WO2020109780A2

WO2020109780A2 - Polypeptide and uses thereof

Info

Publication number: WO2020109780A2
Application number: PCT/GB2019/053341
Authority: WO
Inventors: Mike MCGREW; Helen Sang; Alewo ISAIAH IDOKO-AKOH; Wendy BARCLAY; Jason Long
Original assignee: The University Court Of The University Of Edinburgh; Imperial College Of Science, Technology And Medicine
Priority date: 2018-11-26
Filing date: 2019-11-26
Publication date: 2020-06-04
Also published as: GB201819200D0; WO2020109780A3

Abstract

The present invention relates to genome modified birds adapted to be resistant to influenza virus infection, methods of generating such birds, and nucleotides and polypeptides for use in such methods

Description

Polypeptide and Uses Thereof

Field of the Invention

The present invention relates to genome modified birds adapted to be resistant to influenza virus infection, methods of generating such birds, and nucleotides and polypeptides for use in such methods.

Background

Influenza viruses infect domestic animals including chickens and pigs, causing loss of income, social and economic disruption and threatening food security. In the last decade growth in poultry production accounted for almost half of the increase in total meat production and is projected to be the main driver of global meat production expansion in the next decade (OECD-FAO Outlook 2017). Global poultry production remains at risk of disease outbreaks. Low pathogenic influenza (LPAI) causes respiratory distress and losses in egg production, while high pathogenic strains (HPAI), which can evolve from LPAI during circulation in poultry, are rapidly lethal. Reservoirs of LPAI in wild waterfowl are a continuous risk as they are a source for new emergent variants of LPAI. Current vaccines for LPAI are used in regions with chronic and recurrent outbreaks. 113 billion vaccines were used in poultry in China between 2002 to 2012 (Sun et al. (Sci Rep. (2017); 7:46441). Vaccination of pedigree farms of commercial stock adversely affects the international shipment of poultry products: consequently, avian influenza virus (AIV) vaccinations are not used in commercial pedigree breeding operations.

Current control policies for outbreaks of AIV are not ideal: eradication of flocks by various methods of culling is the method used throughout the world. This is costly and raises concerns of animal welfare.

Furthermore, some strains of AIV can infect humans and genes from AIV also contribute to the emergence of novel influenza pandemics. Since 2013, 1567 laboratory-confirmed human cases of AIV H7N9 have been reported, including at least 615 deaths (WHO, Influenza at the human-animal interface, Summary and assessment, 2 March 2018). All of the pandemics of the 20^th and 21^st century were caused by influenza viruses that acquired novel viral genes from AIV.

Summary of the Invention

As obligate intracellular parasites, all viruses rely on co-opting host cell genes to support their replication. Often viruses that have evolved in one species are exquisitely dependent on the host factors of that species and therefor do not readily infect other hosts. By discovering the identity of these host factors the inventors have determined vulnerabilities in the virus host relationship that can be exploited for their control. Most strains of AIV do not infect humans because of incompatibilities between the virus and the mammalian host. Importantly, the viral polymerase enzyme responsible for replicating the RNA genome functions poorly in human cells if derived from an AIV strain.

Mammalian-adapted viruses such as seasonal strains of human influenza have evolved adaptive mutations that allow their viral polymerase to function. The best known of these adaptive mutations is E627K in the PB2 subunit of the trimeric polymerase, but the molecular basis of the incompatibility of AIV polymerase with the human cell was not understood until recently. The present inventors have previously shown that a family of host proteins, ANP32, are co-opted by influenza virus to support its replication. In mammals, there are three members of the ANP32 family known to be expressed: A, B and E. Knock down of either ANP32A or B compromised replication of human-adapted influenza strains in human cells, suggesting redundancy between these proteins in their support of influenza replication (Sugiyama et al., eLife, 4 (2015), pp. 1-19; Long et al. Nature(2016) 7;529, 101-4). Introduction of the additional avian-derived 33 amino acids into human ANP32A, ANP32B but not ANP32E, facilitated AIV polymerase activity in human cells measured by a minireplicon assay (Long, 2016). Although highly conserved, the proteins are subtly different between species. In particular, all flighted birds have an additional 33 amino acids encoded in their ANP32A gene due to a chromosomal duplication introducing an additional exon (Long, 2016). Only this longer form of the protein can support the replication of AIV in avian cells and, if introduced artificially, in human cells. As described by the inventors, deletion of this 33 amino acid section from avian ANP32 results in a modified ANP32 protein which does not support avian polymerase activity and thus does not support replication of AIV in avian cells. However, while such a deletion could be exploited to generate birds having resistance to avian influenza viruses, such a strategy might not be desirable in the long term, given that such a deletion in the avian ANP32A sequence may drive evolution in chickens of mammalian adapted viruses by the commonly found adaptive mutation E627K in PB2 component of the viral polymerase.

