Modified proteins

Abstract

Disclosed are methods of achieving viral resistance in animals and modified protein sequences which exhibit reduced polymerase activity.

Description

[0001] MODIFIED PROTEINS FIELD The present disclosure provides modified proteins with antiviral properties. The disclosure further provides sequences of (and encoding) three proteins and animals genetically modified to express the same. BACKGROUND The ANP32 family of host proteins (including members: ANP32A, ANP32B and ANP32E) are exploited by influenza A viruses (IAV) in order to replicate their genome in host cells. In avian species (for example chickens), avian influenza A viruses (AIV) rely only on the ANP32A protein and they replicate poorly in human cells as their polymerases cannot efficiently use the mammalian ANP32 proteins. However, AIVs may acquire mammalian adapting mutation such as E627K in the PB2 subunit protein of the viral polymerase; this enables the polymerase to use the mammalian ANP32A and ANP32B proteins. There is a need for further strategies to combat IAV and to provide methods which render avian species resistant to infection. SUMMARY During infection, the polymerases of influenza A viruses (IAV) co-opt host acidic nuclear phosphoproteins (ANP) 32 (ANP32) to facilitate the replication of their genome in host cells. The ANP32 family of proteins contain three members; ANP32A, ANP32B and ANP32E. In mammals, IAV relies redundantly on ANP32A and ANP32B for replication. In avian species, for example chickens, IAV relies only on the ANP32A protein (ANP32B is inactive). Avian (or chicken) ANP32A differs from mammalian (e.g. human) ANP32A in that human ANP32A is shorter. The shorter mammalian ANP32 protein does not efficiently support avian influenza polymerase and this accounts for a host range restriction that limits the infection of humans when exposed to infected birds. As such Avian influenza viruses (AIVs) are adapted to using avian ANP32A and replicate poorly in human cells. However, AIVs can acquire mutations, usually in polymerase PB2 or PA subunits, that adapt polymerase to using mammalian ANP32 proteins or they acquire the relevant genomic segments from a mammalian-adapted virus by a process of viral reassortment. By way of example, AIVs may acquire a E627K mutation (in the PB2 subunit protein of the viral

[0002] 1 55372346-1 polymerase); this renders the polymerase of the AIV able to use mammalian ANP32A and ANP32B proteins. These mechanisms are a prelude for the emergence of influenza strains with pandemic potential. It should be noted that the terms “comprise”, “comprising” and / or “comprises” is / are used to de-note that aspects and embodiments of this invention “comprise” a particular feature or features. It should be understood that this / these terms may also encompass aspects and / or embodiments which “consist essentially of” or “consist of” the relevant feature or features. The present disclosure provides novel and advantageous strategies for establishing resistance to AIV infection in avian species, thereby limiting the risk of the emergence of IAV strains which are capable of infecting mammals (including humans) and which may have pandemic potential. This disclosure is based on the finding that in avian species, for example chickens, if ANP32A is rendered unusable by IAV polymerase (e.g. through mutation), both ANP32E and ANP32B can become proviral; in other words, in the absence of ANP32A, ANP32E and / or ANP32B can be exploited by IAV polymerase to facilitate replication in a host cell. Taking account of this, the disclosure provides modified ANP32 proteins, including modified ANP32E, ANP32B and ANP32A proteins, which modified proteins are anti-viral and cannot be used by IAV polymerase. Accordingly, this disclosure provides modified ANP sequences, which sequences can establish IAV resistance in a host. It should be noted that the term ‘ANP32’ embraces both avian (including chicken) and mammalian (including human) forms of the various ANP32 proteins. Accordingly the term ANP32 embraces the ANP32E, ANP32B and ANP32A family members and the human and / or chicken forms of each. The term “modified” ANP32 sequence embraces ANP32 sequences which contain one or more mutations relative to a reference sequence. The term ‘sequence’ may embrace a nucleic acid or peptide / protein sequence. For example, the term ‘ANP32 sequence’ may embrace a nucleic acid sequence which encodes or provides an ANP32 and / or a peptide sequence which provides an ANP32. A “reference sequence” may be any wild type ANP32 sequence. For example, a reference sequence may comprise, consist essentially of or consist of a wild type ANP32 nucleic acid sequence or the amino acid / peptide sequence of a wild type ANP32. In one teaching, the ‘reference sequence’ may comprise a wild type (avian / chicken or mammalian / human)

[0003] 2 55372346-1 ANP32E, ANP32A or ANP32B nucleic acid sequence or an amino acid / peptide sequence. It should be appreciated that ANP32 sequences which are similar or homologous to the specific ANP32 sequences described herein are to be encompassed within the scope of the term ‘ANP32’ and / or as ‘ANP32 reference sequences’. Accordingly, a modified ANP32 sequence may be derived from a specific or particular wild type ANP32. Moreover, a modified ANP32 sequence may comprise a wild type ANP32 sequence modified to include or comprise one or more mutations. The one or more mutations may be functional – that is to say they may individually (and / or independently) or collectively (for example, synergistically) modulate (alter, improve or suppress / inhibit) one or more of the physiological, biological immunological and / or pharmacological properties characteristic of a wild type ANP32 (for example the wild type ANP32 from which the modified ANP32 is derived). In particular, the one or more mutations may: (i) alter or inhibit / suppress a function of the ANP32; and / or (ii) inhibit, supress or prevent transcription and / or replication of the IAV (or AIV) genome in a host cell; and / or (iii) provide a modified ANP32 which is unable to form a symmetrical or an asymmetric dimer comprising two or more heterotrimeric IAV polymerase molecules. A mutation may not alter or ablate the wild type, host molecular functions of the ANP32 proteins. As such, a modified ANP32 protein of this disclosure may retain important wild- type, host function(s). By way of example, a modified ANP32 protein of this disclosure may retain its role in transcriptional modulation and the ability to interact with histones H3 and H4 and / or H2A.Z. A “mutation” may include any alteration of the wild-type ANP32 sequence. For example, the term “mutation” may embrace, for example: (i) one or more amino acid substitution(s) (where one or more of the wild type nucleic acid bases (nucleobase) or amino acid(s) is / are swapped or changed for another (different) nucleic acid base / amino acid – the term “substitutions” would include conservative amino acid substitutions); and / or (ii) one or more nucleobase or amino acid deletion(s) (where one or more of the wild type nucleobase(s) or amino acid residue(s) are removed); and / or

[0004] 3 55372346-1 (iii) one or more nucleobase / amino acid addition(s) / insertion(s) (where additional nucleobase(s) or amino acid residue(s) are added to a wild type (or reference) sequence); and / or (iv) one or more nucleobase / amino acid / sequence inversions (usually where two or more consecutive nucleobases or amino acids in a sequence are reversed); and / or (v) one or more nucleobase or amino acid / sequence duplications (where a nucleobase or amino acid sequence (for example a stretch of 2-10 nucleobases / amino acids) are repeated). As stated, a modified ANP32 according to this disclosure may comprise, relative to a reference ANP32 sequence, any one or more of the mutations described herein. An exemplary wild type ANP32E (in other words a reference ANP32E sequence from which a modified ANP32E may be derived) has been deposited under accession number Q5F4A3 and is reproduced below as SEQ ID NO:1. MEMKKRINLELRNQAPEEVTELVLDNCKSSNGEIEGLNDSFKELEFLSMANVQLTSLAKLPTLSKLRK LELSDNIISGGLEVLAERCPNLTYLNLSGNKIKDLGTVEALQNLKNLKSLDLFNCEITNLEDYRDSIF DLLQQITYLDGFDQEDNEAPDSEDDDDEGDEDDNDEDEDEAGPPGEYEEEDDEDDGGSDLGEGEEEEE VGLSYLMKEEIQDEDDDDDYVEEGGDEEEEAEGIRGEKRKRDPEDEGEEEDD An exemplary wild type ANP32E nucleic acid sequence may be any which encodes SEQ ID NO: 1. An exemplary wild type ANP32B (in other words a reference ANP32B sequence from which a modified ANP32B may be derived) has been deposited under accession number Q5ZMN0 and is reproduced below as SEQ ID NO: 2. MEMKKRLTLELRNKKPGEVKELVLDNCRSDDGKIVGLSSDFENLEFLSMINVNLLSISNLPKLNKLRK LELSDNRISGGLEVLAERTPNLTHLNLSGNKIKDINTLEPLKKLPNLHSLDLFNCEVTMLINYRESVF TLLPQLTYLDGFDADEQEAPDSDPEADGDGLEDEYENGEGEEEEDDDEEDDLDEEVIDEEDDEDDDLE GEEEEDGVDDEEEDEEEDGEDEEDDEADDDLPRGEKRKRNLEDEGEEDPEDEEDDEDD An exemplary wild type ANP32B nucleic acid sequence may be any which encodes SEQ ID NO: 2.

[0005] 4 55372346-1 An exemplary wild type ANP32A (in other words a reference ANP32A sequence from which a modified ANP32A may be derived) has been deposited under accession number XP_040536190 and is reproduced below as SEQ ID NO: 3. MDMKKRIHLELRNRTPSDVKELVLDNCRSYEGKIEGLTDEFEELEFLSTINVGLASVANLPKLNKLKK LELSDNRVSGGLEVLAEKCPNLTHLNLSGNKIKDLGTIEPLKKLENLKSLDLFNCEVTNLNDYRENVF KLLPQLTYLDGYDRDDKEAPDSDAEGYVEGLDDEEEDEDVLSLVKDRDDKEAPDSDAEGYVEGLDDEE EDEDEEEYDDDAQVVEDEEDEEEEEEGEEEDVSGEEEEDEEGYNDGDVDDDEDEEEPDEERGQKRKRE PEDEGDEDD An exemplary wild type ANP32A nucleic acid sequence may be any which encodes SEQ ID NO: 3. Thus, this disclosure provides ANP32 proteins which comprise modified versions of SEQ ID NOS: 1-3. A modified version of any of SEQ ID NOS: 1-3 may be modified by inclusion of any one of the mutations described herein - the mutations being, for example, nucleobase / amino acid substitutions, additions / insertions, duplications, deletions and / or inversions made relative to any of SEQ ID NOS: 1-3. In one teaching, the disclosure provides a modified ANP32E protein. A modified ANP32E protein may comprise a sequence which, relative to a reference ANP32E (nucleic or amino acid) sequence, comprises one or more nucleic acid or amino acid mutations as described herein. In one teaching, the disclosure provides a modified ANP32E protein comprising a modified version of SEQ ID NO: 1. A modified version of SEQ ID NO: 1 may be modified by inclusion of any one of the mutations described herein - the mutations being, for example, nucleobase / amino acid substitutions, additions / insertions, duplications, deletions and / or inversions made relative to the sequence of SEQ ID NO: 1. As stated, the modified ANP32E proteins of this disclosure exhibit reduced IAV polymerase activity. It should be noted that any reference to ‘reduced IAV polymerase activity’ refers to a reduction as compared to the IAV polymerase activity of a wild-type ANP32 protein, for example a wild-type ANP32E protein, a wild-type ANP32B protein or a wild-type ANP32A protein. While the precise function or functions of the ANP32 proteins (e.g. ANP32E) in viral replication may not be fully known or understood, the present inventors have shown that ANP32E (and ANP32B) can be exploited by IAV polymerase to facilitate replication in a cell. Accordingly, any reduction in ‘IAV polymerase activity’ may manifest as a reduction in the ability of IAV to replicate in a cell. As such, assays which monitor viral replication in host

