All-in-one AAV vector for treating coronavirus-induced diseases

Abstract

The present invention relates to a novel approach to treating coronavirus infections, particularly infections with mutant forms of MERS-CoV, SARS-CoV, and SARS-CoV-2. The present invention provides a Cas13d guide RNA that guides the Cas13d protein to a target site within the genome of a humanized coronavirus that is conserved among MERS-CoV, SARS-CoV, and SARS-CoV-2, based on the ability to effectively target and cleave single-stranded RNA viruses. The disclosed invention further provides an AAV vector comprising such a Cas13d guide RNA expression cassette along with Cas13d for treating coronavirus infections, particularly COVID-19 infections.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedicine. Specifically, the present invention provides an AAV vector comprising a sequence encoding Cas13d for cleaving single-stranded viral RNA and a guide RNA, and includes providing such a guide RNA. [Background technology]

[0002] The ongoing pandemic of the novel coronavirus SARS-CoV-2 has resulted in dramatically high incidences of infection and death worldwide. The virus's basic reproduction number (R0) of approximately 3 will inevitably lead to a further increase in the number of coronavirus cases. SARS-CoV-2 virus strains are known to be highly contagious and spread primarily through the respiratory tract via droplets or respiratory secretions. SARS-CoV-2 infections often result in significant lung damage and severe acute respiratory syndrome (SAR). SARS-CoV-2 is a member of the coronavirus family (Coronavirus family), a widespread family of single-stranded RNA viruses generally thought to cause a variety of human illnesses and diseases, ranging from the common cold to acute illnesses such as MERS (MERS-CoV) and SARS (SARS-CoV) (1). Effective clinical treatment strategies are urgently needed, but there are currently no available curative or preventative therapies or promising drug candidates to treat SARS-CoV-2.

[0003] CRISPR / Cas systems that can effectively target and cleave single-stranded RNA (ssRNA) may offer a potential therapeutic approach against SARS-CoV-2. Considering various Cas proteins, Cas13, particularly the Cas13d mutant, appears to be a promising candidate for such an approach, as recent studies have highlighted the ability of Cas13 to efficiently and specifically target and cleave ssRNA in several model systems, including mammalian cells (2).

[0004] Thus, the present invention provides novel drug candidates and therapeutic regimens for treating infections with viruses of the Coronaviridae family, particularly infections with SARS-CoV, MERS-CoV, and the recently identified SARS-CoV-2, by initiating Cas13-mediated cleavage of single-stranded RNA in infected mammalian cells. Accordingly, the present invention provides guide RNAs that guide the Cas13 protein, preferably the Cas13d protein, to their respective target sites within the genomes of viruses derived from the Coronaviridae family, particularly the family members SARS-CoV, MERS-CoV, and SARS-CoV-2. The present invention further provides AAV vectors comprising the Cas13 guide RNA for introducing Cas13, preferably Cas13d, into human cells. Summary of the Invention

[0005] The present invention describes the use of Cas13 targeting and cleavage of single-stranded RNA to target and cleave the genomes of single-stranded RNA viruses of the Coronaviridae family, particularly family members MERS-CoV, SARS-CoV, and SARS-CoV-2. The guide RNA associated with Cas13, preferably Cas13d, can have target sites located in ORF1ab, S, E, M, and N, regions conserved among members of the Coronaviridae family. An AAV vector containing a guide RNA expression cassette along with Cas13d is used as a vehicle for delivery of Cas13d to virus-infected cells.

[0006] Thus, in one embodiment, a guide RNA is provided for use with a Cas13 of less than 1000 amino acids, preferably in combination with Cas13d, wherein the guide RNA target site is a sequence comprised by the SARS-CoV-2 virus.

[0007] In another embodiment, the guide RNA target site is a sequence conserved among the genomes of human-associated viruses of the Coronaviridae family.

[0008] In a further embodiment, the guide RNA target site is a sequence that is conserved between the genomes of SARS-CoV-2, MERS-CoV and SARS-CoV.

[0009] In a preferred embodiment, the guide RNA target site is a sequence comprised of one or more of the Orf1ab region, the S region, the E region, the M region, and the N region in the genome of each of SARS-CoV-2, MERS-CoV, and SARS-CoV.

[0010] In a more preferred embodiment, the sequence of the guide RNA comprises a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 39.

[0011] Particularly suitable guide RNAs include the spacer sequence of any of SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:27 and SEQ ID NO:31.

[0012] The present invention further provides a nucleic acid molecule comprising a sequence encoding a Cas13d protein and a guide RNA expression cassette encoding a Cas13d guide RNA, the nucleic acid molecule comprising a U6 promoter.

[0013] In another embodiment, the nucleic acid molecule encodes two or more guide RNAs.

[0014] In another embodiment, the nucleic acid molecule encodes a guide RNA comprising the sequence of SEQ ID NO:4, SEQ ID NO:7, and SEQ ID NO:15; SEQ ID NO:15, SEQ ID NO:23, and SEQ ID NO:31; SEQ ID NO:15, SEQ ID NO:27, and SEQ ID NO:31; SEQ ID NO:23, SEQ ID NO:27, and SEQ ID NO:31; SEQ ID NO:4, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:27, and SEQ ID NO:31; SEQ ID NO:4, SEQ ID NO:7 ...15, SEQ ID NO:23, SEQ ID NO:27, and SEQ ID NO:31; or SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31.

[0015] In another embodiment, the nucleic acid molecule encodes a guide RNA comprising the sequences of SEQ ID NO:4, SEQ ID NO:7 and SEQ ID NO:15; SEQ ID NO:4, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:27 and SEQ ID NO:31; and SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:27 and SEQ ID NO:31.

[0016] In another embodiment, the nucleic acid molecule is a plasmid.

[0017] In a preferred embodiment, the nucleic acid molecule is a single plasmid.

[0018] In another embodiment, the Cas13d protein encoded by the above sequence does not contain a nuclear localization signal (NLS).

[0019] In another embodiment, the Cas13d protein encoded by the above sequence is a fusion protein comprising the N-terminal binding domain (N-NTD) of the nucleocapsid protein of SARS-CoV-2.

[0020] In another embodiment, the nucleic acid molecule is obtained by inserting the spacer sequence of a guide RNA into the plasmid pAAV-U6-gRNA-CMV-Cas13d of SEQ ID NO: 40, or by inserting at least the spacer sequence of at least one guide RNA into the plasmid pAAV-U6-gRNA-CMV-Cas13d-array-triguide of SEQ ID NO: 41, the plasmid pAAV-U6-gRNA-quadguide-CMV-Cas13d-V3-basic of SEQ ID NO: 42, the plasmid pAAV-U6-gRNA-CMV-Cas13d-SapI of SEQ ID NO: 43, or the plasmid pAAV-U6-gRNA-CMV-Cas13d-NTD-AarI of SEQ ID NO: 44.