The present invention addresses many of the problems of the prior art. The inventors have surprisingly shown that by modifying avian ANP32A in the LRR5 - central region, the ability of the ANP32A protein to support AIV polymerase is abrogated. The LRR5 - central region typically extends from residue 115 to residue 175 of the avian ANP32A protein, such as of the chicken ANP32A protein. Whilst, there can be provided a gene deleted chicken lacking ANP32A, knocking out chANP32A whilst considered to decrease polymerase activity was believed to be problematic as it was considered that it would completely abrogate the activity, as it was expected that chANP32B would fulfil some of the role of chANP32A in the knockout, as such redundancy has been observed in other species which have been studied. It is additionally considered that alternative functions of chANP32A may be lost in such knock outs that would be disadvantageous to the avians. The invention enables the generation of genome modified birds having resistance to influenza virus infection.

Accordingly, in a first aspect of the invention, there is provided a genome modified (cisgenic) avian, wherein said genome modified avian comprises a nucleic acid encoding a variant avian ANP32A protein, wherein said variant avian ANP32A protein comprises

(i) a sequence having at least 90% homology, (such as at least 92%, at least 95%, at least 98%, at least 99% or 100% homology) to sequence shown as residues 19 to 114 of an avian ANP32A polypeptide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:, 3, and SEQ ID NO: 4 and

(ii) wherein said variant avian ANP32A protein has at least one substitution of a residue in the sequence corresponding to residues 115 to 175 of said avian ANP32A polypeptide sequence Suitably a genome modified avian comprises alteration of a portion of the gene leading to a reduced ability to support AIV polymerase activity or abrogation of activity.

Sequence homology (or identity) may be determined using any suitable homology algorithm, using for example default parameters. For example, the BLAST algorithm may be employed, with parameters set to default values. The BLAST algorithm for nucleotide and protein sequences is described in detail at https://blast.ncbi.nlm.nih.gov/Blast.cgi, which is incorporated herein by reference.

SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 correspond to the wild-type ANP32A protein sequences for chicken, duck, turkey and zebrafinch respectively and are shown below.

1. Gallus gallus (SEQ ID NO: 1)

MDMKKRIHLELRNRTPSDVKELVLDNCRSYEGKIEGLTDE FEELE FLS T I

NVGLASVANLPKLNKLKKLELSDNRVSGGLEVLAEKCPNLTHLNLSGNKI

KDLGT IEPLKKLENLKSLDLFNCEVTNLNDYRENVFKLLPQLTYLDGYDR

DDKEAPDSDAEGYVEGLDDEEEDEDVLSLVKDRDDKEAPDSDAEGYVEGL

DDEEEDEDEEEYDDDAQWEDEEDEEEEEEGEEEDVSGEEEEDEEGYNDG

DVDDDEDEEEPDEERGQKRKREPEDEGDEDD

2. Anas platyrhynchos (SEQ ID NO: 2)

MDMKKRIHLELRNRTPSDVKELVLDNCRSYEGKIEGLTDE FEELE FLS T I

NVGLTSVANLPKLNKLKKLELSDNRI SGGLEVLAEKCPNLTHLNLSGNKI

KDLGT IEPLKKLENLKSLDLFNCEVTNLNDYRENVFKLLPQLTYLDGYDR

DDKEAPDSDAEGYVEGLDDEEEDEDVLSLVKDRDDKEAPDSDAEGYVEGL

DDEEEDEDEEEYDDDAQWEDEEDEEEEEEGEEEDVSGEEEEDEEGYNDG

EVDDDEDEEEPDEERGQKRKREPEDEGDEDD 3. Meleagris gallopavo (SEQ ID NO: 3)

MEQMAEATVCVHLERLAVKELVLDNCRSYEGKIEGLTDE FEELE FLS T IN

VGLTSVANLPKLNKLKKLELSDNRVSGGLEVLAEKCPNLTHLNLSGNKIK

DLGT IEPLKKLENLKSLDLFNCEVTNLNDYRENVFKLLPQLTYLDGYDRD

DKEAPDSDAEGYVEGLDDEEEDEDVLSLVKDRDDKEAPDSDAEGYVEGLD

DEEEDEDEEEYDDDAQWEDEEDEEEEEEGEEEDVSGEEEEDEEGYNDGD

VDDDEDEEEPDEERGQKRKREPEDEGDEDD

4. Taeniopygia guttata (SEQ ID NO: 4)

MEMKRRIHLELRNRTPSDVKELVLDNCRSYEGKIVGLTDE FEELEYLS T I

NVGLTSVANLPKLNKLKKLELGDNRI SGGLEVLAEKCPNLTHLNLSGNKL

KDLGT IEPLKKLENLKSLDLFNCEVTNLNDYRENVFKLLPQLTYLDGYDR

DDKEAPDSDAEGYVEELDDKEEDEDVLSLVKDRDDKEAPDSDAEGYVEGL

DDEEEDEDEEEYDDDAQWEDEEDEEEEEEEGEEEDVSGEEEEDEEGYND

GEVDDEEDEEEPEEERGQKRKREPEDEGDEDD

Suitably, there is provided a genome modified (cisgenic) avian, wherein said genome modified avian comprises a nucleic acid encoding a variant avian ANP32A protein, wherein said variant avian ANP32A protein comprises at least one substitution of a residue in the sequence corresponding to residues 115 to 175 of said avian ANP32A polypeptide sequence, and may further comprise at least one additional, at least two additional at least three additional, at least four additional, at least five additional mutations at a position from 25 to 119

the sequence has at least 90% homology, (such as at least 92%, at least 95%, at least 98%, at least 99% or 100% homology) to sequence shown as residues 19 to 114 of an avian ANP32A polypeptide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:, 3, and SEQ ID NO: 4 and the variant provides a reduced ability to support AIV polymerase activity or abrogates support of influenza polymerase.