[0006] 5 55372346-1 cells (namely a ‘viral replication assay’ or ‘minireplicon assay’) can be used to assess the effect of modifications made to ANP32 proteins (including ANP32E) on viral replication. Useful modified ANP32 proteins, including useful modified ANP32E proteins with reduced IAV polymerase activity, are therefore obtainable by assays of this type. Such assays provide a convenient way of assessing whether or not a modified ANP32 protein (e.g. a modified ANP32E protein of this disclosure) has reduced polymerase activity as any reduction in viral replication (as compared to the level of replication in a host cell having wild- type ANP32 proteins) is an indication that the modified ANP32 protein must have reduced polymerase activity. In one teaching, an assay for determining whether or not a test ANP32 protein (e.g. a modified ANP32E or modified ANP32B protein of this disclosure) exhibits reduced IAV polymerase activity may comprise providing a cell which expresses a modified ANP32 protein, infecting the cell with IAV and determining a level of IAV replication, wherein a reduction in the level of IAV replication indicates that the modified ANP32 protein has reduced IAV polymerase activity. Such an assay may further include a step in which the level of viral replication is compared to the level of viral replication detected in an assay in which the cell comprises wild-type (and not modified) ANP32 protein(s). In one teaching a useful replication assay may exploit cells transfected with an IAV polymerase (e.g. Tky05 PB1577E, PA 556R, PB2) and nucleo protein (NP) and a viral reporter plasmid (for example pPolI-firefly luciferase). A system with these components may then be contacted with a modified ANP32 protein of this disclosure (e.g. a modified ANP32E protein and / or a modified ANP32B protein) and the level of signal from the reporter plasmid used to determine the level of ANP32 polymerase activity. One of skill will appreciate that the lower the signal from the reporter system, the lower the polymerase activity of the modified ANP32 protein. Moreover, the strength of any signal may be compared to the strength of a signal obtained from an assay which comprises the use of a wild-type ANP32 protein. An example of a suitable assay which may be used to obtain modified ANP32 proteins of this disclosure (or by which modified ANP32 proteins (e.g. modified ANP32E proteins) may be obtainable) is described in Aartjan J. W. te Velthuis et al.2018 (Methods in Molecular Biology: Assays to Measure the Activity of Influenza Virus Polymerase: Volume 1836) and Sheppard et al.2023 (Nature Communications: An Influenza A virus can evolve to use human ANP32E through altering polymerase dimerization: 14: article number 6135) – the contents of which are incorporated herein by reference. A modified ANP32E may comprise a sequence in which a residue of one or more of the leucine-rich repeat (LRR) regions (e.g. any one of the regions denoted LRR1, LRR2, LRR3, LRR4 and / or LRR5), has been mutated. In one teaching, a modified ANP32E may comprise (relative to a reference sequence of this disclosure) a modified LRR5 domain. The term ‘modified LRR5 domain’ may comprise an

[0007] 6 55372346-1 LRR5 domain which has been modified by the inclusion of any one or more of the mutations described herein. By way of example, a modified ANP32E may comprise or be substituted for a sequence derived from an ANP32B LRR5 domain. In one teaching, the disclosure provides a modified ANP32E protein in which either or both of residues 129 and 130 has / have been mutated. By way of example, a modified ANP32E protein according to this disclosure may comprise the following substitutions E129I and / or D130N. A modified ANP32E may comprise a sequence in which the wild type LRR5 domain comprises one or more mutations. For example, a modified ANP32E may comprise a sequence in which residues of the wild type LRR5 domain have been replaced with (or substituted for) residues from the LRR5 domain of chicken ANP32B (which domain spans residues 115-141 (aka: chANP32B115 – 141). For example, any one or more of residues 125, 127, 129, 130, 133, 135, 137 and / or 140, may be mutated. A mutation at any of these residues may comprise substitution with the corresponding residue from (wild-type) ANP32B (e.g. the corresponding residue from ANP32B reference SEQ ID NO: 2). A modified ANP32E may comprise the ANP32B LRR5 domain (e.g. the region of the ANP32B protein spanning residues 115-141. A schematic representing an example of a modified ANP32E of this type is given in Figure 7 and is named “chANP32E with chANP32B115-141”. A modified ANP32E may comprise or further comprise a modified central region. By way of example any one or more of residues 141-175 may (relative to the reference sequence of SEQ ID NO: 1) be mutated. A modified ANP32E may comprise or further comprise a modified central region. By way of example any one or more of residues 141-175 may (relative to the reference sequence of SEQ ID NO: 1) be mutated. In one teaching, a modified ANP32E may comprise residues derived from ANP32B, including, for example or all or part of a central region derived from ANP32B. By way of example, any or all of residues 142, 150, 151, 152, 153, 159, 160, 161, 162, 164, 165, 166, 167, 168, 170, 171, 173, 174 and / or 175 may (relative to a ANP32E reference sequence) be mutated; for example, any one of these residues may be modified by substitution with a residue or residues derived from (wild-type) ANP32B (e.g. the corresponding residue from ANP32B reference SEQ ID NO: 3). A modified ANP32E may comprise a central region (residues 142-175) which has been replaced with residues 142- 175 of ANP32B (aka “chANP32B142-175”). A schematic representing an example of a modified ANP32E of this type is given in Figure 7 and is named “chANP32E with chANP32B142-175”. A modified ANP32E may comprise mutations at any residues between residues 115 and 175. For example residues 115-175 of wild type ANP32E (as shown in SEQ ID NO: 1) may

[0008] 7 55372346-1 be replaced with one or more (for example all) of the residues from the corresponding region of ANP32B (aka: “chANP32B115-175”). A schematic representing an example of a modified ANP32E of this type is given in Figure 7 and is named “chANP32E with chANP32B115-175”. A modified ANP32E may comprise a central region (residues 142-175) which has been replaced with residues 142-175 of ANP32B (aka “chANP32B142-175”). A schematic representing this modified ANP32E is given in Figure 7 and is named “chANP32E with chANP32B142-175”. A modified ANP32E may comprise mutations at any one or more of the following residues: 6, 12, 25, 45, 70, 73, 92, 93, 94, 96, 116, 119, 125, 127, 129, 130, 133, 135, 136, 137, 138, 139, 140, 142, 149, 150, 151, 152, 153, 154, 155, 156, 157, 159, 160, 161, 162, 164, 165, 166, 167, 168, 170, 171, 173, 174 and / or 175. Mutations at one or more of these residues may result in an ANP32E molecule which exhibits reduced IAV polymerase activity. A modified ANP32E may comprise mutations at any one or more of the following residues 6, 12, 25, 45, 70, 73, 119, 127, 129, 130, 135, 136, 138, 139, 149, 151, 152, 153, 154, 155, 156, 157, 159. Mutations at one or more of these residues may result in an ANP32E molecule which exhibits reduced IAV polymerase activity. A modified ANP32E may comprise any one or more of the following mutations: R6E; R12E D25A; D25K; E45A; E70A; D73A; D119A; N127M; E129I; D130N; I135G; F136G; L138G; L139G; D149Y; E151A; E151K; D152H; N153A; E154A; E154K; A155K; P156R; P156K; D157A; D157K; and E159K. Each of these mutations independently or in combination with others, results in an ANP32E molecule which exhibits reduced IAV polymerase activity. A modified ANP32E may comprise any one or more of the following mutations: R6E; R12E; D25A; D25K; E151A; E151K; D152H; N153A; E154A; E154K; A155K; P156R; P156K; D157A; D157K; and E159K. Each of these mutations independently or in combination with others, results in an ANP32E molecule which exhibits reduced IAV polymerase activity. A modified ANP32E may comprise mutations at any one or more of the residues 141-170. By way of example, a modified ANP32E may comprise any one or more of the following combinations of mutations: (i) K116H, I125V, N127M, E129I, D130N, D133E, I135V, D137T, Q140P; (ii) R6E, R12E, D25K; (iii) D25A, E45A, E70A, D73A, D119A; (iv) D25A, E45A, E70A, D73A, D119A; (v) D25A, N127M, E129I, D130N; (vi) D25K, N127M, E129I, D130N; (vii) D25K, N127M, E129I,

[0009] 8 55372346-1 D130N, D149Y; (viii) N127M, E129I, D130N; (ix) N127M, E129I, D130N, D149Y; (x) I135G, F136G, L138G, L139G; (xi) D149Y, D152H; (xii) one or more LRR4 substitutions; and (xiii) Y92A, L93A, N94A, S96A. ANP32E proteins modified (relative to SEQ ID NO: 1) to comprise any of these mutation combinations, exhibit reduced IAV polymerase activity. In view of the above, a modified ANP32E protein may comprise, relative to an ANP32E reference sequence, a mutation at any one or more of the following residues: (i) residue 6; (ii) residue 12; (iii) residue 25; (iv) residue 45; (v) residue 70; (vi) residue 73; (vii) residue 92; (viii) residue 93; (ix) residue 94; (x) residue 96; (xi) residue 116; (xii) residue 119; (xiii) residue 125; (xiv) residue 127; (xv) residue 129; (xvi) residue 130; (xvii) residue 133; (xviii) residue 135; (xix) residue 137; (xx) residue 140; (xxi) residue 142; (xxii) residue 150; (xxiii) residue 151; (xxiv) residue 152; (xxv) residue 153; (xxvi) residue 159; (xxvii) residue 160; (xxviii) residue 161; (xxix) residue 162; (xxx) residue 164; (xxxi) residue 165; (xxxii) residue 166; (xxxiii) residue 167; (xxxiv) residue 168; (xxxv) residue 170; (xxxvi) residue 171; (xxxvii) residue 173; (xxxviii) residue 174 and / or (xxxix) residue 175. By way of example, a modified ANP32E protein may comprise, relative to an ANP32E reference sequence, any one or more of the following mutations:(i) R6E; (ii) R12E; (iii) D25K; (iv) D25A; (v) E45A;(vi) E70A; (vii) D73A; (viii) Y92A; (ix) L93A; (x) S96A; (xi) N94A; and (xii) D119A. A modified ANP32E may comprise any one or more mutations selected from the group consisting of R6E; R12E; D25A; D25K; E151A; E151K; D152H; N153A; E154A; E154K; A155K; P156R; P156K; D157A; D157K and D159K. ANP32E proteins with one or more of these mutations exhibit reduced IAV polymerase activity (as evidenced by compromised or reduced polymerase activity in a viral replication or minireplicon assay). A modified ANP32E may comprise any one or more mutations selected from the group consisting of R6E; R12E; D25K; E151A; E151K; N153A; E154A; E154K; A155K; P156R; P156K; D157A; D157K; and D159K. ANP32E proteins with one or more of these mutations exhibit reduced IAV polymerase activity (as evidenced by compromised or reduced polymerase activity in a viral replication or minireplicon assay). A modified ANP32E may comprise any one or more mutations selected from the group consisting of R6E; R12E; D25K; E151A; E151K; N153A; E154A; E154K; A155K; P156R; D157A; D157K; and D159K. ANP32E proteins with one or more of these mutations exhibit reduced IAV polymerase activity (as evidenced by compromised or reduced polymerase activity in a viral replication or minireplicon assay).

