Granted Patent
B2
US 12,600,986 · App. 17/581,203 · Granted Apr 14, 2026
Nucleic acid molecules containing spacers and methods of use thereof
Assignee:
Astellas Gene Therapies, Inc.
C12N15/86C12N15/113A61K48/00C12N2310/141C12N2750/14143C12N2800/70C12N2800/90C12N2830/008C12N2830/38C12N2830/42
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Abstract
The present invention relates nucleic acid molecules and concatemers containing spacer sequences useful for the efficient packaging of viral particles so as to minimize the incorporation of contaminant nucleic acids into these vectors, as well as methods of producing such viral particles.
Claims (27)
1 . A plurality of AAV particles each comprising a nucleic acid molecule comprising:
(i) an SS1;
(ii) an ITR1;
(iii) an HPM;
(iv) an ITR2; and
(v) an SS2;
operably linked to each other in a 5′-to-3′ direction as: SS1-ITR1-HPM-ITR2-SS2, wherein the SS1 and the SS2 each, independently, comprise a portion having a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of any one of SEQ ID NOS: 3-5 and 9-11, and wherein neither the SS1 nor the SS2 comprises an open reading frame that encodes more than 100 amino acids.
2 . A method of producing a plurality of adeno-associated virus (AAV) particles, the method comprising:
(a) introducing into an AAV producer cell in a cell culture medium a nucleic acid molecule comprising
(i) a first spacer (SS1);
(ii) a 5′ AAV inverted terminal repeat (ITR1);
(iii) a heterologous polynucleotide molecule (HPM);
(iv) a 3′ AAV inverted terminal repeat (ITR2); and
(v) a second spacer (SS2);
operably linked to each other in a 5′-to-3′ direction as: SS1-ITR1-HPM-ITR2-SS2, wherein the SS1 and the SS2 each, independently, comprise a portion having a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of any one of SEQ ID NOS: 3-5 and 9-11, and wherein neither the SS1 nor the SS2 comprises an open reading frame that encodes more than 100 amino acids, wherein the AAV producer cell expresses AAV rep and cap proteins; and subsequently (b) isolating the plurality of AAV particles from the cell culture medium.
3 . The method of claim 2 , wherein the SS1 has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 3.
4 . The method of claim 3 , wherein the SS1 has the nucleic acid sequence of SEQ ID NO: 3.
5 . The method of claim 4 , wherein the SS2 has the nucleic acid sequence of SEQ ID NO: 4.
6 . The method of claim 3 , wherein the SS2 has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 4.
7 . The method of claim 2 , wherein the SS2 has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 3.
8 . The method of claim 7 , wherein the SS2 has the nucleic acid sequence of SEQ ID NO: 3.
9 . The method of claim 2 , wherein the SS1 has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 4.
10 . The method of claim 9 , wherein the SS1 has the nucleic acid sequence of SEQ ID NO: 4.
11 . The method of claim 10 , wherein the SS2 has the nucleic acid sequence of SEQ ID NO: 3.
12 . The method of claim 9 , wherein the SS2 has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 3.
13 . The method of claim 2 , wherein the SS2 has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 4.
14 . The method of claim 13 , wherein the SS2 has the nucleic acid sequence of SEQ ID NO: 4.
Assignments (2)
CHANGE OF NAME
Recorded Aug 16, 2023
From: AUDENTES THERAPEUTICS, INC.
To: ASTELLAS GENE THERAPIES, INC.
Reel/Frame 064603/0907 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Sep 14, 2022
From: GRAY, JOHN T.
To: AUDENTES THERAPEUTICS, INC.
Reel/Frame 061094/0858 →
Continuity (3)
Continuation
15768071
Provisional Application
62241483
· Oct 14, 2015
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