Methods to produce rod-derived cone viability factor (RDCVF)
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. 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 rod-derived cone viability factor (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 the signal peptide of insulin growth factor-1 (IGF-1); 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, wherein the signal peptide-encoding sequence and the furin cleavage marker sequence are separated by a nucleotide sequence encoding SEQ ID NO: 1; and
(2) expressing the RdCVF fusion protein at a therapeutically effective amount in the diseased eye to treat the ocular disease.
2. The method of claim 1 , wherein the nucleic acid encoding the RdCVF fusion protein is an expression vector that is a recombinant adeno-associated virus (AAV) or adenovirus (Ad).
3. The method of claim 1 , wherein the ocular disease 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.
4. The method of claim 1 , wherein the RdCVF-encoding nucleotide sequence lacks the initiating methionine coding sequence.
5. 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) a signal peptide-encoding nucleotide sequence positioned upstream of the RdCVF-encoding nucleotide sequence, wherein the signal peptide-encoding sequence encodes the signal peptide of insulin growth factor-1 (IGF-1); 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, wherein the signal peptide-encoding sequence and the furin cleavage marker sequence are separated by a nucleotide sequence encoding SEQ ID NO: 1;
(2) expressing the RdCVF fusion protein at a therapeutically effective amount in the diseased eye to treat the ocular disease.
6. The method of claim 5 , wherein the nucleic acid encoding the RdCVF fusion protein is an expression vector that is a recombinant adeno-associated virus (AAV) or adenovirus (Ad).
7. The method of claim 5 , wherein the ocular disease 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.
8. The method of claim 1 , wherein the RdCVF-encoding sequence encodes the long form of RdCVF (SEQ ID NO: 26) or the short form of RdCVF (SEQ ID NO: 27).
9. The method of claim 1 , wherein the signal peptide is the IGF-1 signal peptide (SEQ ID NO: 30).
10. The method of claim 1 , wherein the nucleotide sequence encoding the furin cleavage marker encodes the amino acid sequence KRIKR (SEQ ID NO: 3).
11. The method of claim 1 , wherein the nucleic acid encoding a RdCVF fusion protein encodes the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 16.
12. The method of claim 1 , wherein the nucleic acid encoding a RdCVF fusion protein comprises the nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 17.
13. The method of claim 5 , wherein the RdCVF-encoding sequence encodes the long form of RdCVF (SEQ ID NO: 26) or the short form of RdCVF (SEQ ID NO: 27) lacking the initiating methionine.
14. The method of claim 5 , wherein the signal peptide is the IGF-1 signal peptide (SEQ ID NO: 30).
15. The method of claim 5 , wherein the nucleotide sequence encoding the furin cleavage marker encodes the amino acid sequence KRIKR (SEQ ID NO: 3).