Methods of packaging multiple adeno-associated virus vectors
Provided herein are methods related to co-packaging of multiple rAAV particles. e.g., by introducing multiple nucleic acid vectors encoding proteins or polypeptides or RNAs of interest into a single cell preparation.
1. A producer cell preparation for producing one or more recombinant adeno-associated virus particles, the producer cell preparation comprising:
a first nucleic acid vector containing a first construct, the first construct comprising a heterologous nucleic acid region encoding a first protein or polypeptide, and
a second nucleic acid vector containing a second construct, the second construct comprising a heterologous nucleic acid region encoding a second protein or polypeptide, wherein the first nucleic acid vector and the second nucleic acid vector are present at an initial ratio,
wherein the producer cell preparation is configured to produce a particle preparation comprising:
a first rAAV particle comprising the first construct, and
a second rAAV particle comprising the second construct, wherein the particle preparation comprises a target ratio of the first rAAV particle to the second rAAV particle within 15% of the initial ratio of the first nucleic acid vector to the second nucleic acid vector.
2. The producer cell preparation of claim 1 , wherein the first and the second nucleic acid vectors each comprise nucleic acid regions comprising an inverted terminal repeat (ITR) flanking each side of the respective heterologous nucleic acid region.
3. The producer cell preparation of claim 1 , wherein the particle preparation comprises a target ratio of the first rAAV particle to the second rAAV particle within 10% of the initial ratio of the first nucleic acid vector to the second nucleic acid vector.
4. The producer cell preparation of claim 1 , wherein the first rAAV particle and the second rAAV particle are each rAAV9 particles.
5. The producer cell preparation of claim 1 , further comprising one or more helper plasmids.
6. The producer cell preparation of claim 1 , further comprising a first helper plasmid comprising a rep gene and a cap gene and a second helper plasmid comprising an E1a gene, an E1b gene, an E4 gene, an E2a gene, and a VA gene.
7. The producer cell preparation of claim 1 , wherein the initial ratio is 1:1, 1:9, or 9:1 of the first nucleic acid vector to the second nucleic acid vector.
8. The producer cell preparation of claim 1 , wherein the first nucleic acid vector and/or the second nucleic acid vector are a first plasmid and/or a second plasmid.
9. The producer cell preparation of claim 1 , wherein the first nucleic acid vector and the second nucleic acid vector are DNA vectors.
10. A cellular composition, the cellular composition comprising:
a producer cell preparation,
a first nucleic acid vector containing a first construct comprising a heterologous nucleic acid region encoding a first protein or polypeptide and nucleic acid regions comprising an inverted terminal repeat (ITR) flanking each side of the heterologous nucleic acid region;
a second nucleic acid vector containing a second construct comprising a heterologous nucleic acid region encoding a second protein or polypeptide and nucleic acid regions comprising an inverted terminal repeat (ITR) flanking each side of the heterologous nucleic acid region; and one or more helper nucleic acids, wherein
the first nucleic acid vector and the second nucleic acid vector are present at a initial ratio,
a first rAAV particle comprising the first construct, and
a second rAAV particle comprising the second construct, wherein
a ratio of the first rAAV particle to the second rAAV particle is within 15% of the initial ratio of the first nucleic acid vector to the second nucleic acid vector.
11. The cellular composition of claim 10 , wherein the first and the second nucleic acid vectors each comprise nucleic acid regions comprising an inverted terminal repeat (ITR) flanking each side of the respective heterologous nucleic acid region.
12. The cellular composition of claim 10 , wherein the ratio of the first rAAV particle to the second rAAV particle is within 10% of the initial ratio of the first nucleic acid vector to the second nucleic acid vector.
13. The cellular composition of claim 10 , wherein the first rAAV particle and the second rAAV particle are each rAAV9 particles.
14. The cellular composition of claim 10 , further comprising one or more helper plasmids.
15. The cellular composition of claim 10 , further comprising a first helper plasmid comprising a rep gene and a cap gene and a second helper plasmid comprising an E1a gene, an E1b gene, an E4 gene, an E2a gene, and a VA gene.
16. The cellular composition of claim 10 , wherein the initial ratio is 1:1, 1:9, or 9:1 of the first nucleic acid vector to the second nucleic acid vector.
17. The cellular composition of claim 10 , wherein the first nucleic acid vector and/or the second nucleic acid vector are a first plasmid and/or a second plasmid.
18. The cellular composition of claim 10 , wherein the first nucleic acid vector and the second nucleic acid vector are DNA vectors.
19. A composition, the composition comprising:
(a) the supernatant or lysate of a producer cell preparation comprising
(i) a first nucleic acid vector containing a first construct comprising a heterologous nucleic acid region encoding a first protein or polypeptide and nucleic acid regions comprising an inverted terminal repeat (ITR) flanking each side of the heterologous nucleic acid region;
(ii) a second nucleic acid vector containing a second construct comprising a heterologous nucleic acid region encoding a second protein or polypeptide and nucleic acid regions comprising an inverted terminal repeat (ITR) flanking each side of the heterologous nucleic acid region; and
one or more helper nucleic acids,
(b) a first rAAV particle comprising the first construct, and
(c) a second rAAV particle comprising the second construct, wherein
a ratio of the first rAAV particle to the second rAAV particle within 15% of the initial ratio of the first nucleic acid vector to the second nucleic acid vector.