Suitably, there is provided a genome modified (cisgenic) avian, wherein said genome modified avian comprises a nucleic acid encoding a variant avian ANP32A protein, wherein said variant avian ANP32A protein comprises at least one substitution of a residue in the sequence corresponding to residues 115 to 175 of said avian ANP32A polypeptide sequence, and further comprise at least one additional, at least two additional at least three additional, at least four additional, at least five additional mutations at a position from 25 to 119

Suitably there is provided a genome modified (cisgenic) avian, wherein said genome modified avian comprises a nucleic acid encoding a variant avian ANP32A protein, wherein said variant avian ANP32A protein has at least one substitution of a residue in the sequence corresponding to residues 115 to 175 of said avian ANP32A polypeptide sequence, suitably wherein a mutation is provided at position 129 or 130 or at both 129 and 130, and optionally further mutations upstream of position 115.

Suitably there is provided a genome modified (cisgenic) avian, wherein said genome modified avian comprises a nucleic acid encoding a variant avian ANP32A protein, wherein said variant avian ANP32A protein has at least two substitutions at positions 129 and 130 and optionally a further mutation(s) upstream of position 115.

Suitably there is provided a genome modified (cisgenic) avian, wherein said genome modified avian comprises a nucleic acid encoding a variant avian ANP32A protein, wherein a mutation is provided at position 129 or 130 or at both 129 and 130, and optionally a further mutation(s) upstream of position 115 and the sequence has at least 90% homology, (such as at least 92%, at least 95%, at least 98%, at least 99% or 100% homology) to sequence shown as residues 19 to 114 of an avian ANP32A polypeptide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:, 3, and SEQ ID NO: 4. Suitably the mutations provide reduced ability to support AIV polymerase activity or completely abrogate support of influenza polymerase but do not prevent ANP binding to polymerase nor to H3 or H4 histones.

It is considered it may be advantageous to provide mutations to provide a variant avian ANP32A protein at two distinct regions of the ANP32A protein to try and mitigate further mutations of the variant which cause gain of function.

Suitably the mutations to provide a variant avian ANP32A protein by modifying at least two distinct regions of the ANP32A may disrupt the interaction of ANP32A with at least two different proteins or disrupt the interaction of ANP32A with two different regions of the same protein. For example a first mutation may cause an allosteric effect to ANP32A and a second mutation may disrupt a direct interaction of ANP32A to another protein.

A second aspect of the invention provides an isolated nucleic acid encoding a variant avian ANP32A protein, wherein said variant avian ANP32A protein comprises (i) a sequence having at least 90% homology to sequence shown as residues 19 to 114 of an avian ANP32A polypeptide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:, 3, and SEQ ID NO: 4 wherein said variant avian ANP32A protein has at least one substitution of a residue in the sequence corresponding to residues 115 to 175 of said avian ANP32A polypeptide sequence.

Suitably any suitably method known in the art may be used to provide a variant of the ANP32A. Suitably CRISPR may be used in provide a variant.

Suitably a variant has at least one substitution of a residue in the sequence

corresponding to residues 115 to 175 of said avian ANP32A polypeptide sequence, and further comprises at least one additional, at least two additional at least three additional, at least four additional, at least five additional mutations at a position from

25 to 119 the sequence has at least 90% homology, (such as at least 92%, at least 95%, at least 98%, at least 99% or 100% homology) to sequence shown as residues 19 to 114 of an avian ANP32A polypeptide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:, 3, and SEQ ID NO: 4 and the variant provides a reduced ability to support AIV polymerase activity or completely abrogates support of influenza polymerase.

Suitably there is provided a genome modified (cisgenic) avian, wherein said genome modified avian comprises a nucleic acid encoding a variant avian ANP32A protein, wherein said variant avian ANP32A protein has at least one substitution of a residue in the sequence corresponding to residues 115 to 175 of said avian ANP32A polypeptide sequence, suitably wherein a mutation is provided at position 129 or 130 or at both 129 and 130, and optionally a further mutation(s) upstream of position 115.

Suitably there is provided a genome modified (cisgenic) avian, wherein said genome modified avian comprises a nucleic acid encoding a variant avian ANP32A protein, wherein a mutation is provided at position 129 or 130 or at both 129 and 130, and optionally a further mutation(s) upstream of position 115 and the sequence has at least 90% homology, (such as at least 92%, at least 95%, at least 98%, at least 99% or 100% homology) to sequence shown as residues 19 to 114 of an avian ANP32A polypeptide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:, 3, and SEQ ID NO: 4.