[0021] The present invention further provides an AAV vector comprising the nucleic acid molecule.

[0022] In a preferred embodiment, the AAV vector is selected from the group AAV1, AAV2, AAV5, AAV6 and AAV9, preferably an AAV2 vector.

[0023] In a further preferred embodiment, the AAV vector is an AAV9 vector.

[0024] In another embodiment, the AAV vector backbone is reduced in size compared to the full-length transcript.

[0025] The present invention further provides an adenoviral vector comprising the nucleic acid molecule.

[0026] The present invention also provides pharmaceutical compositions comprising an AAV vector or an adenovirus vector.

[0027] The present invention also provides a pharmaceutical composition comprising at least one guide RNA as described above and at least one mRNA encoding a Cas13 protein.

[0028] In one embodiment, the Cas13 protein is a Cas13d protein or a Cas13a protein.

[0029] In another embodiment, the Cas13d protein encoded by the mRNA does not contain a nuclear localization signal (NLS).

[0030] In another embodiment, the Cas13d protein encoded by the mRNA is a fusion protein comprising the N-terminal binding domain (N-NTD) of the nucleocapsid protein of SARS-CoV-2.

[0031] The present invention further relates to a method for treating a disease or syndrome induced by a human-associated virus, comprising administering an AAV vector, an adenoviral vector, or a pharmaceutical composition to a patient in need thereof.

[0032] In one embodiment, the virally induced disease or syndrome is the result of infection with a coronavirus genetically related to the group consisting of MERS-CoV, SARS-CoV, and SARS-CoV.

[0033] In a preferred embodiment, the disease is COVID-19.

[0034] In another embodiment, the Cas13d protein cleaves a human-associated virus upon expression.

[0035] In a preferred embodiment, the AAV vector, adenoviral vector or pharmaceutical composition is administered via the upper respiratory tract, preferably by intranasal or intratracheal administration, or in an aerosol composition, for example using an inhaler / nebulizer.

[0036] In another preferred embodiment, the AAV vector, adenoviral vector or pharmaceutical composition is administered to a patient using a ventilator.

[0037] In a further preferred embodiment, the AAV vector, adenoviral vector or pharmaceutical composition is administered to the myocardium of the patient.

[0038] In a further aspect, the present invention also provides an AAV vector, adenovirus vector or pharmaceutical composition as described above for use in the treatment of a disease or syndrome caused by a human-associated virus.

[0039] In one embodiment, the disease or syndrome is the result of infection with a coronavirus that is genetically related to the group consisting of MERS-CoV, SARS-CoV and SARS-CoV-2.

[0040] In a preferred embodiment, the disease is COVID-19.

[0041] In another embodiment, the Cas13d protein cleaves a human-associated virus upon expression.

[0042] In a preferred embodiment, the AAV vector, adenoviral vector or pharmaceutical composition is administered via the upper respiratory tract, preferably by intranasal or intratracheal administration, or in an aerosol composition, for example using an inhaler or nebulizer.

[0043] In another preferred embodiment, the AAV vector, adenoviral vector or pharmaceutical composition is administered to a patient using a ventilator.

[0044] In a further preferred embodiment, the AAV vector, adenoviral vector or pharmaceutical composition is administered to the myocardium of the patient. [Brief explanation of the drawings]

[0045] [Figure 1] Phylogenetic tree of the seven currently known Cas13 proteins, as reported in (3). [Figure 2] Figure 1 shows an alignment of genomes from SARS-CoV-2, SARS-CoV, and MERS-CoV. Short dashes below the alignment indicate the positions of Cas13d guide RNAs designed to target conserved sequence regions between the genomes SARS-CoV-2, SARS-CoV, and MERS-CoV. [Figure 3A]

[0023] Figure 1 shows a vector map of the AAV2 plasmid pAAV2-U6-gRNA-CMV-Cas13d, which contains a Cas13d guide RNA expression cassette along with the Cas13d coding sequence. Vector map featuring a single guide RNA (see SEQ ID NO: 40). [Figure 3B]

[0023] Figure 1 shows a vector map of the AAV2 plasmid pAAV2-U6-gRNA-CMV-Cas13d, which contains a Cas13d guide RNA expression cassette along with the Cas13d coding sequence. The vector map shows three insertion sites for the guide RNA spacer sequence (see SEQ ID NO:41). [Figure 4]FIG. 1 shows the sequence alignment of the genomes from SARS-CoV-2, SARS-CoV and MERS-CoV, as well as the sequences used for the alignment. [Figure 5] FIG. 1 shows the inhibitory efficiency of single guide RNA constructs of the present invention in a luciferase reporter assay. [Figure 6A] FIG. 1 shows a first experimental design for testing guide RNA constructs of the invention in SARS-CoV-2 infected human epithelial lung cells. [Figure 6B] FIG. 6B shows the inhibitory efficiency of several combinations of guide RNA constructs in the experimental design of FIG. 6A. [Figure 7A] FIG. 1 shows a second experimental design for testing guide RNA constructs of the invention in SARS-CoV-2 infected human epithelial lung cells. [Figure 7B] FIG. 7B shows the inhibitory efficiency of several combinations of guide RNA constructs in the experimental design of FIG. 7A. [Figure 8] FIG. 7B shows the inhibitory efficiency of several combinations of guide RNA constructs and multi-guide RNA constructs in the experimental design of FIG. 7A. DETAILED DESCRIPTION OF THE INVENTION

[0046] definition AAV vector: As used herein, the term "AAV vector" refers to an adeno-associated virus (AAV) capable of introducing a nucleic acid sequence into a target cell. This vector contains a sequence encoding Cas13d and / or a guide RNA expression cassette encoding a guide RNA, and may contain a U6 promoter.

[0047] Vector backbone: The term "vector backbone" refers to the native nucleic acid sequences of an AAV vector.

[0048] Cas13d: As used herein, the term "Cas13d" refers to the Cas endonuclease of the CRISPR / Cas13d system, including the RfxCas13d endonuclease.

[0049] Conserved: The term "conserved," as used herein, refers to a sequence or portion of a sequence within the viral genome of a particular member of the Coronaviridae family that is shared by at least two other members of the Coronaviridae family.

[0050] Coronavirus: The term "coronavirus," as used herein, refers to any virus of the Coronaviridae family. Coronaviruses capable of infecting human cells are also referred to herein as human-associated coronaviruses.

[0051] COVID-19: The term "COVID-19," as used herein, refers to the disease known as Coronavirus Disease 2019, which is caused by infection with SARS-CoV-2, a type of coronavirus.

