Methods to produce rod-derived cone viability factor (rdcvf)

Abstract

The invention is related to methods of producing rod-derived cone viability factor (RdCVF). This invention also relates to the treatment of an ocular disease in a mammal using RdCVF. Also provided are expression vectors for high secreted expression of RdCVF of using nucleotide sequences encoding heterologous signal proteins and optionally markers for furin cleavage.

1 . An expression vector for expression of rod-derived cone viability factor (RdCVF) comprising
 a) a RdCVF-encoding nucleotide sequence;   b) a signal peptide-encoding nucleotide sequence positioned upstream of the RdCVF-encoding nucleotide sequence, wherein the signal peptide-encoding sequence encodes an exogenous signal peptide; and   c) a nucleotide sequence encoding a furin cleavage marker located downstream of the signal peptide-encoding nucleotide sequence and upstream of the RdCVF-encoding nucleotide sequence.   
     
     
         2 . The expression vector of  claim 1 , wherein the signal peptide-encoding sequence and the furin cleavage marker sequence are separated by a sequence encoding an amino acid linker. 
     
     
         3 . The expression vector of  claim 1 , wherein the RdCVF-encoding nucleotide sequence lacks the initiating methionine coding sequence. 
     
     
         4 . The expression vector of  claim 1 , wherein the signal peptide-encoding sequence is that of human growth hormone (hGH), brain-derived neurotrophioc factor (BDNF), insulin growth factor-1 (IGF-1), or β-glucoronidase (GUSB). 
     
     
         5 . The expression vector of  claim 2 , wherein the amino acid linker is MPLESGLSSEDSASSESFA (SEQ ID NO:4). 
     
     
         6 . The expression vector of  claim 1 , wherein the RdCVF-encoding sequence encodes the long form of RdCVF (SEQ ID NO:26). 
     
     
         7 . The expression vector of  claim 1 , wherein the RdCVR-encoding sequence encodes the short form of RdCVF (SEQ ID NO:27). 
     
     
         8 . The expression vector of  claim 4 , wherein the signal peptide is the hGH signal peptide (SEQ ID NO:28). 
     
     
         9 . The expression vector of  claim 4 , wherein the signal peptide is the BDNF signal peptide (SEQ ID NO:29). 
     
     
         10 . The expression vector of  claim 4 , wherein the signal peptide is the IGF-1 signal peptide (SEQ ID NO:30). 
     
     
         11 . The expression vector of  claim 4 , wherein the signal peptide is the GUSB signal peptide (SEQ ID NO:31). 
     
     
         12 . The expression vector of  claim 1 , wherein the nucleotide sequence encoding the furin cleavage marker encodes the amino acid sequence KRIKR (SEQ ID NO:3). 
     
     
         13 . The expression vector of  claim 1 , wherein the vector comprises a nucleic acid sequence that codes for the amino acid sequence of SEQ ID NO:6. 
     
     
         14 . The expression vector of  claim 1 , wherein the vector comprises SEQ ID NO:7. 
     
     
         15 . The expression vector of  claim 1 , wherein said vector comprises a nucleic acid sequence that codes for the amino acid sequence of SEQ ID NO:8. 
     
     
         16 . The expression vector of  claim 1 , wherein said vector comprises SEQ ID NO:9. 
     
     
         17 . The expression vector of  claim 1 , wherein said vector comprises a nucleic acid sequence that codes for the amino acid sequence of SEQ ID NO:10. 
     
     
         18 . The expression vector of  claim 1 , wherein said vector comprises SEQ ID NO:11. 
     
     
         19 . The expression vector of  claim 1 , wherein said vector comprises SEQ ID NO:13. 
     
     
         20 . The expression vector of  claim 1 , wherein said vector comprises a nucleic acid sequence that codes for the amino acid sequence of SEQ ID NO:12. 
     
     
         21 . The expression vector of  claim 1 , wherein said vector comprises SEQ ID NO:15. 
     
     
         22 . The expression vector of  claim 1 , wherein said vector comprises a nucleic acid sequence that codes for the amino acid sequence of SEQ ID NO:14. 
     
     
         23 . The expression vector of  claim 1 , wherein said vector comprises SEQ ID NO:17. 
     