As will be appreciated, suitably the mutations abrogate support of influenza polymerase but do not prevent ANP binding to polymerase nor to H3 or H4 histones. It is considered it may be advantageous to provide mutations to provide a variant avian ANP32A protein at two distinct regions of the ANP32A protein to try and mitigate further mutations of the variant which cause gain of function.

Suitably the mutations to provide a variant avian ANP32A protein by modifying at two distinct regions of the ANP32A may disrupt the interaction of ANP32A with at least two different proteins or disrupt the interaction of ANP32A with two different regions of the same protein. For example a first mutation may cause an allosteric effect to ANP32A and a second mutation may disrupt a direct interaction of ANP32A to another protein.

A third aspect of the invention provides an ANP32A variant protein encoded by the nucleic acid according to the second aspect of the invention.

A fourth aspect of the invention provides an avian cell comprising the nucleic acid according to the second aspect of the invention or a protein according to the third aspect of the invention.

In a further aspect there is provided an avian cell comprising an isolated nucleic acid encoding a construct providing a variant as discussed herein which abrogates the ANP32a protein support of avian polymerase. Suitably other functions of ANP32a protein, such as histone binding, are not disrupted.

In one embodiment, the cell may be a germ cell, for example a primordial germ cell. Avian primordial germ cells (PGCs) are a self-renewing cell lineage that can be isolated from chicken embryos early in development, cultured indefinitely in vitro and when injected into a host embryo migrate to the developing gonads, resulting in generation of functional gametes derived from the cultured PGCs (Macdonald et al., 2010, Whyte et al., 2016). PGCs can be gene edited in vitro using TALENS or CRISPR/Cas9 vectors, clonally isolated and expanded, and then used to generate genome-edited offspring (Oishi et al., 2016; Taylor et al., 2017; MM, unpublished data; see Figure 2).

As described herein, the inventors have shown that substitutions in the LRR5-central region portion of the avian ANP32A protein confer resistance to AIV infection. Accordingly in one embodiment of the invention, said variant avian ANP32A protein sequence that abrogates support of avian polymerase includes a substitution of at least 1 residue, at least 2 residues, at least 3 residues, at least 4 residues, at least 5 residues, at least 6 residues, at least 7 residues, at least 8 residues, at least 9 residues, at least 10 residues, for example at least 2 to 10 contiguous residues, of the sequence 115 to 175 of said avian ANP32A polypeptide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:, 3, and SEQ ID NO: 4. In embodiments, there may be at least 10 residues substituted, for example, the variant avian ANP32A protein sequence may have a substitutions of at least 10, at least 20, such as at least 30, for example at least 50 residues, suitably contiguous residues, of the sequence 115 to 175 of said avian ANP32A polypeptide sequence in certain embodiments, and be unable to support avian polymerase. Suitably other functions of ANP32A are not abrogated by the substitutions. Suitably other functions of ANP32A are not inhibited or decreased by the substitutions.

The inventors have also shown that ANP32A variants (mutants) in which, compared to wild-type ANP32A protein sequences, the amino acid residues of the central domain are scrambled, do not support AIV polymerase activity. Accordingly, in embodiments of the invention, amino acid residues 149-175 are scrambled compared to the amino acid residues 149-175 of said avian ANP32A polypeptide sequence. In the context of the invention, a scrambled amino acid sequence, consists of the same amino acid residues as the sequence compared to that which it is scrambled but in which the order of said amino acids differs from that of the sequence to which it is compared. Such scrambled sequences may be considered to be variants provided by at least one substitution, as the amino acid provided at a defined position is substituted with an amino acid from another defined position. Suitably at least one substitution, at least two substitutions, at least three substitutions, at least four substitutions, at least five substitutions, at least six substitutions, at least seven substitutions, at least eight substitutions, at least nine substitutions, at least 10 substitutions, may be provided. Suitably at least fifteen, at least twenty, at least twenty five substitutions may be provided to effectively provide contiguous substitutions or scrambling in a portion of the residues in the region 149 -

175. In one embodiment of any aspect of the invention, said variant avian ANP32A protein sequence consist of the sequence shown as SEQ ID NO: 5:

5. Gallus gallus 149-175 scrambled (SEQ ID NO: 5)

MDMKKRIHLELRNRTPSDVKELVLDNCRSYEGKIEGLTDE FEELE FLS T I

NVGLASVANLPKLNKLKKLELSDNRVSGGLEVLAEKCPNLTHLNLSGNKI

KDLGT IEPLKKLENLKSLDLFNCEVTNLNDYRENVFKLLPQLTYLDGYGA

YDDGEEDEEVSELDEEADDKDDDPRVLSLVKDRDDKEAPDSDAEGYVEGL

DDEEEDEDEEEYDDDAQWEDEEDEEEEEEGEEEDVSGEEEEDEEGYNDG

DVDDDEDEEEPDEERGQKRKREPEDEGDEDD

In one embodiment of any of the aspects of the invention, said variant avian ANP32A protein comprises (i) a sequence having at least 90% homology to sequence shown as residues 19 to 114 of an avian ANP32A polypeptide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:, 3, and SEQ ID NO: 4 wherein said variant avian ANP32A protein has at least one substitution of a residue in the sequence corresponding to residues 115 to 175 of said avian ANP32A polypeptide sequence

As detailed herein, the inventors have shown that mutations in the LRR5 region of the ANP32A protein may lead to a lack of ability to support AIV polymerase activity.

