Methods to produce rod-derived cone viability factor (rdcvf)
AssigneeGenzyme Corporation
InventorsCatherine R. O'RIORDAN, William H. BRONDYK
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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.
Claims55
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.
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).
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.
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.
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.
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.
Dependencies are inferred from the claim text.