Plant Vectors, Compositions and Uses Relating Thereto
The present disclosure relates to a single stranded RNA vector suitable for introducing a therapeutic agent, such as a peptide, a protein or a small RNA, into a host plant. The vector does not encode for any movement protein or coat protein, but is capable of capable of systemic and phloem-limited movement and replication within the host plant.
1 . A ribonucleic acid (RNA) vector comprising a heterologous segment(s), wherein said heterologous element(s) is attached to the RNA vector through a scaffold structure.
2 . The RNA vector of claim 1 , which is a plus-sense singled stranded RNA vector.
3 . The RNA vector of claim 1 , wherein said scaffold structure comprises a lock and dock structure.
4 . The RNA vector of claim 1 wherein said scaffold structure comprises a tetraloop sequence.
5 . The RNA vector of claim 4 , wherein said tetraloop sequence is a GNRA tetraloop sequence, such as GAAA.
6 . The RNA vector of claim 1 , wherein said scaffold structure comprises a branched structure comprising a stem element and a first branch portion attached to the RNA vector and a second branch portion comprising an insert site attached to said heterologous segment(s).
7 . The RNA vector of claim 1 , which comprises a 3′ Cap Independent Translation Enhancer (3′ CITE) comprising the nucleic acid sequence(s) of SEQ ID NO: 4 and/or SEQ ID NO: 5.
8 . The RNA vector of claim 7 , wherein said 3′ CITE comprises the nucleic acid sequence of SEQ ID NO: 3.
9 . The RNA vector of claim 1 , comprising a replication element(s) comprising one or more conserved polynucleotide sequence(s) having the nucleic acid sequence of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and/or SEQ ID NO: 14.
10 . The RNA vector of claim 9 , wherein said replication element(s) further comprises one of more conserved polynucleotide sequence(s) having the nucleic acid sequence of: SEQ ID NO: 15 and/or SEQ ID NO: 16.
11 . The RNA vector of claim 1 , which is derived from citrus yellow vein associated virus (SEQ ID NO:1) or an iRNA relative thereof.
12 . The RNA vector of claim 1 , which comprises a truncated hairpin relative to a corresponding wild-type RNA molecule.
13 . The RNA vector of claim 12 , wherein said scaffold is attached to said truncated hairpin.
14 - 18 . (canceled)
19 . The RNA vector of claim 1 , which is capable of systemic and phloem-limited movement and replication within a host plant.
20 . The RNA vector of claim 1 , which is derived from citrus yellow vein associated virus (CYVaV) having the nucleic acid sequence of SEQ ID NO:1.
21 . The RNA vector of claim 20 , which comprises one of more of said heterologous segment(s) at one or more of positions 2250, 2301, 2319, 2330, 2331, 2336, 2375 and 2083 of a CYVaV based RNA.
22 - 30 . (canceled)
31 . A method of making an RNA-based vector comprising truncating a hairpin of a wild-type RNA molecule and thereby forming an insert site, and attaching a heterologous segment(s) to the insert site.
32 . The method of claim 31 , wherein said heterologous segment(s) is attached to the truncated hairpin via a scaffold structure.
33 - 38 . (canceled)
39 . A method of making an RNA-based vector comprising attaching a heterologous element(s) to an insert site of an RNA molecule via a scaffold structure.
40 . The method of claim 39 , comprising the further step of forming, prior to said attaching step, said insert site by truncating a hairpin of said RNA molecule.