Accordingly, in one such embodiment, said variant avian ANP32A protein sequence has at least one substitution of a residue in the residues corresponding to positions 115 to 140 of said avian ANP32A polypeptide sequence. In a particular embodiment, said variant avian ANP32A protein sequence has a substitution at position 129 of said avian ANP32A polypeptide sequence. In one embodiment, said substitution at position 129 is N129I. In another embodiment, said variant avian ANP32A protein sequence has a substitution at position 130. Suitably the substitution at position 130 is D130N.

In one embodiment, wherein said variant avian ANP32A protein sequence has a substitution at position 129, residues 115-128 and/or 130-175 correspond to the residues shown as residuesll5-128 and or 130-175 of said avian ANP32A polypeptide sequence. In one embodiment, wherein said variant avian ANP32A protein sequence has a substitution at position 130, residues 115-129 and/or 131-175 correspond to the residues shown as residuesll5-129 and or 131-175 of said avian ANP32A polypeptide sequence.

In certain embodiments, said variant avian ANP32A protein sequence comprises a sequence having at least 90% identity with the sequence shown as residues 176-208 of said avian ANP32A polypeptide sequence. This region corresponds to the 33 amino acid repeat region of the avian ANP32A protein sequence, deletion of which has been shown to abrogate the ability of avian ANP32A to support AIV polymerase activity. By retaining such a sequence in the variant ANP protein sequence encoded in a modified avian may help prevent evolution of mammalian adapted viruses by the commonly found adaptive mutation E627K in PB2 component of the viral polymerase.

In one embodiment of any aspect of the invention, said variant avian ANP32A protein sequence comprises or consists of the sequence shown as SEQ ID NO: 6: 6. Gallus gallus N129I (SEQ ID NO: 6)

MDMKKRIHLELRNRTPSDVKELVLDNCRSYEGKIEGLTDE FEELE FLS T I NVGLASVANLPKLNKLKKLELSDNRVSGGLEVLAEKCPNLTHLNLSGNKI KDLGT IEPLKKLENLKSLDLFNCEVTNLIDYRENVFKLLPQLTYLDGYDR

DDKEAPDSDAEGYVEGLDDEEEDEDVLSLVKDRDDKEAPDSDAEGYVEGL DDEEEDEDEEEYDDDAQWEDEEDEEEEEEGEEEDVSGEEEEDEEGYNDG DVDDDEDEEEPDEERGQKRKREPEDEGDEDD In one embodiment of any aspect of the invention, said variant avian ANP32A protein sequence comprises or consists of the sequence shown as SEQ ID NO: 7:

7. Gallus gallus D130N (SEQ ID NO: 7)

MDMKKRIHLELRNRTPSDVKELVLDNCRSYEGKIEGLTDE FEELE FLS T I

NVGLASVANLPKLNKLKKLELSDNRVSGGLEVLAEKCPNLTHLNLSGNKI KDLGT IEPLKKLENLKSLDLFNCEVTNLNNYRENVFKLLPQLTYLDGYDR DDKEAPDSDAEGYVEGLDDEEEDEDVLSLVKDRDDKEAPDSDAEGYVEGL DDEEEDEDEEEYDDDAQWEDEEDEEEEEEGEEEDVSGEEEEDEEGYNDG

DVDDDEDEEEPDEERGQKRKREPEDEGDEDD

A variant deletion mutation of avian ANP32A protein sequence comprises or consists of the sequence shown as SEQ ID NO: 8:

8. Gallus gallus LRR5 deletion (SEQ ID NO: 8)

MDMKKRIHLELRNRTPSDVKELVLDNCRSYEGKIEGLTDE FEELE FLS T I

NVGLASVANLPKLNKLKKLELSDNRVSGGLEVLAEKCPNLTHLNLSGNKI

KDLGT IEPLKKLENLTYLDGYDRDDKEAPDSDAEGYVEGLDDEEEDEDVL

SLVKDRDDKEAPDSDAEGYVEGLDDEEEDEDEEEYDDDAQWEDEEDEEE

EEEGEEEDVSGEEEEDEEGYNDGDVDDDEDEEEPDEERGQKRKREPEDEG

DEDD

This has a deletion of residues KSLDLFNCEVTNLNNYRENVFKLLPQLTYL (SEQ ID NO: 9) between the sequence portions ...KSLDLFNCEVTNLNNYRENVFKLLPQLTYL (SEQ ID NO: 10) and TYLDGYDR...

