCID50 values are then obtained for each virus by determining the virus dilution that reduced the untreated signal by 50%.
  • infection studies are also carried out similarly in at least any other cell line which is successfully used to grow SARS-CoV-2, such as African green monkey kidney VeroE6/TMPRSS2 cells, rhesus monkey kidney LLC-MK2 cells, human Calu-3 cells, and/or human HEK293T cells.
  • any other cell line which is successfully used to grow SARS-CoV-2 such as African green monkey kidney VeroE6/TMPRSS2 cells, rhesus monkey kidney LLC-MK2 cells, human Calu-3 cells, and/or human HEK293T cells.
  • a preliminary study is performed in VeroE6 cells to determine the multiplicity of infection (MOI) ratio and the replication kinetics of SARS-CoV-2 virus. Then, other respiratory system cells susceptible to virus infection are also used with the selected MOI ratio and are infected to study the (prophylactic and therapeutic) antiviral capacity of siRNAs targeting ACE2. Different respiratory system cells are transfected with the ACE2 siRNA candidates and then infected with SARS-CoV-2 for studying viral replication in airway epithelium in vitro and to study the antiviral activity of the selected siRNAs.
  • MOI multiplicity of infection
  • the antiviral SARS-CoV-2 activity of siRNAs targeting ACE2 are investigated in vitro through two different approaches: a prophylactic study (inhibition of ACE2 and SARS-Cov-2 infection) and a therapeutic study (Infection with SARS-Cov-2-infection and inhibition with siRNAs candidates targeting ACE2).
  • a prophylactic study inhibition of ACE2 and SARS-Cov-2 infection
  • a therapeutic study Infection with SARS-Cov-2-infection and inhibition with siRNAs candidates targeting ACE2
  • the supernatant of cultured cells is collected and the RNA are extracted and analysed by relative quantification using RT-qPCR to determine the amount of virus RNA in controls and treatments by the study of viral structural genes to assess the (%) of inhibition.
  • EC50 values are obtained from dose-response curves of the selected ACE2 siRNAs against SARS-CoV-2 for viral RNA and for target mRNA (ACE2). EC50 values are determined at the MOIs selected previously by quantification of viral RNA copy numbers in the cell supernatant 48 h after the infection. The pro-inflammatory cytokines related to the cytokine storm associated with SARS-CoV-2 infection are also evaluated. Levels of interleukin (I L)-1 b, TNF-a, IL-6, IL-18 and interferon-g, are determined both at the mRNA level and at the protein level by detecting them in solution via ELISA assays.
  • I L interleukin
  • Viral RNA is extracted from the supernatant of infected cells using the viral RNA mini kit (QIAGEN, Hilden, Germany) and RNAEasy Kit (QIAGEN, Hilden, Germany and automated nucleic) with the nucleic acid extraction system QIACUBE (QIAGEN, Hilden, Germany) and following the manufacturer’s instructions.
  • Detection of the SARS-CoV-2 virus, the target blocked by ACE2 siRNA candidates and pro-inflammatory biomarkers are performed using the Real-time PCR on the StepOne Plus and Quant Studio 12K Flex real-time PCR systems (Applied Biosystem, USA).
  • Real-time PCR analysis are performed with universal primers and probes targeting SARS-CoV-2 that amplify structural viral proteins encoded by RdRp, E and N genes.
  • Pro-inflammatory cytokines are determined by ELISA in a Microplate reader. All experiments are conducted in triplicates. The relative expression can be determined using the 2-AACt method.
  • lymphocytes kt-3 Cells (94070705, ECACC), T lymphocytes (Primary CD8 + Cytotoxic T Cells (ATCC® PCS-800-017 TM), Primary CD4 + Helper T Cells (ATCC® PCS-800-016 TM), B lymphocytes ( Primary CD19 + B Cells (ATCC® PCS-800-018 TM), HAA1 (ATCC® HB-8534 TM), and Natural Killer (NK) NK92 (NK-92 ® ATCC ® CRL-2407 TM), Primary CD56 + NK Cells (ATCC® PCS-800-019 TM), after transfection of the