[0010] 9 55372346-1 A modified ANP32E may comprise any one or more mutations selected from the group consisting of R6E; R12E; D25K; E151A; E151K; E154A; E154K; A155K; D157A; D157K; and D159K. ANP32E proteins with one or more of these mutations exhibit reduced IAV polymerase activity (as evidenced by compromised or reduced polymerase activity in a viral replication or minireplicon assay). A modified ANP32E may comprise one or more combinations of mutations selected from the group consisting of: (i) D25A, E45A, E70A, D73A, D119A; (ii) D25A, N127M, E129I, D130N; (iii) D25K, N127M, E129I, D130N; (iv) D25K, N127M, E129I, D130N, D149Y; (v) N127M, E129I, D130N; (vi) N127M, E129I, D130N, D149Y; (vii) I135G, F136G, L138G, L139G; and (viii) D149Y, D152H. A modified ANP32E may comprise the following mutations (i) D25K; (ii) D25A, E45A, E70A, D73A, D119A; (iii) D25K, N127M, E129I, D130N; (iv) D25K, N127M, E129I, D130N; D149Y (iv) N127M, E129I, D130N; (v) N127M, E129I, D130N, D149Y and (vi) D149Y, D152HA modified ANP32E may comprise the following sequence (SEQ ID NO: 4): MEMKKEINLELENQAPEEVTELVLK / ANCKSSNGEIEGLNDSFKELAFLSMANVQLTSLAKLPTLSKL RKLALSANIISGGLEVLAERCPNLTAAALAGNKIKDLGTVEALQNLKNLHSLALFNCEVTMLINYRES VFTLLPQITYLDGFDQEDNEAPDSEDDDDEGDEDDNDEDEDEAGPPGEYEEEDDEDDGGSDLGEGEEE EEVGLSYLMKEEIQDEDDDDDYVEEGGDEEEEAEGIRGEKRKRDPEDEGEEEDD A modified ANP32E may comprise the following sequence (SEQ ID NO: 5): MEMKKRINLELRNQAPEEVTELVLKNCKSSNGEIEGLNDSFKELEFLSMANVQLTSLAKLPTLSKLRK LELSDNIISGGLEVLAERCPNLTYLNLSGNKIKDLGTVEALQNLKNLKSLDLFNCEITNLEDYRDSIF DLLQQITYLDGFDQEDNEAPDSEDDDDEGDEDDNDEDEDEAGPPGEYEEEDDEDDGGSDLGEGEEEEE VGLSYLMKEEIQDEDDDDDYVEEGGDEEEEAEGIRGEKRKRDPEDEGEEEDD A modified ANP32E may comprise the following sequence (SEQ ID NO: 6): MEMKKRINLELRNQAPEEVTELVLANCKSSNGEIEGLNDSFKELAFLSMANVQLTSLAKLPTLSKLRK LALSANIISGGLEVLAERCPNLTYLNLSGNKIKDLGTVEALQNLKNLKSLALFNCEITNLEDYRDSIF DLLQQITYLDGFDQEDNEAPDSEDDDDEGDEDDNDEDEDEAGPPGEYEEEDDEDDGGSDLGEGEEEEE VGLSYLMKEEIQDEDDDDDYVEEGGDEEEEAEGIRGEKRKRDPEDEGEEEDD A modified ANP32E may comprise the following sequence (SEQ ID NO: 7):

[0011] 10 55372346-1 MEMKKRINLELRNQAPEEVTELVLKNCKSSNGEIEGLNDSFKELEFLSMANVQLTSLAKLPTLSKLRK LELSDNIISGGLEVLAERCPNLTYLNLSGNKIKDLGTVEALQNLKNLKSLDLFNCEITMLINYRDSIF DLLQQITYLDGFDQEDNEAPDSEDDDDEGDEDDNDEDEDEAGPPGEYEEEDDEDDGGSDLGEGEEEEE VGLSYLMKEEIQDEDDDDDYVEEGGDEEEEAEGIRGEKRKRDPEDEGEEEDD A modified ANP32E may comprise the following sequence (SEQ ID NO: 8): MEMKKRINLELRNQAPEEVTELVLKNCKSSNGEIEGLNDSFKELEFLSMANVQLTSLAKLPTLSKLRK LELSDNIISGGLEVLAERCPNLTYLNLSGNKIKDLGTVEALQNLKNLKSLDLFNCEITMLINYRDSIF DLLQQITYLDGFYQEDNEAPDSEDDDDEGDEDDNDEDEDEAGPPGEYEEEDDEDDGGSDLGEGEEEEE VGLSYLMKEEIQDEDDDDDYVEEGGDEEEEAEGIRGEKRKRDPEDEGEEEDD A modified ANP32E may comprise the following sequence (SEQ ID NO: 9): MEMKKRINLELRNQAPEEVTELVLDNCKSSNGEIEGLNDSFKELEFLSMANVQLTSLAKLPTLSKLRK LELSDNIISGGLEVLAERCPNLTYLNLSGNKIKDLGTVEALQNLKNLKSLDLFNCEITMLINYRDSIF DLLQQITYLDGFDQEDNEAPDSEDDDDEGDEDDNDEDEDEAGPPGEYEEEDDEDDGGSDLGEGEEEEE VGLSYLMKEEIQDEDDDDDYVEEGGDEEEEAEGIRGEKRKRDPEDEGEEEDD A modified ANP32E may comprise the following sequence (SEQ ID NO: 10): MEMKKRINLELRNQAPEEVTELVLDNCKSSNGEIEGLNDSFKELEFLSMANVQLTSLAKLPTLSKLRK LELSDNIISGGLEVLAERCPNLTYLNLSGNKIKDLGTVEALQNLKNLKSLDLFNCEITMLINYRDSIF DLLQQITYLDGFYQEDNEAPDSEDDDDEGDEDDNDEDEDEAGPPGEYEEEDDEDDGGSDLGEGEEEEE VGLSYLMKEEIQDEDDDDDYVEEGGDEEEEAEGIRGEKRKRDPEDEGEEEDD A modified ANP32E may comprise the following sequence (SEQ ID NO: 11): MEMKKRINLELRNQAPEEVTELVLDNCKSSNGEIEGLNDSFKELEFLSMANVQLTSLAKLPTLSKLRK LELSDNIISGGLEVLAERCPNLTYLNLSGNKIKDLGTVEALQNLKNLKSLDLFNCEITNLEDYRDSIF DLLQQITYLDGFYQEHNEAPDSEDDDDEGDEDDNDEDEDEAGPPGEYEEEDDEDDGGSDLGEGEEEEE VGLSYLMKEEIQDEDDDDDYVEEGGDEEEEAEGIRGEKRKRDPEDEGEEEDD ANP32E proteins comprising sequences corresponding to any of SEQ ID NOS: 5-11 above are representative of those modified ANP32Es which exhibit reduced viral polymerase activity (or are unable to support polymerase activity). Such modified proteins are useful as

[0012] 11 55372346-1 they inhibit, supress or prevent transcription and / or replication of the IAV (or AIV) genome in a host (e.g. avian) cell (because the introduced modifications / mutations ensure that the modified ANP32E is unable to support avian influenza polymerase). In one teaching, the disclosure provides a modified ANP32B protein. A modified ANP32B protein may comprise a sequence which, relative to a reference ANP32B (nucleic acid or amino acid) sequence, comprises one or more nucleic acid or amino acid mutations, including any of those described herein. In one teaching, the disclosure provides a modified ANP32B protein comprising a modified version of SEQ ID NO: 2. A modified version of SEQ ID NO: 2 may be modified by inclusion of any one of the mutations described herein - the mutations being, for example, nucleobase / amino acid substitutions, additions / insertions, duplications, deletions and / or inversions made relative to the sequence of SEQ ID NO: 2. A modified ANP32B may comprise a sequence in which a residue of one or more of the leucine-rich repeat (LRR) regions (e.g. any one of the regions denoted LRR1, LRR2, LRR3, LRR4 and / or LRR5), has been mutated. In one teaching, a modified ANP32B may comprise (relative to a reference sequence of this disclosure) a modified central domain. A modified central domain may comprise mutations at any of ANP32B residues 141-175. The term ‘modified central domain’ may comprise a central domain which has been modified by the inclusion of any one or more of the mutations described herein. By way of example, a modified ANP32B may comprise nucleobases and / or amino acid residues derived from ANP32E. By way of example, a sequence encoding a modified ANP32B protein of this disclosure may comprise a sequence encoding a central region which comprises one or more amino acid residues derived from a sequence encoding a ANP32E central domain. Additionally or alternatively, the disclosure provides a modified ANP32B protein comprising a central region comprising amino acid residues derived from an ANP32E central domain. A modified ANP32B may comprise mutations at any one or more of the following residues: 6, 12, 25, 45, 70, 73, 92, 93, 94, 96, 119, 135, 136, 138, 139, 142, 144, 149, 150, 151, 152, 153, 154, 155, 156, 157, 159, 160, 161, 162, 164, 165 and / or 166. Mutations at one or more of these residues may result in an ANP32B molecule which exhibits reduced IAV polymerase activity. A modified ANP32B may comprise mutations at any one or more of the following residues: 6, 12, 25, 70, 142, 144, 150, 151, 154, 155, 156 and / or 157. Mutations at one or more of these residues may result in an ANP32B molecule which exhibits reduced IAV polymerase activity.

[0013] 12 55372346-1 A modified ANP32B may comprise any one or more of the following mutations: R6E; R12E; D25K; E70K; L142I; Y144G; A150R; D151K; E154A; A155E; P156K and D157K. Each of these mutations independently (or in combination with others) result in an ANP32B molecule which exhibits reduced IAV polymerase activity. At least some of these residues may be substituted for the corresponding residue present in ANP32E. It has been shown that mutations at one or more of these residues reduce or eliminate viral polymerase activity. By way of example, a modified ANP32B may comprise any one or more of the following mutations: (i) R6E, R12E, D25K; (ii) D25A, E45A, E70A, D73A, D119A; (iii) one or more LRR4 substitutions; (iv) H92A, L93A, N94A, S96A; (v) D25A, E45A, E70A, D73A, D119A; (vi) D25K, D151K; (vii) V135G, F136G, L138G, L139G; and (viii) D149Y E152H. In view of the above, a modified ANP32B protein may comprise, relative to an ANP32B reference sequence, a mutation at any one or more of the following residues: (i) residue 6; (ii) residue 12; (iii) residue 25; (iv) residue 45; (v) residue 70; (vi) residue 73; (vii) residue 92; (viii) residue 93; (ix) residue 94; (x) residue 96; (xi) residue 119; (xii) residue 142; (xiii) residue 150; (xiv) residue 151; (xv) residue 153; (xvi) residue 159; (xvii) residue 160; (xviii) residue 161; (xix) residue 162; (xx) residue 164; (xxi) residue 165; and (xxii) residue 166. By way of example, a modified ANP32B protein may comprise, relative to an ANP32B reference sequence, any one or more of the following mutations: (i) R6E; (ii) R12E; (iii) D25K; (iv) D25A; (v) E45A; (vi) E70A; (vii) D73A; (viii) H92A; (ix) L93A; (x) N94A; (xi) S96A; and (xii) D119A. A modified ANP32B may comprise any one or more mutations selected from the group consisting of R6E; R12E; D25K; Y144G; A150R; D151K; E154A; A155E; P156K and D157K. Mutations at one or more of these residues may result in an ANP32B molecule which exhibits reduced IAV polymerase activity. ANP32B proteins with one or more of these mutations exhibit reduced IAV polymerase activity (as evidenced by compromised or reduced polymerase activity in a viral replication or minireplicon assay). A modified ANP32B may comprise one or more combinations of mutations selected from the group consisting of: (i) D25A, E45A, E70A, D73A, D119A; (ii) D25K, D151K (iii) V135G; F136G; L138G; L139G; and (iv) D149Y, E152H. ANP32B proteins with any of these mutational combinations exhibit reduced IAV polymerase activity. ANP32B proteins with one or more of these mutations exhibit reduced IAV polymerase activity (as evidenced by compromised or reduced polymerase activity in a viral replication or minireplicon assay).

