Protein production and delivery

The invention relates to novel human DNA sequences, targeting constructs, and methods for producing novel genes encoding thrombopoietin, DNase I, and .beta.-interferon by homologous recombination. The targeting constructs comprise at least: a) a targeting sequence; b) a regulatory sequence; c) an exon; and d) a splice-donor site. The targeting constructs, which can undergo homologous recombination with endogenous cellular sequences to generate a novel gene, are introduced into cells to produce homologously recombinant cells. The homologously recombinant cells are then maintained under conditions which will permit transcription of the novel gene and translation of the mRNA produced, resulting in production of either thrombopoietin, DNase I, or .beta.-interferon. The invention further relates to a methods of producing pharmaceutically useful preparations containing thrombopoietin, DNase I, or .beta.-interferon from homologously recombinant cells and methods of gene therapy comprising administering homologously recombinant cells producing thrombopoietin, DNase I, or .beta.-interferon to a patient for therapeutic purposes.

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Claims

1. A DNA construct which, upon introduction into a cell, alters the expression of a gene encoding thrombopoietin when inserted by homologous recombination into chromosomal DNA of a cell, said construct comprising:

(a) a targeting sequence comprising DNA which selectively promotes homologous recombination with genomic DNA upstream of the thrombopoietin gene;
(b) a regulatory sequence;
(c) a non-coding exon; and
(d) an unpaired splice-donor site,
wherein, upon integration of the construct into chromosomal DNA, the regulatory sequence of (b), the non-coding exon of (c) and the unpaired splice-donor site of (d) are integrated upstream of exon 1 of the thrombopoietin gene, and upon transcription and splicing, the splice-donor site of (d) is spliced to the splice-acceptor site of the second exon of the thrombopoietin gene.

2. The DNA construct of claim 1 wherein the regulatory sequence comprises a promoter.

3. The DNA construct of claim 2 further comprising a selectable marker gene.

4. The DNA construct of claim 2 further comprising an amplifiable marker gene.

5. The DNA construct of claim 1 further comprising a second targeting sequence comprising DNA which selectively promotes homologous recombination with genomic DNA upstream of the thrombopoietin gene.

6. The DNA construct of claim 1 wherein the targeting sequence is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, fragments of SEQ ID NO: 3 which selectively promote homologous recombination with genomic DNA upstream of the thrombopoietin gene and fragments of SEQ ID NO: 4 which selectively promote homologous recombination with genomic DNA upstream of the thrombopoietin gene.

7. The DNA construct of claim 6 wherein the targeting sequence is a fragment of SEQ ID NO: 3 and is at least about 20 base pairs.

8. The DNA construct of claim 6 wherein the targeting sequence is a fragment of SEQ ID NO: 4 and is at least about 20 base pairs.

9. The DNA construct of claim 8 wherein the targeting sequence is at least about 20 base pairs and is a sequence between about nucleotides -1815 to -145, 14 to 245, or 374 to 570 of FIG. 5 (SEQ ID NO: 4).

10. An isolated DNA molecule selected from the group consisting of SEQ ID NO: 3 and fragments of SEQ ID NO: 3 which selectively promote homologous recombination with genomic DNA upstream of the thrombopoietin gene.

11. An isolated DNA molecule which is selected from the group consisting of about nucleotides -1815 to -145 of FIG. 5 (SEQ ID NO: 4), about 14 to 245 of FIG. 5 (SEQ ID NO: 4), and about 374 to 570 of FIG. 5 (SEQ ID NO: 4), and which selectively promotes homologous recombination with genomic DNA within or upstream of the thrombopoietin gene.

12. A method of producing a homologously recombinant cell wherein the expression of the thrombopoietin gene is altered, comprising the steps of:

(a) transfecting a cell containing the thrombopoietin gene with the DNA construct of one of claims 1-9; and
(b) maintaining the transfected cell under conditions appropriate for homologous recombination.

13. A homologously recombinant cell produced by the method of claim 12.

14. A homologously recombinant cell which expresses thrombopoietin, said cell having incorporated therein a new transcription unit, an exogenous regulatory region, an exogenous non-coding exon, and an exogenous unpaired splice-donor site operatively linked to a splice acceptor site of the thrombopoietin gene present in the cell as obtained, wherein the homologously recombinant cell comprises the said exogenous non-coding exon in addition to exons present in the endogenous gene; and upon transcription and splicing, the exogenous unpaired splice-donor site is spliced to the splice-acceptor site of the second exon of the thrombopoietin gene.

15. The homologously recombinant cell of claim 14 wherein the exogenous regulatory region, the exogenous non-coding exon, and the exogenous unpaired splice-donor site are operatively linked to the endogenous splice acceptor site of the second exon of the thrombopoietin gene.

16. A method for producing thrombopoietin comprising maintaining the homologously recombinant cell of claim 14 or 15 under conditions appropriate for the production of thrombopoietin.

17. A method for producing thrombopoietin wherein the expression of the thrombopoietin gene is altered, comprising the steps of:

(a) transfecting a cell containing the thrombopoietin gene with the DNA construct of one of claims 1-9;
(b) maintaining the transfected cell under conditions appropriate for homologous recombination to occur; and
(c) maintaining the homologously recombinant cell produced in step (b) under conditions appropriate for the production of thrombopoietin.
Referenced Cited
U.S. Patent Documents
5272071 December 21, 1993 Chappel
Foreign Patent Documents
WO91/06667 May 1991 WOX
WO91/06666 May 1991 WOX
WO 91/09955 July 1991 WOX
WO 93/03164 February 1993 WOX
WO93/09222 May 1993 WOX
WO 94/05784 March 1994 WOX
WO94/10567 May 1994 WOX
WO94/12650 July 1994 WOX
WO95/18858 July 1995 WOX
WO95/21626 August 1995 WOX
WO95/31560 November 1995 WOX
Other references
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Patent History
Patent number: 5733746
Type: Grant
Filed: Mar 17, 1995
Date of Patent: Mar 31, 1998
Assignee: Transkaryotic Therapies, Inc. (Cambridge, MA)
Inventors: Douglas A. Treco (Arlington, MA), Michael W. Heartlein (Boxborough, MA), Brian M. Hauge (Beverly, MA), Richard F. Selden (Wellesley, MA)
Primary Examiner: James Ketter
Law Firm: Hamilton,Brook,Smith & Reynolds, P.C.
Application Number: 8/406,030
Classifications
Current U.S. Class: 435/696; 435/1723; 435/3201; 536/234; 536/2351
International Classification: C12P 2102;