Compositions and methods useful for prophylaxis of organophosphates
View Patent ↗rAAV compositions useful for preventing the toxicity associated with organophosphate exposure are provided herein. The rAAV co-express a human butyrylcholinesterase and a proline-rich peptide. Each the esterase and the peptide may have an exogenous signal sequence. Also provided are compositions, e.g., aqueous liquid suspensions containing the rAAV.
1. A replication-defective, recombinant AAV (rAAV) which comprises an AAV capsid having a vector genome packaged therein, said vector genome comprising:
(a) an AAV 5′ ITR;
(b) a promoter;
(c) a 5′ UTR;
(d) a first leader sequence operably linked to a codon optimized human butyrlcholinesterase (hBChE) coding sequence, wherein the codon optimized hBChE coding sequence is selected from SEQ ID NOs: 6, 7, 8, 9, 3, 4, 5, 10, 11, 12 or 13;
(e) a linker;
(f) a second leader sequence operably linked to a codon optimized nucleic acid sequence encoding a proline rich peptide;
(g) an optional 3′UTR;
(h) a polyA; and
(i) an AAV3′ ITR.
2. The rAAV according to claim 1 , wherein the leader sequences for expression of hBChE and PRP are selected from a human interleukin-2 leader or a native hBChE leader sequence.
3. The rAAV according to claim 1 , wherein the origin of the proline rich peptide is from Proline-rich membrane anchor 1 (Primal) and lamellipodin (LPDN).
4. The rAAV according to claim 1 , wherein the second leader sequence is the same as the leader sequence which is operably linked to the BChE coding sequence.
5. The rAAV according to claim 1 , wherein the linker is an F2A.
6. The rAAV according to claim 1 , wherein the linker is an IRES.
7. The rAAV according to claim 1 , wherein the vector genome further comprises a 3′ UTR sequence.
8. The rAAV according to claim 7 , wherein the 3′ UTR is a woodchuck post-transcription regulatory element.
9. The rAAV according to claim 1 , wherein the polyA is SV40 or rabbit beta globin.
10. The rAAV according to claim 1 , wherein the vector genome comprises an expression cassette comprising one of:
(a) SEQ ID NO: 34 (C4/UbC-hIL2-BchE C1-IRES-LPDN);
(b) nt 14 to nt 4162 of SEQ ID NO: 30 (UbC-cmyc-IL2-BChE C1-IRES-IL2-PRIMA1);
(c) nt 14 to nt 4874 of SEQ ID NO: 31 (UbC-cmyc-IL2-BChE C1-IRES-IL2-LPDN);
(d) SEQ ID NO: 32 (CB7-cmyc-IL2-BChE C1-IRES-IL2-PRIMA1);
(e) SEQ ID NO: 33 (CB7-cmyc-IL2-BChE C1-IRES-IL2-LPDN); or
(f) SEQ ID NO: 35 (C4/UbC-hIL2-BChE C1-IRES-PRIMA1).
11. The rAAV according to claim 1 , wherein the AAV capsid has tropism for skeletal muscle.
12. The rAAV according to claim 1 , wherein the AAV capsid is AAV8.
13. An aqueous liquid suspension comprising an rAAV according to claim 1 and an aqueous suspension base.