[0014] 13 55372346-1 A modified ANP32B may comprise the following mutations (i) D151K; and (ii) V135G, F136G, L138G, L139G. A modified ANP32B may comprise the following sequence (SEQ ID NO: 12): MEMKKELTLELENKKPGEVKELVLK / ANCRSDDGKIVGLSSDFENLAFLSMINVNLLSISNLPKLNKL RKLALSANRISGGLEVLAERTPNLTAAALAGNKIKDINTLEPLKKLPNLHSLALFNCEVTMLINYRES VFTLLPQITYLDGFDQEENEAPDSEDDDDEGDLEDEYENGEGEEEEDDDEEDDLDEEVIDEEDDEDDD LEGEEEEDGVDDEEEDEEEDGEDEEDDEADDDLPRGEKRKRNLEDEGEEDPEDEEDDEDD A modified ANP32E may comprise the following sequence (SEQ ID NO: 13): MEMKKRLTLELRNKKPGEVKELVLDNCRSDDGKIVGLSSDFENLEFLSMINVNLLSISNLPKLNKLRK LELSDNRISGGLEVLAERTPNLTHLNLSGNKIKDINTLEPLKKLPNLHSLDLFNCEVTMLINYRESVF TLLPQLTYLDGFDAKEQEAPDSDPEADGDGLEDEYENGEGEEEEDDDEEDDLDEEVIDEEDDEDDDLE GEEEEDGVDDEEEDEEEDGEDEEDDEADDDLPRGEKRKRNLEDEGEEDPEDEEDDEDD A modified ANP32E may comprise the following sequence (SEQ ID NO: 14): MEMKKRLTLELRNKKPGEVKELVLDNCRSDDGKIVGLSSDFENLEFLSMINVNLLSISNLPKLNKLRK LELSDNRISGGLEVLAERTPNLTHLNLSGNKIKDINTLEPLKKLPNLHSLDLFNCEVTMLINYRESGG TGGPQLTYLDGFDADEQEAPDSDPEADGDGLEDEYENGEGEEEEDDDEEDDLDEEVIDEEDDEDDDLE GEEEEDGVDDEEEDEEEDGEDEEDDEADDDLPRGEKRKRNLEDEGEEDPEDEEDDEDD ANP32B proteins comprising sequences corresponding to any of SEQ ID NOS: 13-14 above are representative of those modified ANP32Bs which exhibit reduced viral polymerase activity (or are unable to support polymerase activity). Such modified proteins are useful as inhibit, supress or prevent transcription and / or replication of the IAV (or AIV) genome in a host (e.g. avian) cell (because the introduced modifications / mutations ensure that the modified ANP32B is unable to support avian influenza polymerase). It should be noted that any of the modified ANP32B sequences described herein in, including the sequence of SEQ ID NOS: 12-14 may comprise additional mutations, including, for example, additional mutations at one or more of residues 167, 168, 170, 171, 173, 174 and 175. These mutations may swap a ANP32B wild type residue for another residue and (as stated) the effect of this change may be to (i) alter or inhibit / suppress a function of the ANP32B; and / or (ii) inhibit, supress or prevent transcription and / or replication of the IAV (or

[0015] 14 55372346-1 AIV) genome in a host cell; and / or (iii) provide a modified ANP32B which is unable to form a symmetrical or an asymmetric dimer comprising two or more heterotrimeric IAV polymerase molecules. In one teaching, the disclosure may further provide a modified ANP32A protein. A modified ANP32A protein may comprise a sequence which, relative to a reference ANP32A (nucleic or amino acid) sequence, comprises one or more nucleic acid or amino acid mutations as described herein. In one teaching, the disclosure provides a modified ANP32A protein comprising a modified version of SEQ ID NO: 3. A modified version of SEQ ID NO: 3 may be modified by inclusion of any one of the mutations described herein - the mutations being, for example, nucleobase / amino acid substitutions, additions / insertions, duplications, deletions and / or inversions made relative to the sequence of SEQ ID NO: 3. A modified ANP32A may comprise a sequence in which a residue of one or more of the leucine-rich repeat (LRR) regions (e.g. any one of the regions denoted LRR1, LRR2, LRR3, LRR4 and / or LRR5), has been mutated. In one teaching, a modified ANP32A may comprise (relative to a reference sequence of this disclosure) mutations at any one or more of the following residues: 6, 12, 25, 45, 70, 73, 116, 119, 125, 127, 133, 135, 137, 140. It has been shown that mutations at one or more of these residues reduce or eliminate viral polymerase activity. A modified ANP32A may comprise mutations at any one or more of the following residues: (i) R6E, R12E, D25K; (ii) D25A, E45A, E70A, D73A, D119A; (iii) one or more LRR4 substitutions; and (iv) H92A, L93A, N94A, S96A. In view of the above, a modified ANP32A protein may comprise, relative to an ANP32A reference sequence, a mutation at any one or more of the following residues: (i) residue 6; (ii) residue 12; (iii) residue 25; (iv) residue 45; (v) residue 70; (vi) residue 73; (vii) residue 92; (viii) residue 93; (ix) residue 94; (x) residue 96; and (xi) residue 119. By way of example, a modified ANP32A protein may comprise, relative to an ANP32A reference sequence, any one or more of the following mutations: (i) R6E; (ii) R12E; (iii) D25K; (iv) D25A; (v) E45A; (vi) E70A; (vii) D73A; (viii) H92A; (ix) L93A; (x) S96A; (xi) N94A; and (xii) D119A. A modified ANP32A may comprise the following sequence (SEQ ID NO: 15): MDMKKEIHLELENRTPSDVKELVLK / ANCRSYEGKIEGLTDEFEELAFLSTINVGLASVANLPKLNKL KKLALSANRVSGGLEVLAEKCPNLTAAALAGNKIKDLGTIEPLKKLENLKSLALFNCEATALNDYRAN AFALLAQLTYLDGYDRDDKEAPDSDAEGYVEGLDDEEEDEDVLSLVKDRDDKEAPDSDAEGYVEGLDD

[0016] 15 55372346-1 EEEDEDEEEYDDDAQVVEDEEDEEEEEEGEEEDVSGEEEEDEEGYNDGDVDDDEDEEEPDEERGQKRK REPEDEGDEDD In one teaching the modified ANP32, for example the modified ANP32A, the modified ANP32B or the modified ANP32E is not a protein in which the amino acid at position 129 is substituted with isoleucine I, lysine K, aspartic acid D, valine V, proline P, tryptophan W, histidine H, arginine R, glutamine Q, glycine G, or glutamic acid E, the amino acid at position 130 is substituted with asparagine N, phenylalanine F, lysine K, leucine L, valine V, proline P, isoleucine I, methionine M , tryptophan W, histidine H, arginine R, glutamine Q, or tyrosine Y, the amino acid at position 149 is substituted with alanine A, the amino acid at position 151 is substituted with alanine A, and the amino acids at positions 60 and 63, positions 87, 90, 93 and 95, positions 112, 115 and 118 are substituted with alanine. The disclosure may further provide any of the modified ANP32 proteins described herein or sequences providing or encoding the same, for use in medicine. The disclosure also provides any of the modified ANP32 proteins described herein or sequences providing or encoding the same, for use in the treatment or prevention of an IAV infection and / or a disease or condition caused by the same (e.g. influenza’ of ‘flu’). The disclosure further provides the use of any of the modified ANP32 proteins described herein or sequences providing or encoding the same, in the manufacture of a medicament for treating or preventing an IAV infection and / or a disease or condition caused by the same (e.g. influenza’ of ‘flu’). The disclosure further provides functional fragments, variants and / or derivatives of any of the modified proteins / sequences described herein. For example, a functional fragment (which retains any one or more of the functions / properties described herein) may comprise up to about (x) residues of the various proteins / sequences described herein, wherein x is any number of amino acids in a functional fragment derived from any of the modified ANP32 proteins described herein spanning a range from about 5, 6, 7, 8, 9 or 10 amino acids to n-1 amino acids, wherein n is the total number of amino acids in the relevant ANP32 protein. For example, for ANP32E, n=256; for ANP32A, n=281; and for ANP32B n=262. In another teaching, a functional variant or derivative may comprise a sequence which is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or homologous to all or part (e.g. a functional part) of any of the modified ANP32 sequences / proteins described herein. A functional variant of any of the ANP32 sequences / proteins described herein may comprise one or more modifications / mutations (as described herein) relative to the sequence of a modified ANP32

[0017] 16 55372346-1 sequence / protein of this disclosure. Such functional variants and / or derivatives should retain substantially all of the functional properties of the described modified ANP32 proteins (e.g. histone binding). Also described is a method of treating or preventing an IAV infection, said method comprising administering a modified ANP32 protein according to this invention to a subject in need thereof. The term ‘subject’ may comprise any human or animal (including avian e.g. chicken) subject. The subject may have an IAV infection. The subject may be susceptible or at risk of developing or contracting an IAV infection. The disclosure provides genetically modified or edited animals, which modified animals are resistant to IAV. In one teaching, the disclosure provides avian species, for example chickens, which are resistant to IAV. As stated herein, resistance to IAV is brought about via modulation (e.g. reduction) of the expression of the ANP32 proteins (e.g. ANP32E, ANP32B and ANP32A) by knock out, functional deletion, modulation of the function, activity and / or expression of up / downstream expression controllers (e.g. promoters for the ANP32E / B and / or A genes) and / or by the expression (through genetic editing, exogenous expression or gene modification) of modified ANP32 proteins (e.g. any of the disclosed modified ANP32E proteins, modified ANP32B proteins and / or modified ANP32A proteins). As stated, the modified ANP32 proteins described herein exhibit a reduced polymerase activity and cannot support IAV polymerase thereby effecting the ability of IAV to replicate in a host (e.g. an avian species or chicken) cell. In one teaching, the genetically modified animals (e.g. an avian species or chicken) have been modified or edited to express any one or more of the modified ANP32 proteins provided herein, have been genetically modified to lack functional copies of any one of the ANP32 proteins or have been genetically modified to lack the expression of one or more of the ANP32 proteins (e.g. ANP32E). The genetically modified animal may be created by the hand of man – that is to say, the genetically modified or edited animals may be made by a genetic editing processes and not the result of natural breeding or selection. Nevertheless, the disclosure may extend to a population or avian species, e.g. poultry species or chickens which are obtained or obtainable by breeding a genetically modified avian species of this disclosure. The genetically modified / edited animal may be an avian species. For example, the genetically modified / edited animal may be a poultry species, for example a chicken. The disclosure provides a genetically modified or edited chicken, which modified / edited chicken expresses one or more of the modified ANP32 proteins described herein. The genetically modified chicken may not express any of the wild type (or normal) ANP32 proteins.

[0018] 17 55372346-1 An animal, for example a chicken, engineered, modified or edited to expresses one or more of the modified ANP32 proteins of this disclosure may be resistant to AIV infection (because, without wishing to be bound by theory) the IAV polymerase cannot use the modified ANP proteins to replicate in the host cell. In one teaching, the disclosure provides a genetically modified / edited animal or avian species, for example a bird (e.g. a chicken) modified or edited to: lack a functional ANP32E gene / protein; and / or lack a functional ANP32A gene / protein; and / or lack a functional ANP32B gene / protein. In this context, the term ‘functional’ means that the gene is either knocked out (so that it cannot express the relevant ANP32 protein) or it is altered so that the resulting ANP32 protein does not support viral replication (i.e. it is ‘antiviral’ as described herein – e.g. it inhibits, supresses or prevents transcription and / or replication of the IAV (or AIV) genome in a host cell; and / or provide a modified ANP32 which is unable to form a symmetrical or an asymmetric dimer comprising two heterotrimeric IAV polymerase molecules). It should be noted that the term ‘knocked out’ embraces either direct ablation of ANP32E (or ANP32B / A) expression or knocking-out via some other mechanism – e.g. modulation of an up / downstream expression controller – e.g. a promoter; e.g. an ANP32E or ANP32B / A promoter). Where an ANP32 gene is not knocked out, it may be modified or edited to encode any of the modified ANP32 proteins described herein. In one teaching, the disclosure provides a genetically modified / edited animal, for example an avian species (e.g. a chicken) engineered to express any of the modified ANP32E proteins described herein. To achieve this, a genetically modified / edited animal according to this disclosure may comprise an ANP32E gene which has been modified / edited so as to encode any of the modified ANP32E proteins disclosed herein (which modified ANP32E proteins are antiviral). A genetically modified / edited animal according to this disclosure may be modified / edited by removal (knockout) of the gene encoding ANP32E (an ‘ANP32E knock out (KO) animal’)). A genetically modified / edited animal of this type may comprise or further comprise ANP32A and / or ANP32B genes / proteins which have either been modified / edited to render them non- functional (or antiviral as described herein) and / or knocked out entirely. For example, this disclosure may provide modified animals in which the ANP32E gene / protein has been knocked out, the ANP32A gene / protein has been knocked out and the ANP32B gene has been modified in some way (e.g. by any one or more of the modifications described herein). In an alternative teaching, the disclosure provides genetically modified animals in which the ANP32E gene / protein has been modified as described herein and both of the ANP32A / B genes / proteins have been knocked out.