     
         24 . The expression vector of  claim 1 , wherein said vector comprises a nucleic acid sequence that codes for the amino acid sequence of SEQ ID NO:16. 
     
     
         25 . The expression vector of  claim 1 , wherein said vector comprises SEQ ID NO:18. 
     
     
         26 . The expression vector of  claim 1 , wherein said vector comprises a nucleic acid sequence that codes for the amino acid sequence of SEQ ID NO:19. 
     
     
         27 . The expression vector of  claim 1 , wherein said vector comprises SEQ ID NO:20. 
     
     
         28 . The expression vector of  claim 1 , wherein said vector comprises a nucleic acid sequence that codes for the amino acid sequence of SEQ ID NO:21. 
     
     
         29 . The expression vector of  claim 1 , wherein said vector comprises SEQ ID NO:22. 
     
     
         30 . The expression vector of  claim 1 , wherein said vector comprises a nucleic acid sequence that codes for the amino acid sequence of SEQ ID NO:23. 
     
     
         31 . The expression vector of  claim 1 , wherein said vector comprises SEQ ID NO:24. 
     
     
         32 . The expression vector of  claim 1 , wherein said vector comprises a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:25. 
     
     
         33 . An expression vector for expression of rod-derived cone viability factor (RdCVF) comprising
 a) a RdCVF-encoding nucleotide sequence lacking the initiating methionine coding sequence; and   b) an exogenous signal peptide-encoding nucleotide sequence positioned upstream of the RdCVF-encoding nucleotide sequence, wherein the exogenous signal peptide-encoding sequence is selected from the group consisting of: human growth hormone (hGH), brain-derived neurotrophic factor (BDNF), insulin growth factor-1 (IGF-1), or β-glucoronidase (GUSB).   
     
     
         34 . A mammalian cell comprising an expression vector for expression of rod-derived cone viability factor (RdCVF) in a cell, said vector comprising:
 a) a RdCVF-encoding nucleotide sequence;   b) a signal peptide-encoding nucleotide sequence positioned upstream of the RdCVF-encoding nucleotide sequence, wherein the signal peptide-encoding sequence encodes an exogenous signal peptide; and   c) a nucleotide sequence encoding a furin cleavage marker located downstream of the signal peptide-encoding nucleotide sequence and upstream of the RdCVF-encoding nucleotide sequence.   
     
     
         35 . The cell of  claim 34 , wherein the signal peptide-encoding sequence and the furin cleavage marker sequence are separated by a sequence encoding an amino acid linker. 
     
     
         36 . The cell of  claim 34 , wherein the RdCVF-encoding nucleotide sequence lacks the initiating methionine coding sequence. 
     
     
         37 . The cell of  claim 34 , wherein the signal peptide-encoding sequence is that of human growth hormone (hGH), brain-derived neurotrophioc factor (BDNF), insulin growth factor-1 (IGF-1), or β-glucoronidase (GUSB). 
     
     
         38 . A method for expressing of rod-derived cone viability factor (RdCVF), in a host cell, said method comprises:
 providing a mammalian cell comprising a RdCVF expression vector, wherein the vector comprises:
 a) a RdCVF-encoding nucleotide sequence; 
 b) a signal peptide-encoding nucleotide sequence positioned upstream of the RdCVF-encoding nucleotide sequence, wherein the signal peptide-encoding sequence encodes an exogenous signal peptide; and 
 c) a nucleotide sequence encoding a furin cleavage marker located downstream of the signal peptide-encoding nucleotide sequence and immediately upstream of the RdCVF-encoding nucleotide sequence; and 
   culturing the mammalian cell under conditions such that RdCVF is expressed.   
     
     
         39 . The cell of  claim 38 , wherein the signal peptide-encoding sequence and the furin cleavage marker sequence are separated by a sequence encoding an amino acid linker. 
     
     
         40 . The cell of  claim 38 , wherein the RdCVF-encoding nucleotide sequence lacks the initiating methionine coding sequence. 
     
     
         41 . The cell of  claim 38 , wherein the signal peptide-encoding sequence is that of human growth hormone (hGH), brain-derived neurotrophioc factor (BDNF), insulin growth factor-1 (IGF-1), or β-glucoronidase (GUSB). 
     
     
         42 . The method of  claim 38 , wherein the mammalian cell is a CHO cell or a COS cell. 
     
