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Summary for Patent: 5,648,267
|Title:||Impaired dominant selectable marker sequence and intronic insertion strategies for enhancement of expression of gene product and expression vector systems comprising same|
|Abstract:||Disclosed herein are fully impaired consensus Kozak sequences which are most typically used with dominant selectable markers of transcriptional cassettes which are a part of an expression vector. These vectors are most typically utilized in the expression of proteins in mammalian expression systems. As defined, disclosed and claimed herein, a \"fully impaired consensus Kozak\" comprises the following sequence: ##STR1## where: Nat nucleotides 2,3,8 and 9 is a nucleotide selected from the group consisting of adenme (A), quanine (G), cytosine (C) or thymine (T)/uracil (U); Nat nucleotides 1 and 7 is a pyrimidine nucleotide, ie C or T/U; \"ATG\" is a codon encoding for the amino acid methionine, the so-called \"start\" codon; and -3 and +1 are directional reference points vis-a-vis ATG, ie -3 is meant to indicate three nucleotides upstream of ATG and +1 is meant to indicate one nucleotide downstream of ATG. Dominant selectable markers further comprising artificial intronic insertion regions are further disclosed.|
|Inventor(s):||Reff; Mitchell E. (San Diego, CA)|
|Assignee:||IDEC Pharmaceuticals Corporation (San Diego, CA)|
|Patent Claims:||1. An expression vector which expresses at least one protein of interest in a recombinant host cell wherein said expression vector comprises:
(i) a translationally impaired neomycin phosphotransferase (NEO) dominant selectable marker gene which has been translationally impaired by modification of the region of the NEO gene which includes the NEO translation initiation start codon such that said modified region of the NEO gene which includes the NEO translation initiation start codon has the following nucleotide sequence:
which translationally impaired NEO gene is operably linked to a promoter and polyadenylation sequence; and
(ii) at least one heterologous DNA which encodes for at least one protein of interest, wherein said heterologous DNA is operably linked to a promoter and polyadenylation sequence different from the promoter and polydenylation sequence operably linked to the NEO gene, and wherein said heterologous DNA and said promoter and polyadenylation sequence operably linked to said heterologous are inserted into an intronic insertion region contained in the NEO gene.
2. The vector of claim 1 which further comprises a second dominant selectable marker gene.
3. The vector of claim 1 wherein the intronic insertion region is a synthetic intron sequence which is inserted between the CAG which encodes glycine at position 61 and the GAC which encodes aspartic acid at position 62 of the NEO gene.
4. The vector of claim 1 wherein the protein of interest is an antibody.
5. A recombinant host cell which contains the expression vector according to claim 1.
6. A recombinant host cell which contains the expression vector according to claim 2.
7. A recombinant host cell which contains the expression vector according to claim 3.
8. A recombinant host cell which contains the expression vector according to claim 4.
9. The recombinant host cell of claim 5 wherein said host cell is a mammalian cell.
10. The recombinant host cell of claim 9 wherein said mammalian cell is a Chinese hamster ovary cell.
|Applicant||Tradename||Biologic Ingredient||Dosage Form||BLA||Number||Approval Date||Patent No.||Assignee||Estimated Patent Expiration||Status||Orphan||Source|
|Schering||INTRON A||interferon alfa-2b||VIAL||103132||001||1986-06-04||⤷ Free Forever Trial||IDEC Pharmaceuticals Corporation (San Diego, CA)||2014-07-15||RX||search|
|Schering||INTRON A||interferon alfa-2b||VIAL||103132||002||1986-06-04||⤷ Free Forever Trial||IDEC Pharmaceuticals Corporation (San Diego, CA)||2014-07-15||RX||search|
|Schering||INTRON A||interferon alfa-2b||VIAL||103132||003||1986-06-04||⤷ Free Forever Trial||IDEC Pharmaceuticals Corporation (San Diego, CA)||2014-07-15||RX||search|
|>Applicant||>Tradename||>Biologic Ingredient||>Dosage Form||>BLA||>Number||>Approval Date||>Patent No.||>Assignee||>Estimated Patent Expiration||>Status||>Orphan||>Source|
|Country||Patent Number||Estimated Expiration|
|Austria||236987||⤷ Free Forever Trial|
|Australia||5613294||⤷ Free Forever Trial|
|Australia||682481||⤷ Free Forever Trial|
|Canada||2149326||⤷ Free Forever Trial|
|Germany||669986||⤷ Free Forever Trial|
|>Country||>Patent Number||>Estimated Expiration|
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