[0019] 18 55372346-1 In this regard, the disclosure provides a genetically modified / edited avian species – for example a bird (e.g. a chicken) engineered to: express a modified ANP32E protein of this disclosure or to lack a functional ANP32E gene / protein; and / or express a modified ANP32B protein of this disclosure or to lack a functional ANP32B gene / protein; and / or express a modified ANP32A protein of this disclosure or to lack a functional ANP32A gene / protein. In one teaching, the disclosure provides a genetically modified / edited chicken, which modified / edited chicken lacks a functional ANP32A protein (the gene encoding ANP32A in the chicken may have been rendered non-functional (via some loss of function mutation) or fully knocked out) and harbours ANP32B and ANP32E genes which have been modified / edited to encode mutated or modified ANP32B / E proteins which are antiviral (or (i) alter or inhibit / suppress a function of the ANP32B / E; and / or (ii) inhibit, supress or prevent transcription and / or replication of the IAV (or AIV) genome in a host cell; and / or (iii) provide a modified ANP32B / E which is unable to form a symmetrical or an asymmetric dimer comprising two or more heterotrimeric IAV polymerase molecules). In one teaching, the modified ANP32E and / or B proteins may be antiviral (as described herein) but also permissive of any necessary host functions. In one teaching, a genetically modified / edited animal, for example a bird or chicken may comprise: a modified ANP32E gene encoding a modified ANP32E protein according to this disclosure (so that the genetically modified animal expresses an ‘antiviral’ form of ANP32E); and a modified ANP32B gene encoding a modified ANP32B protein according to this disclosure; and a knocked out ANP32A gene (so that the genetically modified animal does not express ANP32A). Genetically modified / edited animals, such as chickens (modified to express a modified ANP32 protein of this invention) may be made using any of the methods described herein (see for example the section below headed “Generation of GE chickens” – which techniques are applicable to many different avian and animal species. By way of example only, standard published techniques permit the reproductive embryonic cells of avian species to be isolated from embryos and propagated in cell culture medium. During the culture period, the reproductive embryonic cells may be genetically modified / edited using site specific nucleases and / or DNA templates to direct sequence specific changes and deletions in the target genetic locus. The modified reproductive embryonic cells are re-introduced into a host avian and the gametes and resulting offspring of this host avian will carry the genetic change in its chromosomes. Standard selective breeding techniques can be used to increase the frequency of this genetic change in the future descendants. These (and / or similar) methods

[0020] 19 55372346-1 may be used to create genetically modified embryos, including avian embryos, which harbour genetic sequences encoding modified ANP32 proteins according to this disclosure. The disclosure provides nucleic acids encoding any of the modified ANP32E, ANP32B and / or ANP32A proteins described herein. The disclosure provides vectors comprising nucleic acid encoding any of the modified ANP32 sequences of this disclosure, including those provided as SEQ ID NOS 4-14 or any functional fragments thereof. The disclosure provides a host cell transformed with a vector of this disclosure. The disclosure provides antibodies or binding agents which bind or which have a specificity, and / or affinity for any of the modified ANP32E, ANP32B or ANP32A proteins described herein. One of skill will appreciate that any of the modified protein described herein may be used as antigens in order to generate antibodies. The term ‘antibody’ should be taken to include any antigen (ANP32E / ANP32B / ANP32A) binding fragments. The disclosure provides a method of making a modified ANP32 protein of this disclosure, said method comprising culturing a cell transformed with a vector of this disclosure under conditions suitable to induce the expression of the relevant ANP32 protein and purifying said protein. The step of purifying may involve lysing the cell and using centrifugation and / or affinity chromatography or other similar technique to extract or purify the modified ANP32 protein. The disclosure further provides a method of rendering an avian species (for example a poultry species or a chicken (Gallus domesticus) resistant to an IAV infection, said method comprising rendering the ANP32E protein unable to support IAV polymerase activity. By way of example, the ANP32E gene may be modified so that it encodes a modified ANP32E protein of this disclosure (which proteins are unable to support IAV polymerase activity). Alternatively, the ANP32E gene may be functionally deleted or knocked out. The method may further comprise rendering the ANP32B protein unable to support IAV polymerase activity. By way of example, the ANP32B gene may be modified so that it encodes a modified ANP32B protein of this disclosure (which proteins are unable to support IAV polymerase activity). Alternatively, the ANP32B gene may be functionally deleted or knocked out. The method may further comprise rendering the ANP32A protein unable to support IAV polymerase activity. By way of example, the ANP32A gene may be modified so that it encodes a modified ANP32A protein of this disclosure (which proteins are unable to support IAV polymerase activity). In one teaching, a method of rendering an avian species (for example a poultry species or a chicken (Gallus domesticus) resistant to an IAV infection may

[0021] 20 55372346-1 comprise modifying the ANP32E gene to encode a modified ANP32E protein which does not support IAV polymerase; and modifying the ANP32B gene to encode a modified ANP32B protein which does not support IAV polymerase; and knocking out the ANP32A gene. It should be noted, that the phrase ‘functionally deleted’ (as it may refer to the functional deletion of any of the ANP32 proteins) may embrace techniques in which the expression of the relevant ANP32E / B or A genes are modulated (e.g. reduced) by modulation of the expression of various up / downstream expression controllers – e.g. promoters and the like. One of skill will appreciate that by modulating the expression of a promoter which controls the expression of an ANP32E, Anp32B or ANP32A protein, it may be possible to modulate (e.g. reduce, the expression of the corresponding protein. Where one is attempting to provide an animal (e.g. an avian species / chicken) which is resistant to IAV, one may modulate the expression of the relevant up / downstream expression controller of ANP32E gene expression as well as the expression of those elements controlling the expression of the ANP32B and A genes. In a further teaching, a user may knockout or functionally delete the ANP32E gene and the ANP32B and / or ANP32B gene and then use plasmids encoding any of the modified proteins described herein to reinstate (modified) ANP32E and ANP32B / A expression. Such methods may use any of the nucleic acids and / or vectors provided herein. Detailed description The present invention will now be described in detail by reference to the following figures which show: Figure 1. Derivation of ANP32A-ANP32B-escape virus: (A) A 6:2 reassortant H5N1 virus (A / turkey / Turkey / 05 / 2005) with PR8 virus HA and NA genes (Tky05) was inoculated on 12 well plates of ehAP1 (control) and ANP32A-ANP32B-double-knockout cells (DKO cells) at an MOI of 0.0005. Samples were titred on MDCK cells via plaque assay at 24 and 48 hours post infection. (B) Following 2 passages through either DKO cells or eHAP1 cells (control), 1000PFU from each population as well as the ancestor (Tky05) were inoculated onto 6 well plates of DKO and eHAP1 cells. After 48 hours, viruses were titred on MDCK cells via plaque assay. (C) Mutations from populations passaged through DKO cells were found using Sanger sequencing. Highlighted mutations were found in the plaque purified virus which was used for future experiments. (D) Growth curve of Tky05 compared to plaque purified virus containing PB1 K577E, PA Q556R on MDCK, eHAP1 and DKO cells.6-well plates were infected at MOI 0.01 and viral titres were measured on MDCK cells at 24, 48 and 72 hours. *=P≤0.05;***=P≤0.001. ‘Tyk05’ is the same as ‘TY05’ used throughout the text.

[0022] 21 55372346-1 Figure 2. (A) H9N2-UDL AIV (A / chicken / Pakistan / UDL01 / 08) minireplicon assay: H9N2-UDL polymerase containing no mutations or harbouring a combination of PA-E349K and PB2- M631L mutations detected in viruses isolated from ANP32A-gene-edited chickens was reconstituted together with NP by plasmid transfection into eHAP1 human cells lacking ANP32 expression and complemented with FLAG-tagged chicken ANP32 proteins. Polymerase activity was assessed at 48 hours post transfection by measuring Firefly luciferase signal generated from the minireplicon normalized to a Renilla luciferase transfection control. N=3 biological replicates. Data shown are Firefly signal normalised to Renilla signal plotted as mean ± SEM and One-way ANOVA with Dunnett’s corrected multiple comparisons to statistical determine mutant polymerase constellations whose activity is significantly different from the wildtype H9N2-UDL polymerase. *=P≤0.05; **=P≤0.01; ****=P≤0.0001. (B) IAV replication in embryonated chicken eggs. Wildtype (WT) H9N2-UDL virus (A / chicken / Pakistan / UDL-01 / 08) or mutant strains (mut) harbouring only PA-E349K mutation or in combination with PB2-M631L mutations (2x mut) were isolated through reverse genetics (RG) or plaque purification (PP). Wildtype or ANP32A-Knockout (AKO) 11-day-old embryonated eggs were inoculated with 100 PFU or 1000 PFU of virus and then incubated for 48 hours. Allantoic fluids were collected, and PFU / ml measured by plaque assay. Figure 3. Using CRISPR / Cas9 gene editing technique to generate chicken cells containing deletions in the ANP32A, B or E proteins. (A) Deletion of ANP32A locus. (B) Deletion of ANP32B locus. (C) Deletion of ANP 32E locus. Figure 4. H9N2-UDL AIV (A / chicken / Pakistan / UDL01 / 08) minireplicon assay. H9N2-UDL polymerase containing no mutations (B) or harbouring a combination of PA-E349K and PB2- M631L mutations (C,D) detected in virus isolated from ANP32A-gene-edited chickens was reconstituted together with NP by plasmid transfection into genome-edited chicken cells. Polymerase activity was assessed at 48 hours post transfection by measuring Firefly luciferase signal generated from the minireplicon normalized to a Renilla luciferase transfection control. N=3 biological replicates. Data shown are Firefly signal normalised to Renilla signal plotted as mean ± SEM and One-way ANOVA with uncorrected multiple comparisons to Wildtype using Fisher’s LSD test. *=P≤0.05; **=P≤0.01; ***=P≤0.001; ****=P≤0.0001. Figure 5. H5N1 AIV (A / turkey / Turkey / 05 / 2005) minireplicon assay. H5N1 / TY05 polymerase containing no mutations (B) or harbouring a combination of PB1-K577E and PA-Q556R mutations (C,D) was reconstituted together with NP by plasmid transfection into genome- edited chicken cells. Polymerase activity was assessed at 48 hours post transfection by

[0023] 22 55372346-1 measuring Firefly luciferase signal generated from the minireplicon normalized to a Renilla luciferase transfection control. N=3 biological replicates. Data shown are Firefly signal normalised to Renilla signal plotted as mean ± SEM and One-way ANOVA with uncorrected multiple comparisons to Wildtype using Fisher’s LSD test. *=P≤0.05; **=P≤0.01; ***=P≤0.001; ****=P≤0.0001. Figure 6. Wildtype 6:2 reassortant H5N1 TY05 virus (A / turkey / Turkey / 05 / 2005) with PR8 virus HA and NA genes or a mutant strain containing PA-Q556R and PB1-K577E mutations was inoculated into wildtype cells, ANP32A-knockout chicken cells (AKO cells), ANP32A- ANP32B-double-knockout chicken cells (AKO / BKO cells), ANP32A-ANP32E-double- knockout chicken cells (AKO / EKO cells) and ANP32A-ANP32B-ANP32E-triple-knockout chicken cells (TKO cells) at an MOI of 0.01. Samples were titred on MDCK cells via plaque assays at 6, 24, 48 and 72 hours post infection (HPI). Figure 7. An illustration of chicken ANP32E protein highlighting the leucine-rich repeat region (LRR), the central domain and the unstructured low complexity acidic region (LCAR). Variants of ANP32E were generated by introducing specific mutations derived from chicken ANP32B or replacing segments of ANP32E with the equivalent region from chicken ANP32B. Figure 8. Alignment of the LRR5 and central domains of chicken ANP32A, ANP32B and ANP32E. Red arrows indicate the residues in LLR5 of ANP32B that were transferred into LRR5 of ANP32E in Figure 7. Figure 9. Alignment of the LRR5 and central domains of chicken ANP32A, ANP32B and ANP32E. Red arrows indicate the residues in the central domain of ANP32B that were transferred into the central domain of ANP32E in Figure 7. Figure 10. H5N1 TY05 virus (A / turkey / Turkey / 05 / 2005) minireplicon assay in genome-edited chicken cells with FLAG-tagged ANP32E or ANP32A expression plasmids (A) and the associated western blots for detection of FLAG (B). Polymerase activity was assessed at 48 hours post transfection by measuring Firefly luciferase signal generated from the minireplicon normalized to a Renilla luciferase transfection control. Cell lysate for western blot analysis was also collected 48 hrs post transfection. N=2 biological replicates. Data shown are Firefly signal normalised to Renilla signal plotted as mean ± SEM and One-way ANOVA with uncorrected multiple comparisons to Wildtype using Fisher’s LSD test. *=P≤0.05; **=P≤0.01; ***=P≤0.001; ****=P≤0.0001. Figure 11: Selected single edits and combinations of edits in chANP32E compromise polymerase activity of H5N1 Tky05 PB1 K577E PA Q556R IAV. A: H5N1 AIV