[0052] Derivative: As used herein, the term "derivative" refers to a virus that is closely related to the viruses described herein. In particular, a virus is a derivative of another virus if the respective genomes show at least 50% sequence similarity.

[0053] Guide RNA: The term "guide RNA" is used herein to refer to a component of the CRISPR / Cas system. In the present invention, "guide RNA" refers to the guide RNA of the CRISPR / Cas13 system, for example, the Cas13d protein. A guide RNA is a short non-coding RNA sequence that "guides" the Cas protein to its target cleavage site. A guide RNA comprises a nucleic acid sequence that binds to a complementary target site in a target nucleic acid sequence. For example, the target nucleic acid of Cas13d is a single-stranded RNA sequence.

[0054] Human-associated virus: The term "human-associated virus" refers to any virus that is capable of infecting human cells.

[0055] Infection: As used herein, the term "infection" refers to the invasion of pathogens into the body tissues of an organism, the proliferation of such pathogens, and the response of host tissues to the pathogens and the toxins they produce. As used herein, the term "infection" refers to a viral infection of a cell.

[0056] Lower Respiratory Tract: As used herein, this term refers to the portion of the larynx below the vocal cords, the trachea, bronchi, bronchioles, and lungs, including the respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli.

[0057] MERS: The term "MERS" refers to the disease Middle East Respiratory Syndrome, a disease or syndrome caused by infection with MERS-CoV, a type of coronavirus.

[0058] SARS: The term "SARS" refers to the disease known as severe acute respiratory syndrome, which results from infection with SARS-CoV, a type of coronavirus.

[0059] Transduction: The term "transduction," as used herein, refers to the deliberate introduction of nucleic acid into a cell using a viral vehicle. In particular, "transduction" refers to the introduction of an AAV vector into a cell.

[0060] Upper respiratory tract: As used herein, this term refers to the nose and nasal cavities, sinuses, pharynx, and the portion of the larynx above the vocal cords.

[0061] Exemplary Advantages of the Invention The disclosed invention relates to the use of Cas13 proteins, preferably Cas13d proteins, to target and cleave viral genomes, such as single-stranded RNA genomes in the case of coronaviruses, thereby inhibiting or eliminating the virus's ability to replicate.

[0062] Thus, the present invention provides guide RNAs for guiding a Cas13 protein, preferably a Cas13d protein, to its respective target cleavage site within the viral genome. The present inventors designed these guide RNAs based on the observation that coronaviruses that have spread from animals to humans share one or more conserved regions, which may be responsible for the high infectivity attributed to such human-associated coronaviruses. Specifically, the present inventors identified and characterized 31 different guide RNA sequences that target these highly conserved regions of each viral sequence (SEQ ID NO: 1 to SEQ ID NO: 31).

[0063] Among the currently known family of Cas proteins, class II type VI Cas13 is the smallest RNA-targeting endonuclease, approximately 930 amino acids in size. Cas13d has been shown to have high levels of catalytic activity and specificity in mammalian cells. Therefore, Cas13d appears particularly suitable for the efficient transduction and specific targeting and degradation of single-stranded RNA viruses such as coronaviruses.

[0064] The present invention further provides AAV vectors containing a sequence encoding a Cas13 protein of less than 1,000 amino acids, preferably Cas13d, along with these guide RNAs. AAV vectors are widely used in gene delivery approaches. However, in contrast to common AAV transduction systems, the AAV vector nucleic acid used in the present invention is a single-component system. As a result, all elements required for target cell transduction and expression of associated proteins are contained in a single vector nucleic acid, simplifying the transduction procedure. To further promote transduction and increase transduction efficiency, the vector nucleic acid is also reduced to a minimum size. Such all-in-one AAV-Cas13-gRNA constructs exhibit superior efficiency and fewer side effects compared to conventional two-vector nucleic acid systems. The AAV vectors disclosed herein are preferably based on AAV2 vectors, as AAV2 vectors have demonstrated high transduction capacity in the lung during Phase III clinical trials.

[0065] The AAV vectors may contain coding sequences for a single Cas13 guide RNA, preferably a Cas13d guide RNA, or a combination of two or more Cas13 guide RNAs, preferably Cas13d guide RNAs, as described herein. The use of two or more guide RNAs can further increase the efficacy of targeting and cleaving the virus. In addition, the combination of several guide RNAs can also increase the efficacy and specificity of targeting additional mutant viruses, such as derivatives of the viruses described herein.

[0066] Suitable guide RNA spacer sequences of the present invention were designed based on sequence alignments of several human-associated coronavirus strains (SEQ ID NO: 1 to SEQ ID NO: 31). By taking MERS-CoV, which shows lower sequence similarity to two other closely related coronaviruses, SARS-CoV and SARS-CoV-2, into consideration for guide RNA design, regions of the viral genome that enable the virus to target human cells can be identified. These guide RNA spacer sequences, and others designed accordingly, enable specific targeting and cleavage even for coronavirus strains that may only become clinically relevant in the future.

[0067] Embodiments of the present invention The current COVID-19 pandemic, caused by infection with the newly identified SARS-CoV-2 virus, has already caused nearly 1,000,000 infections and 50,000 deaths worldwide in just a few months. Despite the clear and urgent need, no effective drugs are available to date, and vaccine development is expected to take approximately 12–18 months. Therefore, promising new therapeutic approaches are desperately needed.

[0068] The newly identified SARS-CoV-2 belongs to the Coronaviridae family, a large family of single-stranded, positive-sense RNA viruses. Viruses in the Coronaviridae family typically infect the respiratory system and are thought to be responsible for numerous human illnesses and diseases, ranging from the common cold to more severe diseases such as MERS (MERS-CoV) and SARS (SARS-CoV) (1). SARS-CoV-2 has been reported to contain 10 distinct proteins (ORF1ab, S, ORF3a, E, M, ORF6, ORF7a, ORF8, N, and ORF10) (GenBank entry MN908947.3). A sequence alignment between genomes derived from MERS-CoV, SARS-CoV, and SARS-CoV-2 is shown in Figure 4.

[0069] The recently identified and developed CRISPR / Cas system has revolutionized gene editing by providing a highly effective, specific, and simple system for genetic modification in eukaryotic cells. Caspases (Cas) as effector proteins and guide RNAs to guide the Cas proteins to specific target sequences within nucleic acids are the only required components of the system, enabling precise cleavage of nucleic acids and / or genetic modification of cells or even entire organisms (4).

