Gene therapy for treating citrullenemia
Compositions and regimens useful in treating type I citrullenemia are provided. The compositions include recombinant adeno-associated virus (rAAV) with a transthyretin enhancer and promoter driving expression of a human Argininosuccinate Synthase 1 (ASS1).
1. A recombinant adeno-associated virus (rAAV) useful as a liver-directed therapeutic for citrullinemia, said rAAV comprising an AAV capsid, and a vector genome packaged therein, said vector genome comprising:
(a) an AAV 5′ inverted terminal repeat (ITR);
(b) a promoter;
(c) a coding sequence encoding a human argininosuccinate synthase 1 (ASS1);
(d) an AAV 3′ ITR,
wherein the coding sequence of (c) is SEQ ID NO: 2.
2. The rAAV according to claim 1 , wherein the AAV capsid is an AAV8 capsid or variant thereof.
3. The rAAV according to claim 1 , wherein the promoter is a thyroxine binding globulin (TBG) promoter.
4. The rAAV according to claim 1 , wherein the promoter is an alpha 1 anti-trypsin (A1AT) promoter.
5. The rAAV according to claim 1 , wherein the promoter comprises SEQ ID NO: 21.
6. The rAAV according to claim 1 , wherein the promoter is a transthyretin promoter (TTR) promoter.
7. The rAAV according to claim 1 , wherein the AAV 5′ ITR and/or AAV 3′ ITR is from AAV2.
8. The rAAV according to claim 1 , wherein the vector genome further comprises a polyadenylation (polyA) sequence.
9. The rAAV according to claim 1 , wherein the polyA sequence is a bovine growth hormone (bGH) polyA sequence.
10. The rAAV according to claim 1 , wherein the vector genome further comprises a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).
11. The rAAV according to claim 1 , wherein the vector genome further comprises an intron.
12. The rAAV according to claim 11 , wherein the intron is human beta globin IVS2 or SV40 intron.
13. The rAAV according to claim 1 , wherein the vector genome further comprises an enhancer.
14. The rAAV according to claim 13 , wherein the enhancer is an APB enhancer, an ABPS enhancer, an alpha mic/bik enhancer, a TTR enhancer, en34, or an ApoE enhancer.
15. The rAAV according to claim 1 , wherein the vector genome is about 3 kilobases to about 5.5 kilobases in size.
16. An aqueous suspension suitable for administration to a citrullinemia patient, said suspension comprising an aqueous suspending liquid and about 1×10 12 genome copies (GC)/mL to about 1×10 14 GC/mL of a rAAV useful as a liver-directed therapeutic for citrullinemia, said rAAV having an AAV capsid, and having packaged therein a vector genome comprising:
(a) an AAV 5′ ITR;
(b) a promoter;
(c) a coding sequence encoding an ASS1; and
(d) an AAV 3′ ITR,
wherein the coding sequence of (c) is SEQ ID NO: 2.
17. The aqueous suspension according to claim 16 , wherein the suspension is suitable for intravenous injection.
18. The aqueous suspension according to claim 16 , wherein the suspension further comprises a surfactant, preservative, and/or buffer dissolved in the aqueous suspending liquid.
19. A method of treating a patient having citrullinemia with an rAAV, the method comprising:
administering to the patient about 1×10 12 to about 1×10 14 GC/kg of the rAAV according to claim 2 in an aqueous suspension,
wherein the GC are calculated by optimized quantitative polymerase chain reaction (oqPCR) or digital droplet polymerase chain reaction (ddPCR).
20. The aqueous suspension according to claim 16 , wherein the AAV capsid is an AAV8 capsid.