[0024] 23 55372346-1 (A / turkey / Turkey / 05 / 2005) minireplicon assay. H5N1 / TY05 polymerase harbouring a combination of PB1 K577E and PA Q556R mutations was reconstituted together with NP and wild-type chicken ANP32E or an edited chicken ANP32 construct or an empty vector (- ANP32) by plasmid transfection into eHAP ANP32A-ANP32B-ANP32E-triple-knockout cells (eHAP-TKO cells). Polymerase activity was assessed at 48 hours post transfection by measuring Firefly luciferase signal generated from the minireplicon normalised to a Renilla luciferase transfection control. N=2 biological replicates. Data shown are Firefly signal normalised to Renilla signal plotted as mean ± SEM and one-way ANOVA with uncorrected multiple comparisons to wild-type chANP32E (WT) using Fisher’s LSD test. *=P≤0.05; **=P≤0.01; ***=P≤0.001; ****=P≤0.0001. B: Combinations of edits are effective in preventing ANP32 support of polymerase activity. H5N1 AIV (A / turkey / Turkey / 05 / 2005) minireplicon assay. H5N1 / TY05 polymerase harbouring a combination of PB1 K577E and PA Q556R mutations was reconstituted together with NP and wild-type chicken ANP32E or an edited chicken ANP32 construct or an empty vector (-ANP32) by plasmid transfection into eHAP ANP32A-ANP32B-ANP32E-triple-knockout cells (eHAP-TKO cells). Polymerase activity was assessed at 48 hours post transfection by measuring Firefly luciferase signal generated from the minireplicon normalised to a Renilla luciferase transfection control. N=2 biological replicates. Data shown are Firefly signal normalised to Renilla signal plotted as mean ± SEM and one-way ANOVA with uncorrected multiple comparisons to wild-type chANP32E (WT) using Fisher’s LSD test. *=P≤0.05; **=P≤0.01; ***=P≤0.001; ****=P≤0.0001; ns=not significant. Figure 12: Selected edits in chANP32B compromise polymerase activity of H5N1 Tky05 PB1 K577E PA Q556R IAV. A: H5N1 AIV (A / turkey / Turkey / 05 / 2005) minireplicon assay. H5N1 / TY05 polymerase harbouring a combination of PB1 K577E and PA Q556R mutations was reconstituted together with NP and wild-type chicken ANP32B or an edited chicken ANP32B construct or an empty vector (-ANP32) by plasmid transfection into eHAP ANP32A- ANP32B-ANP32E-triple-knockout cells (eHAP-TKO cells). Polymerase activity was assessed at 48 hours post transfection by measuring Firefly luciferase signal generated from the minireplicon normalised to a Renilla luciferase transfection control. N=2 biological replicates. Data shown are Firefly signal normalised to Renilla signal plotted as mean ± SEM and one-way ANOVA with uncorrected multiple comparisons to wild-type chANP32B (WT) using Fisher’s LSD test. *=P≤0.05; **=P≤0.01; ***=P≤0.001; ****=P≤0.0001; ns=not significant. Figure 13: Selected edits in chANP32B compromise polymerase activity of H5N1 Tky05 PB1 K577E PA Q556R IAV. A: H5N1 AIV (A / turkey / Turkey / 05 / 2005) minireplicon assay. H5N1 / TY05 polymerase harbouring a combination of PB1 K577E and PA Q556R mutations

[0025] 24 55372346-1 was reconstituted together with NP and wild-type chicken ANP32B or an edited chicken ANP32B construct or an empty vector (-ANP32) by plasmid transfection into eHAP ANP32A- ANP32B-ANP32E-triple-knockout cells (eHAP-TKO cells). Polymerase activity was assessed at 48 hours post transfection by measuring Firefly luciferase signal generated from the minireplicon normalised to a Renilla luciferase transfection control. Data shown are Firefly signal normalised to Renilla signal plotted as mean ± SEM and one-way ANOVA with uncorrected multiple comparisons to wild-type ANP32B (WT) using Fisher’s LSD test. *=P≤0.05; **=P≤0.01; ***=P≤0.001; ****=P≤0.0001; ns=not significant. Example We investigated the possibility of influenza A viruses replicating in mammalian (human) cells lacking both of the essential host factors, ANP32A and ANP32B. Contrary to the established state of the literature, we discovered that competent influenza A viruses capable of replicating in the complete absence of ANP32A and ANP32B arose under selection pressure; following serial passaging in human cells lacking ANP32A and ANP32B (ANP32A-ANP32B-dKO cells), a mutant of a 6:2 reassortant H5N1 virus (A / turkey / Turkey / 05 / 2005) with PR8 virus HA and NA genes (TY05) was isolated that contained mutations in the viral polymerase PB1 and PA genes (Figure 1). In the human system, this virus was found to utilize human ANP32E protein to support its polymerase activity. This implies that edits to ANP32A and B (for example in mammalian cells) will not be robust for producing animals resistant to influenza. Similarly, in the chicken system, we had generated genome-edited birds with mutations that inactivated the chicken ANP32A (N129I-D130N) for support of influenza. During infections with the low pathogenic avian influenza virus H9N2-UDL (A / chicken / Pakistan / UDL01 / 08), mutant viruses with different mutations in the polymerase genes arose. We showed the polymerases of these mutant viruses were able to use the edited chicken ANP32A N129I- D130N protein, and also chicken ANP32B and chicken ANP32E (Figure 2A). We then used CRISPR / Cas9 to generate chick embryos lacking ANP32A (AKO eggs) and infected them with wildtype or mutant H9N2-UDL viruses containing only the PA-E349K mutation or in combination with the PB2-M631L mutation in the viral polymerase. We observed low levels of virus growth in AKO embryonated eggs infected with a high dose (1000PFU) of the wildtype H9N2-UDL virus (Figure 2B). However, the mutant viruses grew efficiently in AKO eggs. This implies that single edits or deletions of single ANP32 proteins is not a robust way to generate genome-edited influenza resistant birds. We next used CRISPR / Cas9 gene editing technique to generate chicken cells containing the loss-of-function mutation (ANP32A 129130) or single or combinations of deletions in the ANP32A, B or E proteins (Figure 3).

[0026] 25 55372346-1 The mutant H9N2-UDL polymerase containing a combination of PA-E349K and PB2-M631L mutations displayed robust activity in both wildtype and N129I-D130N cells (Figure 4C). The activity of this mutant AIV polymerase was significantly reduced but still detectable in chicken cells lacking either only ANP32A (AKO cells) or chicken cells lacking both ANP32A and ANP32B but expressing ANP32E (AKO / BKO cells) (Figure 4C & D). Overall, this result suggests the potential for the polymerases of low pathogenic AIVs to adapt to efficiently use chicken ANP32E if ANP32A is modified to make it non-functional. It was also shown that chicken ANP32B could be co-opted by the mutant TY05 virus. We performed minireplicon assays and observed that the mutant TY05 virus polymerase with PB1-K577E and PA-Q445R mutations functioned efficiently in N129I-D130N chicken cells as well as in AKO and AKO / BKO chicken cells (Figure 5C). We observed significantly reduced but detectable activity of the mutant TY05 polymerase in AKO / EKO chicken cells which express only ANP32B (Figure 5C & D). In chicken cells lacking all three ANP32 proteins (TKO cells), the mutant TY05 polymerase activity was completely absent highlighting the crucial requirement for an ANP32 protein (Figure 5C & D). Virus infection assays we used to assess the growth of the mutant TY05 virus in the gene- edited chicken cells with modified ANP32 proteins. Contrary to the observation in the minireplicon assay, robust virus growth was observed in AKO cells, AK / BKO cells and AKO / EKO cells (Figure 6). Many viral proteins such as NEP and NS1 are not expressed in minireplicon assays but have been reported to enhance polymerase activity and compensate for defective virus replication (Selman et al., 2012; Manz et al., 2012). However, reflecting the observation in the minireplicon assay, virus growth was completely absent in TKO cells again highlighting the crucial requirement for at least one functional ANP32 protein. Contrary to all previous observations, the results disclosed herein suggests that both ANP32E and ANP32B can become proviral if ANP32A is rendered unusable to the IAV polymerase. Consequently, the reliance on ANP32A modifications as a long-term strategy to generate influenza resistance birds may drive the evolution of avian influenza viruses that switch to efficient use of ANP32B and / or ANP32E. Therefore, we propose a Swiss-cheese model for generating influenza resistant animals that includes combined modifications of all three ANP32 proteins to prevent the possibility of selecting for viral strains that escape the modification of only ANP32A (and ANP32B in mammals). ANP32 proteins perform various important molecular functions. ANP32A and ANP32B perform transcriptional modulation functions in cells through interaction with histone H3 and H4. ANP32E also possesses transcriptional modulation activity through its interaction with histone H2A.Z. Therefore, generating animals lacking any or all of these ANP32 proteins may

[0027] 26 55372346-1 negatively impact physiology affecting the viability / fitness of the modified animal. Instead, we suggest a novel strategy for generating influenza resistant animals should constitute specific modifications of ANP32 proteins which inhibit influenza virus activity but preserve the natural molecular functions of the proteins. In view of the above, the defined modification of ANP32E prevents its use by mutant virus polymerases that have evolved to use ANP32E protein. To identify domains of chicken ANP32E (chANP32E) that are critical for supporting the activity of influenza A virus polymerase, FLAG-tagged chANP32E variants containing changes derived from chicken ANP32B (chANP32B) were generated (Figure 7, 8 & 9): 1. chANP32E containing E129I and D130N amino acid substitution derived from chANP32B (E129I / D130N). 2. chANP32E with LRR5 region (residues 115 – 141) of chANP32B; (chANP32B115 – 141). 3. chANP32E with the central region (residues 142 – 175) of chANP32B; (chANP32B142– 175). 4. chANP32E with the LRR5 and central domain (residue 115 – 175) of chANP32B; (chANP32B115 – 175). Minireplicon assays were performed in TKO cells lacking all three ANP32 proteins to assess the ability of the various chANP32E variants to rescue the activity of the reconstituted polymerase of the mutant TY05 virus assessed in Figure 5C. As expected, wildtype chANP32E and chicken ANP32A (chANP32A) significantly rescued polymerase activity in TKO cells (Figure 10). There was small detectable polymerase activity with the chANP32E variant containing the E129I / D130N changes in the LRR5 domain of chANP32E, whereas polymerase activity was abrogated with the chANP32E variant containing replacement of the chANP32E LRR5 domain with that of chANP32B (chANP32B115 – 141) (Figure 10). These observations suggests that the LRR5 domain of chANP32E is proviral and introducing minimal changes into this region will abolish support for the influenza polymerase. Introducing both LRR5 and central domain of chANP32B into chicken ANP32E (chANP32B115– 175) led to small detectable polymerase activity that was similar to levels produced by the chANP32E variant with E129I / D130N changes (Figure 10). Interestingly, it was shown that the chANP32E variant containing only the central domain of chANP32B (chANP32B142 – 175) resulted in significant polymerase support even beyond levels observed with wildtype chANP32E and chANP32A (Figure 10). This suggests that the central domain of chicken ANP32B is strongly proviral and may account for the small traceable level of polymerase activity and robust virus replication supported by wildtype chicken ANP32B in AKO / EKO cells in Figures 5D & 6. In contrast, the central domain of chicken ANP32E is likely not to be proviral