[0070] Within the known family of Cas proteins, class II type VI Cas13 was recently discovered and classified into four distinct subtypes: Cas13a, Cas13b, Cas13c, and Cas13d (5). Cas13d is small in size and exhibits high catalytic activity and specificity in mammalian cells, targeting and cleaving single-stranded RNA. Therefore, Cas13d offers an interesting new approach to combat viral invasion by degrading single-stranded viral RNA.

[0071] However, a major obstacle to using Cas13d to combat viral infection is the delivery of the Cas protein and its guide RNA into (infected) cells.

[0072] Adeno-associated viruses are non-enveloped, single-stranded DNA viruses of the Parvoviridae family. Several serotypes have been identified, of which AAV2 appears to be the best known. Adeno-associated viruses exhibit certain characteristics that make them effective gene delivery tools, such as low pathogenicity and low immunogenicity, while also exhibiting broad tropism (6).

[0073] Thus, the present invention provides a novel therapeutic approach to treat human-associated coronavirus-induced diseases and / or syndromes, particularly COVID-19, by administering to patients AAV vectors containing a sequence encoding the Cas13d protein along with Cas13d guide RNAs that target specific target sites within the viral genome to cleave the target sequence and degrade the virus. The present invention further provides such guide RNAs engineered to interact with highly promising target sites within the viral genome.

[0074] guide RNA The Cas13 guide RNAs of the present invention, preferably Cas13d guide RNAs, are directed to single-stranded RNA target sequences within the genome of human-associated coronaviruses or their derivatives. The target sites of the guide RNAs disclosed herein are specifically directed to conserved sequences and / or sequence portions among several members of the Coronaviridae family, preferably MERS-CoV, SARS-CoV, and SARS-CoV-2.

[0075] Typical Cas13d guide RNAs target a 22-30 nt target sequence (spacer) (2). Therefore, the guide RNAs of the present invention can be directed to any 22-30 nt target sequence within the coronavirus genome. Exemplary guide RNAs used herein are discussed in Konermann et al. (3).

[0076] The guide RNA sequences of the present invention are designed according to one of the following design approaches:

[0077] (1) A sequence alignment of the genome sequences of different coronavirus strains, preferably SARS-CoV-2, SARS-CoV, and MERS-CoV, is generated. A 22-30 nt spacer sequence is designed so that the seed region perfectly matches the 100% overlap region between the aligned viral genome sequences. The remainder of the spacer sequence is identical to at least one of the viral sequences, preferably SARS-CoV2, but may only partially match the remaining sequences. Therefore, this approach results in particularly high cleavage efficiency for all three coronaviruses, and the affinity of the guide RNA is maximized for viruses with perfectly matched spacer sequences.

[0078] (2) A sequence alignment of the genome sequences of different coronavirus strains, preferably SARS-CoV-2, SARS-CoV, and MERS-CoV, is generated. A 22-30 nt spacer sequence is designed so that the seed region perfectly matches at least one of the viral sequences, preferably SARS-CoV-2. In contrast to the first approach, mismatches between the seed region of the spacer sequence and each target sequence in some viral sequences are tolerated. Guide RNA spacer sequences designed using this approach have very high binding affinity but reduced cleavage efficiency for viruses with incompletely matched spacer sequences.

[0079] (3) A sequence alignment of the genome sequences of different coronavirus strains, preferably SARS-CoV-2, SARS-CoV, and MERS-CoV, is generated. A 22- to 30-nt spacer sequence is designed to ensure that the guide RNA spacer sequence has the best overall match with all members of the Coronaviridae family subjected to sequence alignment. Based on the requirement that the guide RNA spacer sequence have 100% sequence similarity in the seed region to each target sequence of all aligned coronavirus members, a maximum overall sequence similarity of 95% for the guide RNA spacer sequence to each sequence in the aligned coronavirus members is achievable. This approach allows for the highest probability that the guide RNA will also target future coronavirus variants.

[0080] Thus, in one embodiment, the guide RNA is directed to a target sequence within a conserved region of the genome of a virus of the Coronaviridae family, preferably the genome of a human-associated coronavirus.

[0081] In a preferred embodiment, the guide RNA is directed to a target sequence within a conserved region of the genome of MERS-CoV, SARS-CoV and SARS-CoV-2, or their derivatives.

[0082] In a preferred embodiment, the guide RNA is directed to a target sequence within the SARS-CoV-2 genome, or a derivative thereof.

[0083] In a preferred embodiment, the guide RNA comprises a spacer sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 39 or a combination thereof, as shown in Table 1 herein.

[0084] The guide RNA can include, for example, a spacer sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, 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, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39, or any combination thereof.

[0085] In another preferred embodiment, the guide RNA comprises a spacer sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 31 or a combination thereof.

[0086] The guide RNA spacer sequence can include, for example, the spacer sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, 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, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31, or any combination thereof.

[0087] In a more preferred embodiment, the guide RNA comprises a spacer sequence of any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11 to SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 29, and SEQ ID NO: 31, or a combination thereof.

[0088] The guide RNA spacer sequence can include, for example, the spacer sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:29, or SEQ ID NO:31, or a combination thereof.

[0089] In a most preferred embodiment, the guide RNA comprises a spacer sequence of any of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:29, or a combination thereof.

[0090] The guide RNA spacer sequence can include, for example, the spacer sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:29, or a combination thereof.

[0091] In another most preferred embodiment, the guide RNA comprises a spacer sequence of any of SEQ ID NOs: 13 to 18 or SEQ ID NO: 31.

[0092] In another most preferred embodiment, the guide RNA comprises a spacer sequence of any of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:11 to SEQ ID NO:16, and SEQ ID NO:31.

[0093] Particularly suitable guide RNAs include the spacer sequence of any of SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:27 and SEQ ID NO:31.

[0094] The guide RNA spacer sequence can include, for example, the spacer sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 31, or a combination thereof.

[0095] [Table 1] TIFF0007756284000002.tif29170

[0096] Among the spacer sequences listed above, those designated as gRNA_XX-O-1 to gRNA_XX-O-18 target the ORF1ab gene, those designated as gRNA_XX-S-1 to gRNA_XX-S-4 target the "S" spike protein gene, those designated as gRNA_XX-E-1 to gRNA_XX-E-4 target the envelope protein gene, those designated as gRNA_XX-M-1 to gRNA_XX-M-2 target the membrane protein gene, and those designated as gRNA_XX-N-1 to gRNA_XX-N-3 target the nucleocapsid protein gene.

[0097] Cas13d According to the present invention, Cas13, preferably Cas13d, is selected as the endonuclease for cleaving and targeting the coronavirus genome. This CRISPR / Cas member appears to be particularly well suited for antiviral approaches due to its small size and high targeting and cleavage potency and specificity in mammalian cells.

