JP-2022512395-A5 -

Dates

Publication Date

20221220

Application Date

20191212

Description

The secondary structure of the YNPX 2 DXGX 2 N motif (SEQ ID NO: 829) in the N22 domain of ORF1 also has a conserved surrounding secondary structure. Starting with a 5-6AA β chain, this is cleaved after the tyrosine (Y) at position 1 of the motif, and most of the motif is sequenced as an 8-9AA random coil to the terminal asparagine (N), at which point another β chain of 7-8AA is initiated. An alignment of an exemplary anelovirus ORF1 N22 motif sequence is shown in Figure 48. The tyrosine in this motif cleaves the β chain, and the second β chain is initiated at the terminal asparagine of the motif. In certain embodiments, for example, the following are provided: (Item 1) below: (I) Below: (a) Promoter element and (b) A nucleic acid sequence encoding an external effector, wherein the nucleic acid sequence is operably linked to the promoter element, and the external effector is a growth hormone (e.g., hGH); (c) Below: A 5' UTR domain containing the nucleic acid sequence of nucleotides 323-393 of sequence number 54, or a nucleic acid sequence that is at least 90% identical thereto, Genetic elements containing; (II) Protein outer layer containing polypeptides encoded by the anelovirus ORF1 nucleic acid, such as the ORF1 molecule. In synthetic anesomes containing; The gene element is confined within the proteinaceous outer layer; and A synthetic anesome capable of delivering the gene element into a human cell. (Item 2) The synthetic anestroma according to item 1, wherein the ORF1 molecule contains the amino acid sequence of SEQ ID NO: 217, or an amino acid sequence having at least 90% identity thereto. (Item 3) The synthetic anestroma according to any one of items 1 to 2, wherein the ORF1 molecule is encoded by nucleotides 612 to 2612 of sequence number 54. (Item 4) The synthetic anestroma according to any one of items 1 to 3, wherein the gene element comprises the nucleic acid sequence of nucleotides 2868 to 2929 of SEQ ID NO: 54, or a nucleic acid sequence having at least 85% sequence identity thereto. (Item 5) The synthetic anestroma according to any one of items 1 to 4, wherein the ORF1 molecule includes an amino acid sequence comprising one or more of the amino acid sequences of the arg-rich region, jelly-roll domain, hypervariable domain, N22 domain, and/or C-terminal domain listed in Table 16, or an amino acid sequence having at least 85% identity thereto. (Item 6) The synthetic anestroma according to any one of items 1 to 5, wherein the ORF1 molecule contains the amino acid sequence of SEQ ID NO: 58, or a nucleic acid sequence having at least 85% sequence identity thereto. (Item 7) A synthetic anestroma according to any one of items 1 to 6, further comprising a polypeptide containing the amino acid sequence of ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 listed in Table 16, or an amino acid sequence having at least 85% identity thereto. (Item 8) A synthetic anestrome according to any one of items 1 to 7, wherein the gene element codes for the amino acid sequence of ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 listed in Table 16, or an amino acid sequence having at least 85% identity thereto. (Item 9) The synthetic anestrome according to any one of items 1 to 8, wherein the synthetic anestrome does not contain a polypeptide comprising the amino acid sequence of ORF2, ORF2/2, ORF2/3, TAIP, ORF1/1, or ORF1/2 listed in Table 16, or an amino acid sequence having at least 85% identity thereto. (Item 10) A synthetic anestrome according to any one of items 1 to 9, wherein the gene element does not encode the amino acid sequence of ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 listed in Table 16, or an amino acid sequence having at least 85% identity thereto. (Item 11) The synthetic anestroma according to any one of items 1 to 10, wherein the ORF1 molecule contains the amino acid sequence YNPX 2 DXGX 2 N (SEQ ID NO: 829) (where X and n are each independently a sequence of any n amino acids). (Item 12) The synthetic anestronomy according to item 11 , wherein the ORF1 molecule further comprises a first β-chain and a second β-chain flanking with the amino acid sequence YNPX 2 DXGX 2 N (SEQ ID NO: 829), for example, the first β-chain comprising a tyrosine (Y) residue of the amino acid sequence YNPX 2 DXGX 2 N (SEQ ID NO: 829), and/or the second β-chain comprising a second asparagine (N) residue (N to C) of the amino acid sequence YNPX 2 DXGX 2 N (SEQ ID NO: 829). (Item 13) The synthetic anestroma according to any one of items 1 to 12, wherein the ORF1 molecule comprises, in order from the N-terminus to the C-terminus, a first β-chain, a second β-chain, a first α-helix, a third β-chain, a fourth β-chain, a fifth β-chain, a second α-helix, a sixth β-chain, a seventh β-chain, an eighth β-chain, and a ninth β-chain. (Item 14) The synthetic anestroma according to any one of items 1 to 13, wherein the gene element can be amplified by rolling circle replication within a host cell to produce, for example, at least eight copies. (