[0028] 27 55372346-1 and introducing it into chicken ANP32B may abrogate mutant polymerase support (Figure 10). Therefore, it is suggested that the central domain of chicken ANP32B must also be edited to prevent potential polymerase interactions. There is the potential to identify additional specific residues within the central domain of ANP32E to prevent unforeseen AIV polymerase adaptation. Thus the following actions that could be taken to generate a bird that is completely resistant to avian influenza: 1. The following residues in chicken ANP32E could be mutated to reduce or eliminate viral polymerase activity: 116, 125, 127, 129, 130, 133, 135, 137, 140. Specifically, we have shown that concurrently introducing the following changes derived from chicken ANP32B into chicken ANP32E results in antiviral activity: K116H, I125V, N127M, E129I, D130N, D133E, I135V, D137T, Q140P (Figure 8 & 10). 2. The following residues in chicken ANP32B could be mutated to reduce or eliminate potential viral polymerase support: 142, 150, 151, 153, 159, 160, 161, 162, 164, 165, 166, 167, 168, 170, 171, 173, 174 and 175. Specifically, we have shown that concurrently introducing the following changes derived from chicken ANP32B into chicken ANP32E was proviral and must be addressed: L142I, A150Q, D151E, E152D, Q153N, D159E, P160D, E161D, A162D, G164E, D165G, G166D, L167E, E168D, E170N, Y171D, N173D, G174E, E175D (Figure 9 & 10). 3. To reduce or eliminate viral polymerase activity, the following residues in chicken ANP32A could be mutated: 116, 125, 127, 133, 135, 137, 140. These mutations may be combined with others, including others known in the prior art. 4. All three ANP32 proteins (ANP32A, ANP32B and ANP32E) may be concurrently edited by mutating the residues highlighted in actions 1, 2 and 3 in order to completely eliminate viral polymerase activity and the potential for adaptation. 5. Two ANP32 proteins (ANP32A and ANP32B) may be concurrently deleted and ANP32E is concurrently edited by mutating the residues highlighted in action 1 in order to completely eliminate viral polymerase activity and the potential for adaptation. 6. Two ANP32 proteins (ANP32A and ANP32E) may be concurrently deleted and ANP32B is concurrently edited by mutating the residues highlighted in action 2 in order to completely eliminate viral polymerase activity and the potential for adaptation. 7. ANP32A may be deleted while ANP32B and ANP32E are concurrently edited by mutating the residues highlighted in actions 1 and 2 in order to completely eliminate viral polymerase activity and the potential for adaptation.

[0029] 28 55372346-1 8. All three ANP32 proteins are concurrently deleted and replaced with a novel synthetic ANP32 protein that cannot support viral polymerase activity but retains ANP32 molecular functions. Generation of GE chickens Male and female GE PGCs were micro-injected into stage 15-16+HH (ED 2.5) surrogate iCaspase9 host embryos as described previously14. Briefly, 1.0 µl of 25 mM B / B compound (in DMSO) (Takara Bio) was added to a 50 µl cell suspension (5000 PGCs / µl) and maintained at room temperature.1.0 µl of the B / B compound-PGC mixture was injected into the dorsal aorta of embryos in windowed eggs. Egg shells were sealed with medical Leukosilk tape (BSN Medical) and then incubated until hatch. Surrogate males and female chickens (G0) were mated in pens to produce homozygous GE offspring. To screen for homozygous ANPN129I-D130Nembryos or chicks, chorioallantoic membrane or blood were analysed by PCR amplification of genomic DNA and Sanger sequencing using primers (5’ – ACTCCTTTTGTCACGAGAAGC – 3’, 5’ – TTCCTCCTCATCGTCTAAGCC – 3’). To screen for homozygous AKO embryos, chorioallantoic membrane or blood were analysed by PCR amplification of genomic DNA and Sanger sequencing using primers (5’ – TCAAAGTCCCTTATTACCGCG – 3’, 5’ – CCTTTCACTCCCCATCTTTCA – 3’) that bind to areas outside the deleted 15-kb region and amplify a PCR product of approximately 220 bp only if the deletion is successful but fail to yield a product if there is no deletion. GE chickens received routine vaccinations and blood samples were sent to Sci-Tech Labs (Cawood Scientific), Dublin, Ireland to perform ELISA tests to assess response to vaccines. References Staller, E., Sheppard, C. M., Neasham, P. J., Mistry, B., Peacock, T. P., Goldhill, D. H., ... & Barclay, W. S. (2019). ANP32 proteins are essential for influenza virus replication in human cells. Journal of virology, 93(17), e00217-19. Xiaojun Wang, Haili Zhang, Zhenyu Zhang (2019). Use of anp32 protein in maintaining influenza virus polymerase activity in host. EP3763730A4 Han Jae Yong, Park Young Hyun, Seo Jung Yong, Im Jung Mook (2021). Method for producing genetically edited birds having resistance to avian influenza viruses. WO2021141421A1 Long, J. S., Giotis, E. S., Moncorgé, O., Frise, R., Mistry, B., James, J., ... & Barclay, W. S. (2016). Species difference in ANP32A underlies influenza A virus polymerase host restriction. Nature, 529(7584), 101-104. Long, J. S., Idoko-Akoh, A., Mistry, B., Goldhill, D., Staller, E., Schreyer, J., ... & Barclay, W. (2019). Species specific differences in use of ANP32 proteins by influenza A virus. Elife, 8, e45066. Yu, M., Qu, Y., Zhang, H., & Wang, X. (2022). Roles of ANP32 proteins in cell biology and viral replication. Animal Diseases, 2(1), 1-14.

[0030] 29 55372346-1 Mänz, B., Brunotte, L., Reuther, P., & Schwemmle, M. (2012). Adaptive mutations in NEP compensate for defective H5N1 RNA replication in cultured human cells. Nature communications, 3(1), 1-11. Park, Y. H., Chungu, K., Lee, S. B., Woo, S. J., Cho, H. Y., Lee, H. J., ... & Han, J. Y. (2020). Host-specific restriction of avian influenza virus caused by differential dynamics of ANP32 family members. The Journal of Infectious Diseases, 221(1), 71-80. Park, Y. H., Woo, S. J., Chungu, K., Lee, S. B., Shim, J. H., Lee, H. J., ... & Han, J. Y. (2021). Asp149 and Asp152 in chicken and human ANP32A play an essential role in the interaction with influenza viral polymerase. The FASEB Journal, 35(6), e21630. Reilly, P. T., Yu, Y., Hamiche, A., & Wang, L. (2014). Cracking the ANP32 whips: important functions, unequal requirement, and hints at disease implications. Bioessays, 36(11), 1062-1071. Selman, M., Dankar, S. K., Forbes, N. E., Jia, J. J., & Brown, E. G. (2012). Adaptive mutation in influenza A virus non-structural gene is linked to host switching and induces a novel protein by alternative splicing. Emerging microbes & infections, 1(1), 1-10. Zhang, H., Zhang, Z., Wang, Y., Wang, M., Wang, X., Zhang, X., ... & Wang, X. (2019). Fundamental contribution and host range determination of ANP32A and ANP32B in influenza A virus polymerase activity. Journal of virology, 93(13), e00174-19. Numbered references Long, J. S., Mistry, B., Haslam, S. M. & Barclay, W. S. Host and viral determinants of influenza A virus species specificity. Nature Reviews Microbiology 1 (2018). Fasanmi, O. G. et al. National surveillance and control costs for highly pathogenic avian influenza H5N1 in poultry: A benefit-cost assessment for a developing economy, Nigeria. Research in Veterinary Science 119, 127–133 (2018). Thompson, J. M. & Seitzinger, A. H. Economic evaluation of low pathogenic avian influenza in northeastern US live bird markets. Journal of Applied Poultry Research 28, 78–84 (2019). Wang, D., Zhu, W., Yang, L. & Shu, Y. The Epidemiology, Virology, and Pathogenicity of Human Infections with Avian Influenza Viruses. Cold Spring Harbor Perspectives in Medicine 11, a038620 (2021). Peters, A. R. & Guyonnet, V. Are current avian influenza vaccines a solution for smallholder poultry farmers? Gates Open Research vol.4 Preprint at https: / / doi.org / 10.12688 / gatesopenres.13171.1 (2020). JM Wandzik, T. K. S. C. Structure and function of influenza polymerase. Cold Spring Harb. Perspect. Med.10, a038372 (2020). Long, J. S. et al. Species difference in ANP32A underlies influenza A virus polymerase host restriction. Nature 529, 101–104 (2016). Staller, E. et al. ANP32 Proteins Are Essential for Influenza Virus Replication in Human Cells. Journal of Virology 93, (2019). Long, J. S. et al. Species specific differences in use of ANP32 proteins by influenza A virus. eLife 8, (2019).

[0031] 30 55372346-1 Carrique, L. et al. Host ANP32A mediates the assembly of the influenza virus replicase. Nature 587, 638–643 (2020). Zhang, H. et al. Fundamental Contribution and Host Range Determination of ANP32A and ANP32B in Influenza A Virus Polymerase Activity. Journal of Virology 93, (2019). Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8, 2281–2308 (2013). Idoko-Akoh, A., Taylor, L., Sang, H. M. & McGrew, M. J. High fidelity CRISPR / Cas9 increases precise monoallelic and biallelic editing events in primordial germ cells. Sci Rep 8, 15126 (2018). Ballantyne, M. et al. Direct allele introgression into pure chicken breeds using Sire Dam Surrogate (SDS) mating. Nature Communications 12, 1–10 (2021). Cornelis, F. M. F. et al. ANP32A regulates ATM expression and prevents oxidative stress in cartilage, brain, and bone. Science Translational Medicine 10, 8426 (2018). Monteagudo, S. et al. ANP32A represses Wnt signaling across tissues thereby protecting against osteoarthritis and heart disease. Osteoarthritis and cartilage 30, (2022). Peacock, T. P., James, J., Sealy, J. E. & Iqbal, M. A Global Perspective on H9N2 Avian Influenza Virus. Viruses 2019, Vol.11, Page 62011, 620 (2019). Zhao, Y. et al. Adaptive amino acid substitutions enhance the virulence of a novel human H7N9 influenza virus in mice. Veterinary Microbiology 187, 8–14 (2016). Zhang, X. et al. Enhanced pathogenicity and neurotropism of mouse-adapted H10N7 influenza virus are mediated by novel PB2 and NA mutations. Journal of General Virology 98, 1185–1195 (2017). Choi, W. S. et al. Rapid acquisition of polymorphic virulence markers during adaptation of highly pathogenic avian influenza H5N8 virus in the mouse. Scientific Reports 20177:17, 1–13 (2017). Chen, K. Y., Afonso, E. D. S., Enouf, V., Isel, C. & Naffakh, N. Influenza virus polymerase subunits co- evolve to ensure proper levels of dimerization of the heterotrimer. PLOS Pathogens 15, e1008034 (2019). Brown, E. G., Liu, H., Chang Kit, L., Baird, S. & Nesrallah, M. Pattern of mutation in the genome of influenza A virus on adaptation to increased virulence in the mouse lung: Identification of functional themes. Proceedings of the National Academy of Sciences 98, 6883–6888 (2001). Park, Y. H. et al. Asp149 and Asp152 in chicken and human ANP32A play an essential role in the interaction with influenza viral polymerase. The FASEB Journal 35, e21630 (2021). Camacho-Zarco, A. R. et al. Molecular basis of host-adaptation interactions between influenza virus polymerase PB2 subunit and ANP32A. Nature Communications 202011:111, 1–12 (2020). Sugiyama, K., Kawaguchi, A., Okuwaki, M. & Nagata, K. PP32 and APRIL are host cell-derived regulators of influenza virus RNA synthesis from cRNA. eLife (2015) doi:10.7554 / eLife.08939. Fan, H. et al. Structures of influenza A virus RNA polymerase offer insight into viral genome replication. Nature 2019573:7773573, 287–290 (2019). Chang, S. et al. Cryo-EM Structure of Influenza Virus RNA Polymerase Complex at 4.3 Å Resolution. Molecular Cell 57, 925–935 (2015).