[0098] Cas13d, like other Cas13 family enzymes, has the property of independently processing its own CRISPR array into a mature guide RNA containing a 30 base pair 5' direct repeat followed by a variable 3' spacer ranging in length from 22 bp to 30 bp.

[0099] To date, seven distinct Cas13d proteins have been identified: EsCas13d, RffCas13d, UrCas13d, RaCas13d, P1E0 Cas13d, Adm Cas13d, and RfxCas13d (Figure 1). Among these Cas13d variants, RfxCas13d has been reported to exhibit high RNA knockdown efficacy with minimal off-target activity (2).

[0100] Thus, in some embodiments, any Cas13d endonuclease can be used.

[0101] In a preferred embodiment, the RfxCas13d endonuclease is used.

[0102] AAV vector containing a Cas13d-encoding sequence and a guide RNA expression cassette The AAV vector of the present invention contains a Cas13d guide RNA expression cassette and a sequence encoding the Cas13d protein, and serves as a vehicle for delivering the CRISPR / Cas13d system into cells, including those infected with the virus.

[0103] AAV vectors are known gene delivery tools suitable for a variety of applications. AAV vectors exhibit significant tropism, depending on the respective AAV serotype, allowing for directional transduction of target cells. For example, AAV9 has been shown to be tropic for cardiomyocytes, and AAV9-based vectors are considered suitable for gene delivery to these cells (see, e.g., European Patent No. 3132041 to Kupatt et al.). Similarly, AAV2-based vectors have shown potential for treating cystic fibrosis in humans, as discussed by Guggino et al. (7) (see also (8)).

[0104] AAV vectors have a packaging limit of only about 4.7 kb, so most transduction approaches involve the use of two or more vectors to allow for the introduction of all the required genetic elements.

[0105] However, by selecting a relatively small Cas13d and, if necessary, further removing non-essential elements from the AAV vector, the present invention provides a single AAV vector that contains all elements required for expression of Cas13d and its guide RNA in transduced cells.

[0106] Similarly, other Cas13 proteins can be used that do not exceed the packaging size of an AAV vector, and thus, Cas13 proteins of less than 1000 amino acids are well suited for vectors according to the present invention.

[0107] An exemplary schematic map of an AAV2 vector plasmid encoding Cas13d and guide RNA is shown in Figure 3A and Figure 3B.

[0108] AAV vectors of the invention can be based on a number of AAV serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9.

[0109] In preferred embodiments, the AAV vector is based on AAV1, AAV2, AAV5 or AAV9.

[0110] In a further preferred embodiment, the AAV vector is based on AAV2.

[0111] In another more preferred embodiment, the AAV vector is based on AAV9.

[0112] In a most preferred embodiment, the AAV vector plasmid is pAAV2-U6-gRNA-CMV-Cas13d (see SEQ ID NO: 40).

[0113] In another preferred embodiment, the AAV vector plasmid is pAAV2-U6-gRNA-CMV-Cas13d-array-triguide, which allows for the insertion of three gRNA sequences (see SEQ ID NO: 41).

[0114] In another preferred embodiment, the AAV vector plasmid is pAAV-U6-gRNA-quadguide-CMV-Cas13d-V3-basic, which allows for the insertion of four guide RNA sequences (see SEQ ID NO: 42). The vector plasmid of SEQ ID NO: 42 is used to generate an AAV vector, resulting in packaging of a 5064 bp DNA having the sequence of SEQ ID NO: 45 into the AAV vector.

[0115] The absence of a nuclear localization sequence (NLS) in the coding sequence of the Cas13d protein can reduce the size of the AAV vector cargo. SARS-CoV-2 propagation occurs in the cytosol. Therefore, the use of an NLS-free Cas13d protein can enhance the accumulation of Cas13d in the cytosol while simultaneously reducing the size of the protein and the construct encoding it. A suitable vector plasmid encoding the NLS-free Cas13d protein is pAAV-U6-gRNA-CMV-Cas13d-SapI (see SEQ ID NO: 43). When the vector plasmid of SEQ ID NO: 43 is used to generate an AAV vector, a 4833-bp DNA having the sequence of SEQ ID NO: 46 is packaged into the AAV vector.

[0116] AAV vectors preferably contain less than 5 kb of DNA, as sizes greater than 5 kb result in significantly reduced packaging efficiency and intracellular expression (11).

[0117] To further promote binding of the Cas13d protein to the SARS-CoV-2 genome, an N-terminal RNA-binding domain (N-NTD) can be fused to the Cas13d protein. The N-NTD is the RNA-binding domain of the SARS-CoV-2 nucleocapsid (N) protein. The primary function of the nucleocapsid protein during infection is to bind to viral RNA and form a helical ribonucleoprotein (RNP) complex to protect the viral genome and maintain reliable viral replication. The N-terminal binding domain (N-NTD) of the nucleocapsid protein captures the viral RNA genome, and the C-terminal domain anchors the RNP complex to the viral membrane through its interaction with the M protein. Therefore, fusion of the N-NTD to the Cas13d protein is expected to promote the formation of a complex containing Cas13d and the viral genome, thereby guiding Cas13d into spatial proximity with the viral genome. A suitable vector plasmid encoding a Cas13d protein fused to the N-NTD at its C-terminus is pAAV-U6-gRNA-CMV-Cas13d-NTD-AarI (see SEQ ID NO: 44). The vector plasmid of SEQ ID NO: 44 is used to generate an AAV vector, and a 5238 bp DNA having the sequence of SEQ ID NO: 47 is packaged into the AAV vector.

[0118] Depending on the respective serotype on which the AAV vectors of the present invention are based, the AAV vectors can exhibit tropism for certain cell types, tissues and organs that harbor these cell types.

[0119] Therefore, the AAV vectors of the present invention can be directed to various cell types and organs within the human body by selecting a particular serotype of the AAV vector.

[0120] In a preferred embodiment, the AAV vector is tropic for and directed to cells of the human respiratory system.

[0121] In a further preferred embodiment, the AAV vector is an AAV2 vector directed to cells of the human respiratory system.

[0122] In another preferred embodiment, the AAV vector is directed to human cardiomyocytes.

[0123] In a further preferred embodiment, the AAV vector is an AAV9 vector directed to cells of the human cardiac muscle.

[0124] The AAV vectors of the invention can encode a single Cas13d guide RNA or a combination of several guide RNAs, such that an AAV vector can encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 guide RNAs.

[0125] In a preferred embodiment, the AAV vector encodes a single guide RNA.

[0126] In another preferred embodiment, the AAV vector encodes two guide RNAs.

[0127] In the most preferred embodiment, the AAV vector encodes three, four or five guide RNAs, each with a spacer sequence that preferably targets a different gene in the SARS-CoV-2 genome.

