Methods and compositions for targeted trans-splicing
The present disclosure provides compositions and methods of use thereof for targeting trans-splicing of a pre-mRNA in a cell.
1 . A method for trans-splicing one or more pre-mRNA target sequences in a eukaryotic cell comprising:
(a) contacting the eukaryotic cell comprising the one or more pre-mRNA target sequences with one or more trans-splicing RNA molecules wherein each trans-splicing RNA molecule comprises:
(i) at least one sequence comprising a non-coding RNA (ncRNA), wherein the ncRNA is a small nucleolar RNA (snoRNA) sequence of 50 to 300 nucleotides in length, wherein the snoRNA sequence comprises:
a C box having the polynucleotide sequence of RUGAUGA, wherein R is A or G,
a D box having the polynucleotide sequence of CUGA,
a C′ box having the polynucleotide sequence of RUGAUGA, wherein
R is A or G, and
a D′ box having the polynucleotide sequence of CUGA;
(ii) at least one splice acceptor site or splice donor site;
(iii) at least one exonic sequence, wherein the at least one splice acceptor site or splice donor site is located at a 3′ boundary of the at least one exonic sequence, and wherein the at least one splice acceptor site or splice donor site and the at least one exonic sequence is located 5′ of the snoRNA sequence; and
(iv) one or more binding domains, each comprising a nucleic acid sequence of 4 to 300 nucleotides in length, and having at least 95% complementarity to the one or more pre-mRNA target sequences, wherein the snoRNA sequence comprises at least one binding domain positioned (i) upstream of the C box; (ii) between the C box and the D′ box; (iii) between the C′ box and the D box; (iv) downstream of the D box; or (v) a combination of (i)-(iv);
(b) binding at least a portion of the one or more binding domains of the one or more trans-splicing RNA molecules to the one or more pre-mRNA target sequences via complementary base pairing;
(c) recruiting a ribonucleoprotein (RNP) to direct splicing of the at least one exonic sequence into the one or more pre-mRNA target sequences; and
(d) splicing the at least one exonic sequence into the one or more pre-mRNA target sequences.
2 . The method of claim 1 , wherein the one or more binding domains comprise 2 binding domains, or wherein the one or more binding domains comprise more than 2 binding domains.
3 . The method of claim 1 , wherein the one or more binding domains are each 5 to 20, 5 to 30, 5 to 40, 5 to 50, 10 to 50, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 50 to 150, 50 to 200, 50 to 250, 100 to 150, 100 to 200, 100 to 250, or 100 to 300 nucleotides in length, and each have 95% complementarity to the one or more pre-mRNA target sequences.
4 . The method of claim 1 , wherein the one or more binding domains comprise 4 to 30 nucleotides having at least 95% complementarity to the one or more pre-mRNA target sequences.
5 . The method of claim 1 , wherein the one or more pre-mRNA target sequences comprises an USH2A pre-mRNA sequence, or wherein the one or more pre-mRNA target sequences comprises intron 12 and/or exon 13 of an USH2A pre-mRNA.
6 . The method of claim 1 , wherein the snoRNA sequence comprises a nucleic acid sequence of SNORD45A, SNORD51, SNORD10, or SNORD70.
7 . The method of claim 1 , wherein the one or more trans-splicing RNA molecules further comprises one or more splicing signals.
8 . The method of claim 7 , wherein the one or more splicing signals are selected from an exonic splicing enhancer (ESE), an intronic splicing enhancer (ISE), an exonic splicing silencer (ESS), intronic splicing silencer (ISS), a polypyrimidine tract, a branch point, and a combination thereof.
9 . The method of claim 1 , wherein the one or more trans-splicing RNA molecules comprises one or more polypyrimidine tracts, one or more branch points, and at least one splice acceptor site or splice donor site and is suitable for 5′ editing of the one or more pre-mRNA target sequences.
10 . The method of claim 1 , wherein the contacting step comprises contacting the cell with a plasmid, viral vector, or non-viral vector encoding the one or more trans-splicing RNA molecules; and/or wherein the contacting step comprises using a delivery vehicle comprising a lipid nanoparticle (LNP) or a polymeric nanoparticle.
11 . The method of claim 1 , wherein the contacting step comprises contacting the cell with multiple plasmids, viral vectors, or non-viral vectors encoding distinct trans-splicing RNA molecules; and/or wherein the contacting step comprises using a delivery vehicle comprising a lipid nanoparticle (LNP) or a polymeric nanoparticle.