[0032] 31 55372346-1 Chen, K. Y., Afonso, E. D. S., Enouf, V., Isel, C. & Naffakh, N. Influenza virus polymerase subunits co- evolve to ensure proper levels of dimerization of the heterotrimer. PLOS Pathogens 15, e1008034 (2019). Tait-Burkard, C. et al. Livestock 2.0 - Genome editing for fitter, healthier, and more productive farmed animals. Genome Biology 19, 1–11 (2018). Knap, P. W. & Doeschl-Wilson, A. Why breed disease-resilient livestock, and how? Genetics Selection Evolution 202052:152, 1–18 (2020). Lyall, J. et al. Suppression of avian influenza transmission in genetically modified chickens. Science 331, 223–226 (2011). Schusser, B. & Doran, T. Advances in genetic engineering of the avian genome. Avian Immunology 559–572 (2022) doi:10.1016 / B978-0-12-818708-1.00022-1. Whyte, J. et al. FGF, Insulin, and SMAD Signaling Cooperate for Avian Primordial Germ Cell Self- Renewal. Stem Cell Reports 5, 1171–1182 (2015). Gu, B. et al. Opposing Effects of Cohesin and Transcription on CTCF Organization Revealed by Super- resolution Imaging. Mol Cell 80, 699-711.e7 (2020). Labun, K., Montague, T. G., Gagnon, J. A., Thyme, S. B. & Valen, E. CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Research 44, W272–W276 (2016). Giotis, E. S. et al. Chicken interferome: Avian interferon-stimulated genes identified by microarray and RNA-seq of primary chick embryo fibroblasts treated with a chicken type i interferon (IFN-α). Veterinary Research 47, 1–12 (2016). Giotis, E. S., Montillet, G., Pain, B. & Skinner, M. A. Chicken Embryonic-Stem Cells Are Permissive to Poxvirus Recombinant Vaccine Vectors. Genes 2019, Vol.10, Page 23710, 237 (2019). Long, J. S. et al. The Effect of the PB2 Mutation 627K on Highly Pathogenic H5N1 Avian Influenza Virus Is Dependent on the Virus Lineage. Journal of Virology (2013) doi:10.1097 / GCO.0000000000000038. World Health Organisation. Collecting, preserving and shipping specimens for the diagnosis of avian influenza A(H5N1) virus infection^: guide for field operations. ‘October 2006’ Preprint at (2006). Pedersen, J. C. Hemagglutination-Inhibition Test for Avian Influenza Virus Subtype Identification and the Detection and Quantitation of Serum Antibodies to the Avian Influenza Virus. Methods in Molecular Biology 436, 53–66 (2008).

[0033] 32 55372346-1

Claims

Claims 1. A modified ANP32E protein with reduced IAV polymerase determinable by minireplicon assay, wherein relative to ANP32E SEQ ID NO: 1, the modified ANP32E protein comprises a mutation or mutations.

2. The modified ANP32E protein of claim 1, wherein the modified ANP32E protein comprises a mutation or mutations at any one or more of the following residues: 6, 12, 25, 45, 70, 73, 92, 93, 94, 96, 116, 119, 125, 127, 129, 130, 133, 135, 136, 137, 138, 139, 140, 142, 149, 150, 151, 152, 153, 154, 155, 156, 157, 159, 160, 161, 162, 164, 165, 166, 167, 168, 170, 171, 173, 174 and / or 175 3. A modified ANP32E protein with reduced IAV polymerase activity, wherein relative to ANP32E SEQ ID NO: 1, the modified ANP32E protein comprises a mutation or mutations at any one or more of the following residues: 6, 12, 25, 45, 70, 73, 92, 93, 94, 96, 116, 119, 125, 127, 129, 130, 133, 135, 136, 137, 138, 139, 140, 142, 149, 150, 151, 152, 153, 154, 155, 156, 157, 159, 160, 161, 162, 164, 165, 166, 167, 168, 170, 171, 173, 174 and / or 175 and further wherein the polymerase activity is reduced as compared to the polymerase activity of wild-type ANP32E.

4. The modified ANP32E protein of any preceding claim, wherein the modified ANP32E protein comprises a mutation or mutations at any one or more of the following residues: 6, 12, 25, 45, 70, 73, 119, 127, 129, 130, 135, 136, 138, 139, 149, 151, 152, 153, 154, 155, 156, 157 and / or 159.

5. The modified protein of any preceding claim, wherein the modified ANP32E protein comprises a mutation selected from the group consisting of: (i) R6E; (ii) R12E; (iii) D25A; (iv) D25K; (v) E151A; (vi) E151K; (vii) D152H; (viii) N153A; (ix) E154A; (x) E154K; (xi) A155K; (xii) P156R; (xiii) P156K; (xiv) D157A; (xv) D157K; (xvi) E159K; (xvii) D25A, E45A, E70A, D73A, D119A; (xviii) D25A, N127M, E129I, D130N; (xix) D25K, N127M, E129I, D130N; (xx) D25K, N127M, E129I, D130N, D149Y; (xxi) N127M, E129I, D130N; N127M, E129I, D130N, D149Y; (xxii) I135G, F136G, L138G, L139G; and (xxiii) D149Y, D152H.

6. The modified ANP32E protein of any preceding claim, wherein the modified ANP32E protein comprises a mutation selected from the group consisting of: (i) R6E; (ii) R12E; (iii) D25A; (iv) D25K; (v) E151A; (vi) E151K; (vii) D152H; (viii) N153A; (ix) E154A; (x) E154K; (xi) A155K; (xii) P156R; (xiii) P156K; (xiv) D157A; (xv) D157K; (xvi) D159K.33 55372346-17. The modified ANP32E protein of any preceding claim, wherein the modified ANP32E protein comprises a mutation selected from the group consisting of: (i) R6E; (ii) R12E; (iii) D25K; (iv) E151A; (v) E151K; (vi) N153A; (vii) E154A; (viii) E154K; (ix) A155K; (x) P156R; (xi) P156K; (xii) D157A; (xiii) D157K and (xiv) D159K.

8. The modified ANP32E protein of any preceding claim, wherein the modified ANP32E protein comprises a mutation or mutations selected from the group consisting of: (i) R6E; (ii) R12E; (iii) D25K; (iv) E151A; (v) E151K; (vi) N153A; (vii) E154A; (viii) E154K; (ix) A155K; (x) P156R; (xi) D157A; (xii) D157K and (xiii) D159K.

9. The modified ANP32E protein of any preceding claim, wherein the modified ANP32E protein comprises a mutation selected from the group consisting of: (i) R6E; (ii) R12E; (iii) D25K; (iv) E151A; (v) E151K; (vi) E154A; (vii) E154K; (viii) A155K; (ix) D157A; (x) D157K; (xi) D159K.

10. The modified ANP32E protein of any preceding claim, wherein the modified ANP32E protein comprises mutations selected from the group consisting of: (i) D25A, E45A, E70A, D73A, D119A; (ii) D25A, N127M, E129I, D130N; (iii) D25K, N127M, E129I, D130N; (iv) D25K, N127M, E129I, D130N, D149Y; (v) N127M, E129I, D130N; (vi) N127M, E129I, D130N, D149Y; (vii) I135G, F136G, L138G, L139G; and (viii) D149Y, D152H.

11. The modified ANP32E protein of any preceding claim, wherein the modified ANP32E protein comprises combinations of mutations selected from the group consisting of: (i) D25K; (ii) D25A, E45A, E70A, D73A, D119A; (iii) D25K, N127M, E129I, D130N; (iv) D25K, N127M, E129I, D130N; D149Y (iv) N127M, E129I, D130N; (v) N127M, E129I, D130N, D149Y and (vi) D149Y, D152H.

12. The modified ANP32E protein of any preceding claim, wherein the modified ANP32E protein comprises a sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9,10 or 11.

13. The modified ANP32E protein of any preceding claim, wherein the modified ANP32E protein comprises:34 55372346-1(a) a sequence in which a residue of one or more of the leucine-rich repeat (LRR) regions has been mutated; and / or (b) a sequence in which either or both of residues 129 and 130 has / have been mutated or E129I and / or D130N substitutions; and / or (c) a sequence in which the LRR5 domain comprises one or more mutations; and / or (d) a sequence wherein residues of LRR5 have been substituted with residues from the LRR5 domain of chicken ANP32B (SEQ ID NO: 2); and / or (e) a sequence in which residues 125, 127, 129, 130, 133, 135, 137 and / or 140 of the LRR5 domain have been mutated and / or substitution with the corresponding residue from the ANP32B reference sequence of SEQ ID NO: 2; and / or (f) a sequence in which the central region has been modified; and / or (g) a sequence in which one or more of the central region residues 141-175 of the modified ANP32E protein, have been mutated; and / or (h) a sequence in which the central region of the modified ANP32 protein comprises one or more residues derived from ANP32B; and / or (i) a sequence in which the central region of the modified ANP32E protein comprises mutations at any or all of residues 142, 150, 151, 152, 153, 159, 160, 161, 162, 164, 165, 166, 167, 168, 170, 171, 173, 174 and / or 175; and / or (j) a sequence in which there are mutations at any residues between residues 115 and 175 of the modified ANP23E protein; and / or (k) a sequence in which any one or more of residues 115-175 of the modified ANP32E protein have been substituted with one or more residues from the corresponding region of ANP32B.

14. A nucleic acid encoding the modified ANP32E protein of any preceding claim.

15. A vector comprising the nucleic acid of claim 14.

16. A host cell transformed with the nucleic acid or vector of claims 14-15.

17. A genetically modified avian species in which the ANP32E gene has been edited to encode a modified ANP32E protein according to any one of claims 1-13.35 55372346-118. A genetically modified avian species in which the ANP32E gene has been edited to encode a ANP32E protein which does not support IAV polymerase or which, compared to wild-type ANP32E, has reduced IAV polymerase activity.

19. A genetically modified avian species, in which the ANP32E gene has been functionally deleted or knocked out.

20. The genetically modified avian species of any one of claims 17-19, wherein the avian species further comprises ANP32B and / or ANP32A gene(s) which have been edited to encode ANP32B and / or ANP32A protein(s) which do not support IAV polymerase or which, compared to wild-type ANP32B / A, has / have reduced IAV polymerase activity.

21. The genetically modified avian species of claim 20 wherein the ANP32B and / or ANP32A gene(s) have / has been functionally deleted or knocked out.

22. A genetically modified avian species, comprising: an ANP32E gene which has been (i) edited to encode a modified ANP32E protein which does not support IAV polymerase or which, compared to wild-type ANP32E, has reduced IAV polymerase activity; or (ii) functionally deleted or knocked out; and an ANP32B gene which has been (i) edited to encode a modified ANP32B protein which does not support IAV polymerase or which, compared to wild-type ANP32B, has reduced IAV polymerase activity; or (ii) functionally deleted or knocked out; and an ANP32A gene which has been (i) edited to encode a modified ANP32A protein which does not support IAV polymerase or which, compared to wild-type ANP32A, has reduced IAV polymerase activity; or (ii) functionally deleted or knocked.

23. The genetically modified avian species of claim 22 wherein the edited ANP32E gene encodes a modified ANP32E protein according to any one of claims 1-13.

24. The genetically modified avian species of claims 22-24 wherein the avian species is a poultry species or a chicken (Gallus domesticus).

25. The genetically modified avian species of claims 22-24 wherein relative to ANP32E SEQ ID NO: 1, the avian species comprises a ANP32E gene encoding a ANP32E protein with mutations or amino acid substitutions at any one or more of the following residues: 6, 12, 25, 45, 70, 73, 92, 93, 94, 96, 116, 119, 125, 127, 129, 130, 133, 135, 136, 137, 138, 139, 140, 142, 149, 150, 151, 152, 153, 154, 155, 156, 157, 159, 160, 161, 162, 164, 165, 166, 167, 168, 170, 171, 173, 174 and / or 175.36 55372346-126. A modified ANP32B protein which cannot be used by IAV polymerase or which, relative to a wild-type ANP32B exhibits reduced IAV polymerase activity, wherein relative to ANP32B SEQ ID NO: 2, the modified ANP32B protein comprises a mutation or mutations at any one or more of the following residues: 6, 12, 25, 45, 70, 73, 92, 93, 94, 96, 119, 135, 136, 138, 139, 142, 144, 149, 150, 151, 152, 153, 154, 155, 156, 157, 159, 160, 161, 162, 164, 165 and / or 166.

27. A genetically modified avian species expressing a ANP32B gene edited to encode a ANP32B protein according to claim 26.

28. A genetically modified avian species expressing a ANP32E gene edited to encode an ANP32E protein according to any one of claims 1-13 and a ANP32B gene edited to encode modified ANP32B protein according to claim 26.37 55372346-1

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