Expression system incorporating a capsid promoter sequence as an enhancer
View Patent ↗The invention provides a method for enhancing expression of a transgene in a host cell, which includes the steps of inserting a capsid promoter element (Pcap), or a sequence comprising the reverse complement of the capsid promoter element (PcapR), into a mammalian expression cassette upstream (5′) of a cytomegalovirus immediate/early enhancer/promoter region (Pcmv); inserting the transgene into the expression cassette downstream (3′) of the cytomegalovirus immediate/early enhancer/promoter region; inserting a vector containing the expression cassette into the host organism; and causing expression of the transgene. The capsid promoter element (Pcap) is typically from a circovirus, parvovirus or anellovirus. The transgene is typically expressed at a higher level than when expressed by a vector containing the expression cassette without the transcriptional control element. An expression cassette, vector, DNA vaccine, pharmaceutical composition and method of treatment are also claimed.
1. A method for enhancing expression of a transgene from a cytomegalovirus immediate/early promoter (Pcmv) in a host cell comprising the steps of:
(a) inserting a porcine circovirus type 1 capsid gene promoter (Pcap) or a Pcap reverse complement sequence (PcapR) as shown in SEQ ID NOs: 1 or 2 into a mammalian expression cassette upstream (5′) of the Pcmv promoter element;
(b) inserting the transgene into the mammalian expression cassette downstream (3′) of the Pcmv element;
(c) inserting the expression cassette into the host organism; and
(d) causing expression of the transgene.
2. The method of claim 1 , wherein a cytomegalovirus intron is inserted downstream (3′) of the Pcmv and a bovine growth hormone polyadenylation site (bgh polyA) is inserted downstream (3′) of the transgene.
3. The method of claim 1 , wherein the Pcap or PcapR is inserted immediately upstream (5′) of the Pcmv element.
4. The method of claim 1 , wherein the Pcap or PcapR is inserted up to 1100 base pairs upstream (5′) of the Pcmv element.
5. The method of claim 1 , wherein the host cell is a mammalian cell line for in vitro transgene expression or a cell of a mammalian host organism for in vivo transgene expression.
6. A mammalian expression cassette comprising:
(a) a cytomegalovirus immediate/early promoter (Pcmv) element; and
(b) a sequence that is the Pcap or PcapR located upstream (5′) of the Pcmv element, wherein the Pcap or PcapR comprises SEQ ID NO: 1 or SEQ ID NO: 2;
wherein the Pcap or PcapR enhances the Pcmv promoter activity.
7. The mammalian expression cassette of claim 6 , which includes a transgene downstream (3′) of the cytomegalovirus immediate/early promoter element.
8. A vector including the expression cassette of claim 6 .
9. An isolated host cell comprising the vector of claim 8 .
10. A DNA vaccine comprising the expression cassette or vector as claimed in claims 6 or 8 .