In an embodiment of any of the aspects of the invention, said variant avian ANP32A protein, when compared to wild-type ANP32A protein of the same species, has reduced ability to support AIV polymerase activity. In one embodiment, said variant avian ANP32A protein supports at least 50% less, such as at least 60% less, such as at least 75% less, such as at least 80% less, for example at least 90% or at least 95% less AIV polymerase activity. In one embodiment, said variant avian ANP32A protein supports 100% less AIV polymerase activity. The ability of a variant ANP32A protein to support AIV polymerase activity may be measured using any suitable assay, for example an assay as described herein. Thus, in one embodiment, the ability of a variant ANP32A protein to support AIV polymerase activity may be measured using the plasmid -based minigenome assay such as that described in Example 3.

In the invention, the nucleic acid construct of and for use in the invention may be targeted to germ cells. In one aspect of the invention there is provided a method of producing a genome modified avian, said method comprising inserting a nucleic acid construct comprising the nucleic acid of the second aspect of the invention into germ cells. The modified germ cells are introduced into fertilised eggs of an avian and then the method comprises incubating said eggs to hatching, wherein said nucleic acid is integrated into the introduced germ cells of said host embryo., Said construct is integrated not only into the germ cells of said embryo but also the germ cells and somatic cells of all offspring produced subsequently from the bird resulting from said embryo. Suitably the method may further comprise crossing male and female offspring from one or more of said host avian to produce offspring avians with somatic cells and germ cells having the genetic characteristics of the transplanted germ cells. Detection of offspring from the transplanted germ cells can be identified by standard genomic sequencing techniques.

In the invention, the modified avian may be any suitable bird. For example, the avian may be of the order galliformes, aseriformes, passeriformes, gruiformes,

Struthioniformes, rheiformes, casuariformes, apyerygiformes, otidiformes,

columbiformes, sphenisciformes, cathartiformes, accipitriformes, strigiformes, psittaciformes, or falconiformes. Suitably, the avian is a chicken, turkey, duck, goose, quail, pheasant, grouse, guinea fowl, pigeon, ostrich, emu, song bird, parrot, finch, sparrow, or falcon. In one embodiment, the avian is a chicken.

Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying figures in which:

Figure 1 a) shows a structural schematic of chicken ANP32A and human ANP32A/B or chicken ANP32B.

Figure 1 b) shows diagram of the chicken ANP32A gene displaying the exons in boxes and highlighting the duplicated sequence that partially makes up the 33aa insertion that is not observed in human ANP32A/B or chicken ANP32B. Figure 1 c) illustrates GE Human eHAP knockout cells lacking ANP32A and ANP32B. Polymerase assay of H5N1 50-92 polymerase with either no PB2, PB2 627E or 627K (human adapted), co-expressed with either human ANP32A or B and chicken ANP32A or B.

Figure 2 shows schematically a protocol to differentiate PGCs into fibroblasts which will support viral replication a) PGCs can isolated from chicken embryos and cultured indefinitely in serum-free medium b) PGCs can be modified in vitro using CRISPR/Cas9 vectors c) The inventors have established protocol to sort single PGCs. The single cell clones are sequenced to identify PGCs with the desired genetic mutation d) The inventors have developed a protocol to differentiate the PGCs into fibroblasts which can then be infected with AIV and transfected at high efficiency e) The GE PGCs are injected into host embryos from the inventors' DDX4 null line of chickens, hatched and raised to sexual maturity. The host birds are crossed to produce G1 edited offspring.

Figure 3 illustrates influenza virus replication in fibroblasts derived from GE PGCs. a) Western blot of WT, D33 or KO PGC fibroblasts probing for chicken ANP32A protein. b) infected WT, D33 or KO PGC fibroblasts with PR8 at MOI 0.001. c) WT or KO PGC fibroblasts infected with H5N1 50-92 virus with PR8 HA and NA external genes at MOI 0.001. d) Polymerase activity of reconstituted viral polymerase bearing either PB2 627E (filled) or PB2 627K (hatched) in fibroblasts derived from GE PGCs, co-expressed with empty vector or full length chicken ANP32A.

Figure 4 shows that human and chicken cells expressing altered or variant ANP32A proteins restrict AIV polymerase activity and virus replication. a) Schematic of the ANP32A protein structure illustrating 5 domains within the LRR. b) Polymerase activity of H5N1 PB2 E627 AIV in human 293T cells with co expressed with ANP32 constructs: huANP32B and chANP32A substituted for the indicated domains from chANP32B (line over graph portion) and containing the 33 amino acid domain (33) where indicated c) Human 293T cells expressing mCherry tagged ANP32A (red), nuclear staining in blue, 1. Empty vector, 2. WT chANP32A, 3. chANP32A N129I, 4. chANP32A D130N. d) Interaction between reconstituted influenza polymerase tagged on PB1 and chANP32A or chANP32A N129I mutant measured in a split luciferase assay e) H5N2 E627 polymerase activity in KO fibroblast PGC cells with co-expressed chANP32A or the N129I mutant f) GE Human eHAP knockout cells lacking huANP32A and B. Polymerase assay of H5N1 50-92 polymerase with either no PB2, PB2 627E or 627K (human adapted), co-expressed with chicken ANP32A and scrambled mutants or chimeric human ANP32B with 33aa insertion and the central domain of chicken ANP32B.

