	US 12,084,703 B2
	Synthetic self-amplifying mRNA molecules with secretion antigen and immunomodulator
Yingzhong Li, Reading, MA (US); and Libin Zhang, Lynnfield, MA (US)
Assigned to SunVax mRNA Therapeutics Inc., Beverly, MA (US)
Filed by SunVax mRNA Therapeutics Inc., Beverly, MA (US)
Filed on May 11, 2023, as Appl. No. 18/316,033.
Claims priority of provisional application 63/393,688, filed on Jul. 29, 2022.
Claims priority of provisional application 63/341,018, filed on May 12, 2022.
Prior Publication US 2023/0366001 A1, Nov. 16, 2023
Int. Cl. C12P 19/34 (2006.01); C12N 15/88 (2006.01)

CPC C12P 19/34 (2013.01) [C12N 15/88 (2013.01)]

29 Claims

1. An in vitro method of increasing the copy number of a nucleic acid comprising:

a) contacting cells with a nucleic acid encoding two expression units, the nucleic acid comprising:

i) an origin of replication sequence (Ori);

ii) a first expression unit encoding a first nucleotide sequence that is operably linked to a first promoter; and

iii) a second expression unit encoding a second nucleotide sequence that is operably linked to a second promoter, wherein the second promoter comprises an engineered T7 promoter comprising the nucleotide sequence of SEQ ID NO: 47 (TAATACGACTCACTATAGG) operably linked to a 5′ UTR and wherein the 5′ UTR is 3′ to SEQ ID NO: 47;

wherein the first expression unit encodes a selectable marker and the second expression unit encodes a self-amplifying mRNA (sa-mRNA);

b) selecting cells that express the selectable marker;

c) subculturing the selected cells to obtain a population of cells that express the selectable marker; and

d) propagating the population of cells to increase the copy number of the nucleic acid.