  • siRNA compound of the invention bioconjugated or formulated in nanoparticles can be also evaluated in vivo to study their antiviral capacity against SARS-CoV-2 and to prevent the spread of infection in the host for it.
  • the efficacy can be studied for the most effective candidate obtained from the in vitro studies alone/or in combination with other drugs in an animal model that reproduces the signs and symptoms of SARS-CoV-2 infection (COVID-19).
  • the first approach is based on two different mouse models: a mouse model that expresses the human ACE2 receptor (K18-hACE2) and/or the MERS-CoV mouse model.
  • the second approach is based on a Rhesus macaque model of SARS-CoV-2 infection.
  • mice are infected with a mouse-adapted strain of the beta-coronavirus (BtCoV HKU5- SE), which replicates efficiently in mice and induces alveolar damage.
  • BtCoV HKU5- SE beta-coronavirus
  • Humanized K18- hACE2 mice are infected with SARS-CoV-2 virus.
  • Humanized ACE2 mice are intranasally infected with SARS-CoV-2 10 5 (TCID50) pfu in PBS. 24 h previous to infection or after infection, a group of animals are treated with 3 different doses of the treatments.
  • the infection can be performed both in epithelial cells of the upper respiratory tract (nasopharynx and trachea) as well as the lower respiratory tract (bronchia and lung) to analyse the efficacy of the treatments developed both in the preventive and therapeutic regimes.
  • CBC Complete blood count
  • WBC White blood count
  • Lymphocyte subsets in peripheral blood can be analysed to study Th1 and Th2 immune response type markers.
  • the perturbations in immune cell subsets and the frequencies of T/NK cells and lymphocyte subsets in peripheral blood can be also analysed. Lymphocyte subsets in peripheral blood will be analysed to study Th1 and Th2 immune response type markers.
  • the perturbations in immune cell subsets and the frequencies of T/NK cells and lymphocyte subsets in peripheral blood can be also analysed.
  • the measurement of an effective SARS-CoV-2 specific antibody response will be performed and IgG, IgM levels will be determined in mice sera samples.
  • specific TLRs, RNA-sensors and downstream pathways of type I/ III IFN and/or the inflammasome pathways will be determined, as well as molecules involved in autophagy processes and endosomal trafficking.
  • Detection of the SARS-CoV-2 virus the target blocked by ACE2 siRNA candidates and pro-inflammatory biomarkers are performed using the Real-time PCR on the StepOne Plus and Quant Studio 12K Flex real-time PCR systems (Applied Biosystem, USA). Real time PCR analysis are performed with universal primers and probes targeting SARS- CoV-2 that amplify structural viral proteins encoded by RdRp, E and N genes. Pro- inflammatory cytokines are determined by ELISA in a Microplate reader. All experiments will be conducted in triplicates. The relative expression will be determined using the 2- AACt method.
  • RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15(2): 188-200.
  • RNA interference medicines the clinical landscape of synthetic gene silencing drugs. Saiide & Tecnologia, 21, p. 05-17.
  • Elevated plasma angiotensin converting enzyme 2 activity is an independent predictor of major adverse cardiac events in patients with obstructive coronary artery disease.

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Abstract

L'invention concerne des procédés de prévention ou de traitement d'une infection virale chez un sujet causée par le coronavirus 2 du syndrome respiratoire aigu sévère (SARS-CoV-2), les procédés comprenant l'administration audit sujet d'un ARNsi ciblant ACE2. L'invention concerne également des ARNsi spécifiques ciblant l'ACE2.
PCT/EP2021/066665 2020-06-19 2021-06-18 Arnsi et compositions pour le traitement prophylactique et thérapeutique des maladies virales WO2021255262A1 (fr)

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