Figure 5 shows that chicken cells expressing chANP32A with either a deletion of the LRR5 domain (residues 115-141 deleted) or an N129I residue change restrict AIV polymerase activity.

KO PGC fibroblast-like cells were transfected with H5N2 E627 polymerase together with Firefly minigenome reporter, Renilla expression plasmid and either: empty vector, chANP32A, chANP32AALRR5 or chANP32AN129l and luciferase activity measured after 24 hours. Data are firefly activity normalised to Renilla ±SEM. One way ANOVA, ****= p<0.0001.

Figure 6 illustrates a two amino acid (N129I,D130N) genome edit of ANP32A in chicken cells where a change in inhibition of viral polymerase activity occurs.

Figure 7 illustrates five loss of activity mutations in huANP32 in the LRR region at residues D25A, E45A, E70A, E73A and D119A which abrogate support of influenza polymerase but do not prevent ANP binding to polymerase nor to H3 or H4 histones. Such mutations are considered advantageous over simple truncations as they do not abrogate H3 and H4 binding. The binding of the huANP32B protein is indicative of that expected from the chicken homologue. Figure 8 illustrates an expanded portion of figure 7

Figure 9 illustrates that huANP32B proteins with the loss of activity mutations maintains interaction with human histone H3 and H4. The behaviour of the huANP32B protein is indicative of that expected from the chicken homologue.

Figure 10 provides an illustrations of SEQ ID Nos: 1, 2, 3, 4, 6 and 7 aligned and which is used as the reference for the numbering discussed in this document.

Examples

Example 1

The requirements within ANP32 to support influenza polymerase were investigated using human cells gene edited to lack endogenous ANP32 proteins. Human cells lacking both human ANP32A or ANP32B do not support polymerase activity of either AIV or of a mammalian adapted influenza virus (Fig lc). Complementation with human ANP32A or B can rescue a human-adapted viral polymerase (PB2 627K), complementation of chicken ANP32A can rescue both avian (PB2 627E) or human adapted (PB2 627K) polymerase, whereas chicken ANP32B could not support either polymerase (Figure lc). Interestingly, introduction of the 33 amino acids into chicken ANP32B did not support activity of AIV polymerase in human cells (Figure 4b).

Example 2

Chicken cells lacking ANP32A are viable and refractory to AIV replication

Cultured PGCs, like many stem cell populations, are not permissive for AIV replication (Wu et al., 2018; MM, N. Smith, P. Digard (Roslin Institute), unpublished results). The inventors have established a protocol to differentiate PGCs into fibroblasts which will support viral replication (Figure 2). This protocol has allowed us to genetically edit ANP32A in PGCs, differentiate them into fibroblasts, and investigate the function of ANP32A in vitro (Figures 3 and 4). The edited PGCs may be used in the future to generate chickens carrying ANP32A mutations present in the edited PGCs (Figure 2).

The inventors used CRISPR/Cas9 to delete 8 bp of exon 1 of the chicken ANP32A gene. This deletion causes a frame shift in the coding sequence of ANP32A and no detectable ANP32A protein (KO) (Figure 3a). The inventors also used two separate CRISPR/Cas9 molecules to delete exon 5, and this removed the 33 amino acid domain which is required to support the unadapted AIV RdRp function (D33). The inventors

differentiated the ANP32A-edited PGCs into fibroblasts and infected the fibroblasts with influenza virus PR8 (Figure 3b). PR8 is a human adapted laboratory strain of influenza virus that contains human-adapted viral polymerase (PB2K627). The inventors observed that PR8 virus did not replicate in ANP32A-knockout cell line (KO PGCs) (red line Figure 3b). In the ANP32A-exon5-deleted cell line (D33, blue lines), the human-adapted PR8 virus replicated to the same extent as in unedited WT cells, since PB2 K627 can co-opt shorter forms of ANP32 which lack the 33a insert. An avian H5N1 virus, 50-92, that carries the external HA and NA genes of PR8 did not replicate in the KO PGCs (red line Figure 3c), suggesting that in the absence of chANP32A, AIV cannot replicate in chicken cells.

