Small angiotensin peptide expression system in mammalian cells
The present invention is directed to a delivery vector for transferring a small peptide coding sequence to a cell for expression of the small peptide coding sequence within the cell. The delivery vector comprises a secretory signal sequence; a sequence encoding a carrier protein operatively associated with the secretory signal sequence; a sequence encoding a cleavage site operatively associated with the sequence encoding a carrier protein; and a sequence encoding a small peptide operatively associated with the sequence encoding a cleavage site.
1. An adeno-associated virus (AAV) vector for transferring a small peptide coding sequence to a first cell for expression of the small peptide within the first cell and delivery of the small peptide to surrounding cells in a tissue, wherein the tissue is a retinal tissue, the AAV vector comprising:
(i) a secretory signal sequence encoding a secretory signal peptide, wherein the secretory signal sequence consists of SEQ ID NO: 4;
(ii) a sequence encoding a carrier protein;
(iii) a sequence encoding a cleavage site; and
(iv) a sequence encoding a small peptide less than 50 amino acids in length, wherein said small peptide is Angiotensin (Ang)1-7,
wherein the secretory signal sequence, the sequence encoding the carrier protein and the sequence encoding the cleavage site are operatively associated with the sequence encoding the small peptide, and
further wherein the AAV vector provides continuous secretion of the small peptide to cells distant from the first cell.
2. The AAV vector of claim 1 , wherein the secretory signal sequence is 5′ to the sequence encoding the carrier protein, the sequence encoding the cleavage site, and the sequence encoding the small peptide.
3. The AAV vector of claim 1 , wherein the sequence encoding the cleavage site encodes a furin cleavage site.
4. The AAV vector of claim 1 , wherein said carrier protein is albumin.
5. The AAV vector of claim 1 , wherein said carrier protein is alpha-1-antitrypsin.
6. The AAV vector of claim 1 , wherein said carrier protein is a heat shock protein, an Advanced Glycation End-product, or a pro-renin receptor.
7. The AAV vector of claim 1 , wherein elements (i)-(iv) are arranged in a 5′-3′ order on a polynucleotide molecule.
8. The AAV vector of claim 1 , wherein said cleavage site is a site cleaved by PC1/3, PC2, PO4, P05/6, PACE4, SKI-1/S1P, NARC-1/PCSK9, thrombin or factor Xa.
9. A pharmaceutical composition comprising the AAV vector of claim 1 in a pharmaceutically acceptable carrier.
10. The AAV vector of claim 1 , wherein the AAV vector is contained within an AAV viral particle.
11. The AAV vector of claim 1 , wherein the cells distant from the first cell are retinal vascular endothelial cells.