[0128] Suitable combinations of guide RNAs for use in the present invention have spacer sequences of SEQ ID NO:4, SEQ ID NO:7 and SEQ ID NO:15; SEQ ID NO:15, SEQ ID NO:23 and SEQ ID NO:31; SEQ ID NO:15, SEQ ID NO:27 and SEQ ID NO:31; SEQ ID NO:23, SEQ ID NO:27 and SEQ ID NO:31; SEQ ID NO:4, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:27 and SEQ ID NO:31; SEQ ID NO:4, SEQ ID NO:7 ...15, SEQ ID NO:23, SEQ ID NO:27 and SEQ ID NO:31; and SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31.

[0129] Particularly preferred guide RNAs for use in the present invention have the spacer sequences SEQ ID NO:4, SEQ ID NO:7 and SEQ ID NO:15; SEQ ID NO:4, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:27 and SEQ ID NO:31; and SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:27 and SEQ ID NO:31.

[0130] In a preferred embodiment, the AAV vector encodes a combination of guide RNAs under the same promoter.

[0131] AAV vectors encoding Cas13d and Cas13d guide RNAs for the treatment of viral infections The present invention provides a novel therapeutic approach to treating human-associated coronavirus infections. By introducing an AAV vector into cells, Cas13 expression allows the guide RNA to guide Cas13 to a target sequence within the viral genome for cleavage and, therefore, destruction of the genomic sequence. In cells already infected with coronavirus, the viral sequence is cleaved, degrading the virus and preventing any further spread of viral contamination. In cells that have not yet been infected, expression of the Cas13 guide RNA system provides a protective mechanism for such cells by immediately degrading viral genetic material after it enters the cell.

[0132] To reach target cells, AAV vectors are delivered to tissues and organs that harbor the target cells.

[0133] According to reports to date, coronavirus-induced diseases primarily manifest in the respiratory system of infected subjects, causing mild to severe respiratory symptoms and reactions. In particular, MERS, SARS, and COVID-19 coronavirus variants commonly cause pulmonary inflammation, pulmonary distress, and acute respiratory syndromes, often resulting from or driven by a cytokine storm caused by an overactive immune system. Other scientific literature has reported findings suggesting that at least COVID-19 variants may also adversely affect the myocardium in some patients. Consequently, during patient treatment, AAV vectors are delivered to the respiratory system, particularly the lungs, and / or myocardium, as indicated.

[0134] Thus, in one embodiment, an AAV vector for the treatment of human-associated coronavirus-induced disease is administered to the respiratory system of a patient.

[0135] In another embodiment, the AAV vector is administered to the lower respiratory tract of the patient.

[0136] In a preferred embodiment, the AAV vector is administered to the upper respiratory tract by inhalation.

[0137] AAV vectors can be administered directly to tracheal tissue.

[0138] In one embodiment to specifically treat MERS, SARS and / or COVID-19, the AAV vector may be administered to the upper respiratory tract of the patient.

[0139] In another embodiment for treating MERS, SARS and / or COVID-19, the AAV vector may be administered to the lower respiratory tract of the patient.

[0140] The AAV vector containing the guide RNA expression cassette and encoding the Cas13d sequence may be composed of a composition.

[0141] In one embodiment, the composition may be in the form of a tablet, capsule, syrup, film, liquid, solution, powder, paste, aerosol, injection, cream, gel, lotion, or drops.

[0142] In another embodiment, the composition is in the form of an aerosol and is administered by an inhaler, nebulizer, or vaporizer.

[0143] In a preferred embodiment, the composition is in the form of an aerosol and is administered by inhaler.

[0144] In a more preferred embodiment, the composition is in the form of an aerosol and is administered to the upper respiratory tract by means of an inhaler.

[0145] In another preferred embodiment, the AAV vector or a composition comprising same is administered to the patient via a ventilator.

[0146] Adenovirus vectors All aspects of the present invention can also be practiced using adenoviral vectors instead of AAV vectors, which have the advantage of a higher packaging size limit.

[0147] Thus, adenoviral vectors can contain a sequence encoding Cas13d and a guide RNA expression cassette, which can be delivered to target cells and used to treat human-related coronavirus infections, such as SARS-CoV-2 infection.

[0148] Viral vector-independent delivery of guide RNA and Cas13d protein The guide RNA of the present invention can also be delivered to target cells without using a viral vector. For example, the method described in Reference (10) can be used, which involves the delivery of Cas13 mRNA produced by in vitro transcription simultaneously with synthetic guide RNA. A molar ratio of 50:1 between guide RNA and Cas13 mRNA can be used. The mRNA and guide RNA can be delivered using a vesicle.

[0149] The present invention provides a method for treating human-associated coronavirus infections, such as SARS-CoV-2 infection, by delivering a synthetic guide RNA and mRNA encoding a Cas13 protein to the respiratory tract of a patient in need thereof. Details of this method are described in reference (10). The Cas13 protein may be a Cas13a protein. The Cas13 protein may be a Cas13d protein. The mRNA and guide RNA can be delivered using a vesicle. [Example]

[0150] Example 1 The use of the AAV vectors disclosed herein represents a promising new therapeutic approach for the prevention and / or treatment of coronavirus-induced diseases and syndromes. Accordingly, numerous clinical trials are currently underway to test the suitability of these vector vehicles in the treatment of various diseases. The clinical trials and the respective AAV vectors tested are listed in Table 2 below.

[0151] [Table 2]

[0152] Example 2 - Design of guide RNA sequences To generate AAV vectors encoding Cas13d and guide RNA for the treatment of human-associated coronavirus-induced diseases, we designed guide RNA spacer sequences.

[0153] Similar to the cases of SARS-CoV and MERS-CoV, SARS-CoV-2, the pathogen that causes COVID-19, has successfully survived transmission from animals to humans. Based on this observation, the inventors hypothesized that there must be highly conserved regions among the genomes of SARS-CoV, MERS-CoV, and SARS-CoV-2 that account for the high pathogenicity and infectious properties of these viruses in humans. The inventors also concluded that such conserved genomic regions provide promising target sites for CRISPR / Cas-mediated cleavage, including guide RNAs, that guide the Cas13 protein, preferably the Cas13d protein, to its target and cleavage site, thereby cleaving and degrading viral nucleic acid.

[0154] To this end, we performed a sequence alignment of nucleic acid sequences derived from SARS-CoV, MERS-CoV, and SARS-CoV-2 coronavirus variants and identified five highly conserved regions: ORF1ab, S, E, M, and N (see Figure 4). By targeting these conserved regions, we identified 31 guide RNA spacer sequences (SEQ ID NO: 1 to SEQ ID NO: 31) that are highly promising targets for pathogen cleavage and degradation.

