JP-2022532139-A5 -

Dates

Publication Date

20230516

Application Date

20200508

Description

This disclosure includes the following embodiments.Embodiment 1A method for editing nucleic acid bases of a hepatitis B virus (HBV) genome, the method comprising contacting the HBV genome with one or more guide RNAs and a base-edited organism comprising a polynucleotide programmable DNA-binding domain and an adenosine deaminase or cytidine deaminase domain, wherein the guide RNA targets the base-edited organism to result in modification of the nucleic acid bases of the HBV genome.Embodiment 2The method according to Embodiment 1, wherein the nucleic acid base is located in a polynucleotide encoding the HBV protein.Embodiment 3The method according to Embodiment 1 or 2, wherein the contact is performed in eukaryotic cells, mammalian cells, or human cells.Embodiment 4The method according to any one of Embodiments 1 to 3, wherein the cells are in vivo or ex vivo.Embodiment 5The method according to any one of Embodiments 1 to 4, wherein the cytidine deaminase converts target C in the HBV genome to U.Embodiment 6The method according to any one of Embodiments 1 to 4, wherein the cytidine deaminase converts target C and G within the polynucleotide encoding the HBV protein to T and A.Embodiment 7The method according to any one of Embodiments 1 to 4, wherein the adenosine deaminase converts target A and T within the polynucleotide encoding the HBV protein to G and C.Embodiment 8The method according to any one of Embodiments 2 to 7, wherein the modification of the nucleic acid base in the polynucleotide encoding the HBV protein produces an immature stop codon.Embodiment 9The method according to Embodiment 8, wherein the modification of the nucleic acid base causes an R87* or W120* termination in the HBV X protein.Embodiment 10The method according to Embodiment 8, wherein the modification of the nucleic acid base produces W35* or W36* in the HBV S protein.Embodiment 11The method according to any one of Embodiments 2 to 7, wherein the modification of the HBV polynucleotide is a missense mutation.Embodiment 12The method according to Embodiment 11, wherein the missense mutation is located in the HBV pol gene.Embodiment 13The method according to Embodiment 12, wherein the missense mutation produces E24G, L25F, P26F, R27C, V48A, V48I, S382F, V378I, V378A, V379I, V379A, L377F, D380G, D380N, F381P, R376G, A422T, F423P, A432V, M433V, P434S, D540G, A688V, D689G, A717T, E718K, P713S, P713L, or L719P in the HBV polymerase protein encoded by the HBV pol gene.Embodiment 14The method according to Embodiment 11, wherein the missense mutation is located in the HBV core gene.Embodiment 15The method according to Embodiment 14, wherein the missense mutation causes T160A, T160A, P161F, S162L, C183R, or *184Q in the HBV core protein encoded by the HBV core gene.Embodiment 16The method according to Embodiment 11, wherein the missense mutation is located in the HBV X gene.Embodiment 17The method according to Embodiment 16, wherein the missense mutation causes H86R, W120R, E122K, E121K, or L141P in the HBV X protein encoded by the HBV X gene.Embodiment 18The method according to Embodiment 11, wherein the missense mutation is located in the HBV S gene.Embodiment 19The method according to Embodiment 18, wherein the missense mutation causes S38F, L39F, W35R, W36R, T37I, T37A, R78Q, S34L, F80P, or D33G in the HBV S protein encoded by the HBV S gene.Embodiment 20The method according to any one of Embodiments 1 to 19, wherein the polynucleotide programmable DNA-binding domain is Cas9 selected from Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), Steptococcus canis Cas9 (ScCas9), or a variant thereof.Embodiment 21The method according to Embodiment 18, wherein Cas9 has protospacer adjacent motif (PAM) specificity for nucleic acid sequences selected from 5'-NGG-3', 5'-NAG-3', 5'-NGA-3', 5'-NAA-3', 5'-NNAGGA-3', or 5'-NNACCA-3'.Embodiment 22The method according to any one of Embodiments 1 to 20, wherein the polynucleotide programmable DNA-binding domain comprises a modified Cas9 having modified protospacer adjacent motif (PAM) specificity.Embodiment 23The method according to Embodiment 19, wherein the nucleic acid sequence of the modified PAM is selected from 5'-NNNRRT-3', NGA-3', 5'-NGCG-3', 5'-NGN-3', NGCN-3', 5'-NGTN-3', or 5'-NAA-3'.Embodiment 24The method according to any one of Embodiments 1 to 23, wherein the polynucleotide programmable DNA-binding domain is a nuclease-inactive variant or a nickas variant.Embodiment 25The method according to Embodiment 24, wherein the nuclease-inactivating variant or nickase variant is a nuclease-inactivating Cas9 (dCas9) comprising an amino acid substitution D10A or a corresponding amino acid substitution.Embodiment 26The method according to any one of Embodiments 1 to 25, wherein the adenosine deaminase domain can deaminate adenine in deoxyribonucleic acid (DNA).Embodiment 27The method according to any one of Embodiments 1 to 26, wherein the