     
         43 . The method of  claim 38 , wherein the RdCVF-encoding nucleotide sequence encodes the long form of RdCVF. 
     
     
         44 . The method of  claim 38 , wherein the RdCVF-encoding nucleotide sequence encodes the short form of RdCVF. 
     
     
         45 . The method of  claim 38 , wherein the nucleotide sequence the furin cleavage marker is a sequence encoding the amino acid sequence KRIKR. 
     
     
         46 . The method of  claim 38 , further comprising the step of isolating the expressed RdCVF protein. 
     
     
         47 . A method of treating an ocular disease in a mammal, the method comprising:
 (1) administering to a diseased eye of the mammal a nucleic acid encoding a RdCVF fusion protein, where the nucleic acid encoding the RdCVF fusion protein comprises:
 a) a RdCVF-encoding nucleotide sequence; 
 b) a signal peptide-encoding nucleotide sequence positioned upstream of the RdCVF-encoding nucleotide sequence, wherein the signal peptide-encoding sequence encodes an exogenous signal peptide; and 
 c) a nucleotide sequence encoding a furin cleavage marker located downstream of the signal peptide-encoding nucleotide sequence and upstream of the RdCVF-encoding nucleotide sequence; and 
   (2) expressing the RdCVF fusion protein at a therapeutically effective amount in the diseased eye to treat the ocular disease.   
     
     
         48 . The method of  claim 47 , wherein the nucleic acid encoding the RdCVF fusion protein is an expression vector that is a recombinant adeno-associated virus (AAV) or adenovirus (Ad). 
     
     
         49 . The method of  claim 47 , wherein the ocular diseases is Stargardt's disease, Retinitis Pigmentosa, Dry Age-related Macular Degeneration (Dry AMD), Wet Age-related Macular Degeneration (Wet AMD), Glaucoma/Ocular Hypertension, Diabetic Retinopathy, Thyroid related eye disease, Grave's disease, a diseases associated with Retinal Pigmented Epithelial Cells, Anterior segment disease, Lens disease/Cataracts, an Eye cup disorder, or Uveitis. 
     
     
         50 . The method of  claim 47 , wherein the signal peptide-encoding sequence and the furin cleavage marker sequence are separated by a sequence encoding an amino acid linker. 
     
     
         51 . The method of  claim 47 , wherein the RdCVF-encoding nucleotide sequence lacks the initiating methionine coding sequence. 
     
     
         52 . The method of  claim 47 , wherein the signal peptide-encoding sequence is that of human growth hormone (hGH), brain-derived neurotrophioc factor (BDNF), insulin growth factor-1 (IGF-1), or β-glucoronidase (GUSB). 
     
     
         53 . A method of treating an ocular disease in a mammal, the method comprising:
 (1) administering to a diseased eye of the mammal a nucleic acid encoding a RdCVF fusion protein, where the nucleic acid encoding the RdCVF fusion protein comprises:
 a) a RdCVF-encoding nucleotide sequence lacking the initiating methionine coding sequence; 
 b) an exogenous signal peptide-encoding nucleotide sequence positioned upstream of the RdCVF-encoding nucleotide sequence, wherein the exogenous signal peptide-encoding sequence is selected from the group consisting of: human growth hormone (hGH), brain-derived neurotrophic factor (BDNF), insulin growth factor-1 (IGF-1), or β-glucoronidase (GUSB); and 
   (2) expressing the RdCVF fusion protein at a therapeutically effective amount in the diseased eye to treat the ocular disease.   
     
     
         54 . The method of  claim 53 , wherein the nucleic acid encoding the RdCVF fusion protein is an expression vector that is a recombinant adeno-associated virus (AAV) or adenovirus (Ad). 
     
     
         55 . The method of  claim 53 , wherein the ocular diseases is Stargardt's disease, Retinitis Pigmentosa, Dry Age-related Macular Degeneration (Dry AMD), Wet Age-related Macular Degeneration (Wet AMD), Glaucoma/Ocular Hypertension, Diabetic Retinopathy, Thyroid related eye disease, Grave's disease, a diseases associated with Retinal Pigmented Epithelial Cells, Anterior segment disease, Lens disease/Cataracts, an Eye cup disorder, or Uveitis.