[0155] To identify similar sequences between all three SARS-CoV-2, SARS-CoV and MERS-CoV variants, three main approaches are used.

[0156] (1) In the first approach, we identified a spacer sequence containing a seed sequence that contains a 7-base-pair region that precisely overlaps the sequences of all three viral genomes. This sequence was included as the seed region of the guide RNA spacer sequence (bases 15–21 of the guide RNA spacer sequence; (2)). The remainder of this spacer sequence perfectly matches the SARS-CoV-2 sequence but only partially matches the MERS-CoV and SARS-CoV variants. The seed region is considered essential for the targeting specificity of the guide RNA by Cas13d. Consequently, if there is a mismatch between the essential seed sequence and the target sequence, Cas13d will be unable to cleave the target sequence. Therefore, this approach maximizes the affinity of the guide RNA for SARS-CoV-2 while achieving the best cleavage efficiency for all three coronaviruses.

[0157] (2) In the second approach, the binding affinity of the spacer sequence to viral RNA is increased by increasing the sequence similarity between the guide RNA spacer sequence and its respective target sequence over the entire length of the spacer sequence. Unlike the first approach, mismatches in the seed region of the spacer sequence to the respective target sequences in SARS-CoV and MERS-CoV are allowed. However, this assumes 100% sequence similarity between the seed region sequence and the respective target sequences in SARS-CoV-2. The guide RNA spacer sequences designed by this approach have very high binding affinity but exhibit reduced cleavage efficiency against MERS-CoV and SARS-CoV.

[0158] (3) The third approach identifies guide RNAs that provide the best overall match for all three members of the coronavirus family. Based on the requirement that the guide RNA spacer sequence have 100% sequence similarity to each target sequence in all three coronavirus members in the seed region, a maximum of 95% overall sequence similarity of the guide RNA spacer sequence to each sequence in the three coronavirus members was achieved. This approach allows for the highest probability that the guide RNA will also target future coronavirus variants.

[0159] The identified guide RNA spacer sequences were aligned with all known human-associated SARS-CoV-2 viral transcripts. Spacer sequences showing high target sequence specificity and potentially reducing the risk of treatment-induced side effects were selected and inserted into the pAAV-U6-gRNA-CMV-Cas13d plasmids, which contain the Cas13d protein-encoding sequence along with the U6 promoter of the guide RNA expression cassette (see also SEQ ID NOs: 1 to 40).

[0160] Example 3 - Evaluation of the efficiency of single guide RNA sequences The inhibitory potency of the 39 gRNAs in Table 1 was assessed in a luciferase assay by co-transfection of the pMir reporter and all-in-one Cas13 guide constructs. A guide RNA targeting LacZ was used as a negative control.

[0161] The initial screening experiment was performed using non-infectious material. Thirty-nine guide RNAs (gRNAs) targeted six distinct regions: gRNA_XX-O-1 through gRNA_XX-O-18 targeted the ORF1ab gene, gRNA_XX-S-1 through gRNA_XX-S-4 targeted the "S" spike protein gene, gRNA_XX-E-1 through gRNA_XX-E-4 targeted the envelope protein gene, gRNA_XX-M-1 through gRNA_XX-M-2 targeted the membrane protein gene, and gRNA_XX-N-1 through gRNA_XX-N-3 targeted the nucleocapsid protein gene. To this end, we cloned the six regions by PCR and inserted them into the pMIR vector (3'-UTR luciferase vector). This resulted in six luciferase constructs designated pMIR-report-SARS-COV-2-fragment1 through 6. To assess the inhibitory potency of each gRNA, human embryonic kidney (HEK293) cells were co-transfected with the all-in-one Cas13 guide construct and its corresponding pMIR reporter construct. The LacZ guide was used as a negative control. Of the 39 gRNAs, seven guides were ultimately selected for further experiments (highlighted in red in Figure 5).

[0162] Example 4 - Testing the Cas13-guide RNA system in SARS-CoV-2 infected human epithelial lung cells Experimental Design 1 To assess the inhibitory effects of our system, we first loaded several guide RNA constructs into AAV2 viruses. Each AAV was applied at a titer of 10,000 vg / cell (number of viral genomes per cell), and different combinations of guide RNA constructs were transduced into human bronchial epithelial Calu-3 cells (40,000 cells / well), a typical cell line for coronavirus in vitro research. After 72 hours, the AAV-transduced cells were further infected with SARS-CoV-2 virus at an MOI (multiplicity of infection, or the number of viral particles per cell) of 0.01. After 1 hour of incubation at 37°C, the infection medium was removed, and the cells were washed twice with DPBS. The culture medium was then collected at 24 hpi (hours post-infection) and 48 hpi. The experimental schedule is shown in Figure 6A.

[0163] The infectivity of SARS-CoV-2 was measured by plaque assay. The results are shown in Figure 6B. The plaque assay indicates that, in most cases, effective single-guide RNA combinations enhance the ability to inhibit viral replication. All guide RNAs identified as efficient in the luciferase assay (Example 3) and tested in combinatorial approaches were found to be highly efficient at suppressing viral replication in live human epithelial lung cells. Therefore, in principle, any combination of guide RNAs that showed degradation efficiencies approaching 50% or less can be used in combinatorial approaches.

[0164] Experimental Design 2 A new experimental design was developed in which higher AAV titers (100,000 vg / cell per construct) were used to transduce Calu-3 cells (30,000 cells / well) (see Figure 7A). After 48 hours, the AAV-transduced cells were further infected with SARS-CoV-2 virus, and culture medium was collected at 24 and 48 hpi.

[0165] SARS-CoV-2 infectivity was measured by plaque assay. The results are shown in Figure 7B. All three samples (D–F) treated with different gRNA combinations showed significant effects compared to the untreated control sample. Three-guide RNA combination D showed a 93% reduction in SARS-CoV2 titer within 24 hours and a 94% reduction within 48 hours. Four-guide RNA combination F showed a 98% reduction within 24 hours and a 95% reduction within 48 hours. Five-guide RNA combination E showed a 94% reduction within 24 hours and a 100% reduction within 48 hours.

[0166] The experiment was repeated including multi-guide RNA constructs capable of delivering several guide RNAs with one AAV. These included the quadguide construct of SEQ ID NO: 42 and the construct of SEQ ID NO: 44 encoding Cas13d with the NTD fused to its C-terminus. The results are shown in Figure 8.