Example 3

The inventors then performed an influenza virus plasmid-based minigenome replication assay to analyse RdRp function with both an avian and a human adapted polymerase. The inventors used reconstituted viral polymerase derived from H5N1 AIV with either PB2 E627 (avian-like) or PB2 K627 (human-like) and with or without rescue by co expression of full length chANP32A (Figure 3d). As expected, both PB2 E627 and K627 were active in WT fibroblasts (Figure 3d). In AN32PA-exon5-deleted cells (D33), PB2 E627 polymerase activity was dramatically decreased; activity was rescued by co expression of full length chANP32A in these cells. Polymerase activity with PB2 K627 was not reduced in ANP32A-exon5-deleted cell line (D33), since the PB2K mutation adapts the polymerase for use of a shorter ANP32A protein. Strikingly, activity of either polymerase was completely lost in the ANP32A-knockout line (KO PGCs), in line with the failure of viral replication in these cells. Activity could be rescued by co-expressed chANP32A. Complete loss-of-expression of ANP32A prevents avian influenza virus (non- adapted and mammalian-adapted) replication in chicken cells due to failure of viral polymerase function.

Example 4

As described above, the inventors have shown that chANP32B does not support AIV polymerase activity, even when the 33 amino acids from chANP32A are inserted (Figure 4b, chANP32B³³). By generating chimeric human and chicken ANP32B proteins together with the 33 amino acid insertion, the inventors found that the 5th leucine rich repeat (LRR) of chicken ANP32B was responsible for this loss of activity (Figure 4b,

huANP32Bi__RR5 ³³) and the central domain of chicken ANP32B caused a partial loss in polymerase activity (Figure 4b&f, huANP32Bc_ENT ³³). When the inventors introduced each of the 5 amino acid differences of the 5th LRR of chANP32B into chANP32A the inventors found that a single amino acid change, N129I, abrogated the ability of ANP32A to bind to and support AIV 627E polymerase (Figure 4 c,d&e). The inventors used a novel assay (split luciferase) to probe binding between PB1 and chicken ANP32A. PB1 interacted with full length ANP32A but not with the single amino acid mutant N129I. In addition to changes in LRR5, the inventors found that scrambling amino acids 149-175 (the sequence from which the duplicated 33aa insertion is derived) or 176-208 (the 33aa insertion) was sufficient to prevent these altered ANP32A proteins from supporting AIV polymerase activity. Moreover, scrambling amino acids 149-175 could also prevent activity of human adapted influenza virus polymerase (Figure 4f). In fact, using ANP32A knockout chicken cells, reintroducing a chANP32A protein lacking either the LRR5 domain (residues 115-141) or the single N129I residue change could not support viral polymerase activity (Figure 5). The results indicate that targeted engineering of the chANP32A gene between amino acids 115-175, will prevent AIV replication, with minimal impact on the natural function of chANP32A. All documents referred to in this specification are herein incorporated by reference. Various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.

Claims

1. An isolated nucleic acid encoding a variant avian ANP32A protein, wherein said variant avian ANP32A protein comprises (i) a sequence having at least 90% homology to sequence shown as residues 19 to 114 of an avian ANP32A polypeptide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:, 3, and SEQ ID NO: 4 wherein said variant avian ANP32A protein has at least one substitution of a residue in the sequence corresponding to residues 115 to 175 of said avian ANP32A polypeptide sequence

2. The isolated nucleic acid according to claim 1, wherein said variant avian ANP32A protein sequence has at least one substitution of a residue in the residues corresponding to positions 115 to 140 of said avian ANP32A polypeptide sequence.

3. The isolated nucleic acid according to claim 1 or claim 2 wherein said variant avian ANP32A protein sequence has a substitution at position 129 of said avian ANP32A polypeptide sequence or a substitution at position 130 of said avian ANP32A polypeptide sequence or a combination thereof.

4. The isolated nucleic acid according to claim 3, wherein said substitution at position 129 is N129I and at position 130 is D130N.

5. The isolated nucleic acid according to claim 4, wherein when the substitution is N129I, residues 115-128 and or 130-175 correspond to the residues shown as residues 115-128 and or 130-175 of said avian ANP32A polypeptide sequence and wherein the substitution is D130N residues 115-129 and or 131-175 correspond to the residues shown as residues 115-129 and or 131-175 of said avian ANP32A polypeptide sequence, or where both substitutions N129I and D130N are made residues 115-128 and or 131-175 correspond to the residues shown as residues 115-128 and or 131-175 of said avian ANP32A polypeptide sequence .

6. The isolated nucleic acid according to any one of the preceding claims, wherein said variant avian ANP32A protein sequence comprises a sequence having at least 90% identity with the sequence shown as residues 176-281 of said avian ANP32A polypeptide sequence.

7. The isolated nucleic acid according to claim 6, wherein said variant avian ANP32A protein sequence consists of the amino acid sequence shown as SEQ ID NO: 5.

8. The isolated nucleic acid according to claim 6, wherein said variant avian ANP32A protein sequence consists of the amino acid sequence shown as SEQ ID NO: 6.

9. An ANP32A protein encoded by the nucleic acid according to any one of the preceding claims.

10. An avian cell comprising the nucleic acid according to any one of claims 1 to 10 or the protein according to claim 9.

11. The cell according to 10, wherein said cell is a primordial germ cell.

12. A genome modified avian comprising the nucleic acid according to any one of claims 1 to 7, the protein according to claim 9, or the cell according to any of claims 10 or 11.