Fusion proteins comprising PDGF and VEGF binding portions and methods of using thereof
The present provides fusion proteins comprising PDGF and VEGF binding portions, and recombinant viral particles encoding the fusion proteins. Compositions comprising the fusion proteins and viral particles as well as methods of using the same are also provided.
1. An rAAV particle comprising a nucleicacid encoding a fusion protein comprising (a) an extracellular portion of a PDGF receptor comprising at least Ig-like domains D1-D3 of a PDGF receptor beta (PDGFRβ), (b) an extracellular portion of a VEGF receptor 1 (Flt-1) comprising at least an Ig-like domain D2, and (c) a multimerization domain, wherein the fusion proteins bind to a PDGF and a VEGF, and the fusion protein is arranged from N-terminus to C-terminus in the following order: (a), (b) and (c) and wherein the fusion protein does not comprise an Ig-like domain D3 of a VEGFR2.
2. The rAAV particle of claim 1 , wherein the rAAV particle comprises capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, or AAVrh10.
3. The rAAV particle of claim 2 , wherein the nucleic acid comprises an ITR from a serotype different from the serotype of the capsid.
4. The rAAV particle of claim 3 , wherein the ITR is an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, or AAVrh10.
5. A method of producing an rAAV particle, comprising: culturing a host cell under a condition that rAAV particles are produced, wherein the host cell comprises (i) one or more AAV package genes, wherein each said AAV packaging gene encodes an AAV replication or encapsidation protein; (ii) an rAAV pro-vector comprising the nucleotide encoding the fusion protein comprising (a) an extracellular portion of a PDGF receptor comprising Ig-like domains D1-D3 of a PDGF receptor beta (PDGFRβ), (b) an extracellular portion of a VEGF receptor I (Flt-I) comprising an Ig-like domain D2, and (c) a multimerization domain, wherein the fusion proteins bind to a PDGF and a VEGF, and the fusion protein is arranged from N-terminus to C-terminus in the following order: (a), (b) and (c) flanked by at least one AA V ITR and wherein the fusion protein does not comprise an Ig-like domain D3 of a VEGFR2, and (iii) an AAV helper function; and recovering the rAAV particles produced by the host cell.
6. The method of claim 5 , wherein the rAAV particles are purified.
7. A method of delivering a viral vector to a subject, comprising administering the rAAV particle of claim 1 to the subject, wherein the fusion protein encoded by the rAAV particle is expressed in the subject.
8. The method of claim 7 , wherein the subject has macular degeneration or proliferative diabetic retinopathy.
9. The method of claim 8 , wherein the macular degeneration is wet age-related macular degeneration or dry age-related macular degeneration.
10. The method of claim 7 , wherein the rAAV particle is administered by intravitreal injection to the subject.
11. The method of claim 7 , wherein the subject has cancer.
12. The method of claim 7 , wherein the subject has rheumatoid arthritis, osteoarthritis, or asthma.
13. The method of claim 7 , wherein the subject has uveitis or corneal neovascularization.
14. The rAAV particle of claim 1 , wherein (a) comprises:
(a) Ig-like domains D1-D4 of a PDGF receptor beta;
(b) Ig-like domains D1-D5 of a PDGF receptor beta; or
(c) amino acid sequence SEQ ID NO: 1, 2, or 3.
15. The rAAV particle of claim 1 , wherein (b) comprises:
(a) Ig-like domains D1-D3 of a VEGFR1 (Flt-1); or
(b) amino acid sequence of SEQ ID NO: 4 or 5.
16. The rAAV particle of claim 1 , wherein the fusion protein further comprises at least one of a linker peptide between the extracellular portion of the PDGF receptor and the extracellular portion of the VEGF receptor and a peptide linker between the extracellular portion of the VEGF receptor and the multimerization domain.
17. The rAAV particle of claim 16 , wherein the peptide linker comprises the amino acid sequence selected from the group consisting of SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50.
18. The rAAV particle of claim 1 , wherein the multimerization domain is:
(a) a Fc region of an antibody;
(b) a Fc region of an antibody comprising a CH3 region of IgG1, IgG2, IgG3, or IgG4, or a CH2 and a CH3 region of IgG1, IgG3, or IGG4; or
(c) a Fc region of an antibody comprising the amino acid sequence of SEQ ID NO:6.
19. The rAAV particle of claim 1 , wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:13 or 15 or an amino acid sequence having at least 85% identity to SEQ ID NO:13 or 15.
20. The rAAV particle of claim 1 , wherein the fusion protein is in a dimeric or a multimeric form.
21. A composition comprising the rAAV particle of claim 14 and a pharmaceutically acceptable carrier.