[0167] References [1] Coronaviridae Study Group of the International Committee on Taxonomy of Viruses, "The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2", Nature Microbiology (5): 536-544, 2020. [2] Wessels HH et al., "Massively parallel Cas13 screens reveal principles for guide RNA design", Nature Biotechnology, 2020. [3] Konermann S et al., "Transcriptome engineering with RNA-targeting Type VI-D CRISPR effectors", Cell (173(3)): 665-676, 2018 [4] Cong L, "Multiplex genome engineering using CRISPR / Cas systems", Science (339(6121)):819-23, 2013. [5] Shmakov S et al., "Discovery and functional characterization of diverse class 2 CRISPR-Cas systems", Molecular Cell (69):385-397, 2015. [6] Colella P et al., "Emerging issues in AAV-mediated in vivo gene therapy",Molecular Therapy: Methods & Clinical Development (8):87-104, 2018. [7] Guggino WB et al., "AAV gene therapy for cystic fibrosis: current barriers and recent developments", Expert Opinion on Biological Therapy (17(10)):1265-1273, 2017. [8] Moss RB et al., "Repeated Adeno-Associated Virus serotype 2 aerosol-mediated cystic fibrosis transmembrane regulator gene transfer to the lungs of patients with cystic fibrosis", CHEST (125(2)):509-521, 2004. [9] Abbott TR et al., "Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza", bioRxiv, 2020.

[10] Blanchard EL et al., "Treatment of influenza and SARS-CoV-2 infections via mRNA-encoded Cas13a in rodents", Nature Biotechnology, doi: 10.1038 / s41587-021-00822-w, 2021.

[11] Grieger CJ & Samulski RJ, "Packaging capacity of adeno-associated virus serotypes: Impact of larger genomes on infectivity and postentry steps", Journal of Virology79(15):9933-9944, 2005.

Claims

1. A guide RNA used in combination with a Cas13 protein, wherein the guide RNA target site is a sequence comprised by the SARS-CoV-2 genome; A guide RNA, wherein the sequence of the guide RNA comprises a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:27 and SEQ ID NO:

31.

2. A nucleic acid molecule comprising a sequence encoding a Cas13d protein and a guide RNA expression cassette encoding one or more guide RNAs according to claim 1, and comprising a U6 promoter.

3. 3. The nucleic acid molecule of claim 2, encoding a guide RNA comprising the sequences of SEQ ID NO:4, SEQ ID NO:7 and SEQ ID NO:15; SEQ ID NO:15, SEQ ID NO:23 and SEQ ID NO:31; SEQ ID NO:15, SEQ ID NO:27 and SEQ ID NO:31; SEQ ID NO:23, SEQ ID NO:27 and SEQ ID NO:31; SEQ ID NO:4, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:27 and SEQ ID NO:31; SEQ ID NO:4, SEQ ID NO:7 ...15, SEQ ID NO:23, SEQ ID NO:27 and SEQ ID NO:31; or SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:

31.

4. 3. The nucleic acid molecule of claim 2, encoding a guide RNA comprising the sequences of SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 27 and SEQ ID NO:

31.

5. The nucleic acid molecule according to any one of claims 2 to 4, which is a plasmid.

6. The nucleic acid molecule according to any one of claims 2 to 4, which is a single plasmid.

7. the Cas13d protein encoded by said sequence does not contain a nuclear localization signal (NLS), and / or 7. The nucleic acid molecule of any one of claims 2 to 6, wherein the Cas13d protein encoded by the sequence is a fusion protein comprising the N-terminal binding domain (N-NTD) of the nucleocapsid protein of SARS-CoV-2.

8. 8. The nucleic acid molecule of any one of claims 5 to 7, obtained by inserting a spacer sequence of a guide RNA of claim 1 into the plasmid pAAV2-U6-gRNA-CMV-Cas13d of SEQ ID NO: 40, or by inserting at least one spacer sequence of at least one guide RNA of claim 1 into the plasmid pAAV2-U6-gRNA-CMV-Cas13d-array-trigguide of SEQ ID NO: 41, the plasmid pAAV-U6-gRNA-quadguide-CMV-Cas13d-V3-basic of SEQ ID NO: 42, the plasmid pAAV-U6-gRNA-CMV-Cas13d-SapI of SEQ ID NO: 43, or the plasmid pAAV-U6-gRNA-CMV-Cas13d-NTD-AarI of SEQ ID NO:

44.

9. An AAV vector comprising the nucleic acid molecule of any one of claims 2 to 8.

10. is an AAV2 vector or an AAV9 vector, and / or The AAV vector backbone is reduced in size; An AAV vector comprising the nucleic acid molecule of any one of claims 2 to 8.

11. An adenoviral vector comprising the nucleic acid molecule of any one of claims 2 to 8.

12. A pharmaceutical composition comprising an AAV vector according to claim 9 or 10 or an adenoviral vector according to claim 11.

13. A pharmaceutical composition comprising at least one guide RNA according to claim 1 and at least one mRNA encoding a Cas13 protein.

14. The pharmaceutical composition of claim 13, wherein the Cas13 protein is a Cas13d protein or a Cas13a protein and does not contain a non-localization signal (NLS).

15. The pharmaceutical composition of claim 13 or 14, wherein the Cas13d protein encoded by the mRNA is a fusion protein comprising the N-terminal binding domain (N-NTD) of the nucleocapsid protein of SARS-CoV-2.

16. An AAV vector according to claim 9 or 10, an adenoviral vector according to claim 11, or a pharmaceutical composition according to any one of claims 12 to 15, for use in treating a disease or syndrome caused by a human-associated virus.

17. 17. The AAV vector, adenoviral vector, or pharmaceutical composition of claim 16, wherein the disease or syndrome is the result of infection with a coronavirus genetically related to the group consisting of MERS-CoV, SARS-CoV, and SARS-CoV-2.

18. The AAV vector, adenovirus vector, or pharmaceutical composition of claim 17, wherein the disease is COVID-19.

19. The AAV vector, adenovirus vector, or pharmaceutical composition according to any one of claims 16 to 18, wherein the human-associated virus is cleaved by expression of Cas13.

20. The AAV vector, adenoviral vector, or pharmaceutical composition according to any one of claims 16 to 19, wherein the AAV vector, adenoviral vector, or pharmaceutical composition is administered in an aerosol composition via the upper respiratory tract.

21. The AAV vector, adenovirus vector, or pharmaceutical composition according to any one of claims 16 to 19, wherein the AAV vector or pharmaceutical composition is administered intranasally or intratracheally, or in an aerosol composition by inhaler or nebulizer.

22. The AAV vector, adenovirus vector or pharmaceutical composition according to any one of claims 16 to 21, wherein the AAV vector or pharmaceutical composition is administered by mechanical ventilation.

23. The AAV vector, adenoviral vector or pharmaceutical composition according to any one of claims 16 to 22, wherein the AAV vector, adenoviral vector or pharmaceutical composition is administered to the myocardium.

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