US 6,270,989 B1 · Protein production and delivery

Abstract

The invention relates to novel human DNA sequences, targeting constructs, and methods for producing novel genes encoding thrombopoietin, DNase I, and β-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 β-interferon. The invention further relates to a methods of producing pharmaceutically useful preparations containing thrombopoietin, DNase I, or β-interferon from homologously recombinant cells and methods of gene therapy comprising administering homologously recombinant cells producing thrombopoietin, DNase I, or β-interferon to a patient for therapeutic purposes.

Claims356

Dependent claims

A DNA construct which alters the expression of a targeted gene in a cell when the DNA construct is homologously recombined with a target site within the chromosomal DNA of the cell, the DNA construct comprising:

(a) a targeting sequence homologous to the target site;
(b) an exogenous regulatory sequence;
(c) an exon;
and
(d) an unpaired splice-donor site at the 3′ end of the exon;
wherein following homologous recombination of the targeting sequence with the target site, the chromosomal DNA of the cell comprises the construct-derived exon in addition to all endogenous exons of the targeted gene.

3 more elements

(a) a targeting sequence homologous to the target site;
(b) an exogenous regulatory sequence;
(c) an exon;
and
(d) an unpaired splice-donor site at the 3′ end of the exon,
wherein following homologous recombination of the targeting sequence with the target site, (b)-(d) are positioned upstream of the endogenous transcription initiation site of the targeted gene.

3 more elements

A method of altering the expression of a targeted gene in a cell comprising the steps of:

(a) providing a DNA construct comprising:
(i) a targeting sequence;
(ii) an exogenous regulatory sequence;
(iii) an exon;
and
(iv) an unpaired splice-donor site at the 3′ end of the exon,
(b) providing a cell, the genome of which comprises
(i) a target site homologous to the targeting sequence, and
(ii) a targeted gene having an endogenous regulatory region;
(c) transfecting the cell with the DNA construct, thereby producing a transfected cell;
(d) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell the genome of which comprises the exogenous regulatory sequence, the construct-derived exon, and the construct-derived splice-donor site, in addition to all endogenous exons of the targeted gene;
and
(e) maintaining the homologously recombinant cell under conditions appropriate for transcription under the control of the exogenous regulatory sequence, to produce a transcript of the construct-derived exon, the targeted gene, and any sequence lying between the construct-derived exon and the targeted gene, wherein the RNA of the transcript corresponding to the construct-derived splice-donor site directs splicing to a splice-acceptor site in the transcript which corresponds to a site within the targeted gene.

10 more elements

A method of altering the expression of a gene in a cell comprising the steps of:

(a) providing a cell, the genome of which comprises
(i) a targeted gene having an endogenous regulatory region;
(ii) a target site;
(b) providing a DNA construct comprising:
(i) a targeting sequence homologous to the target site;
(ii) an exogenous regulatory sequence;
(iii) an exon;
and
(iv) an unpaired splice-donor site at the 3′ end of the exon,
(c) transfecting the cell with the DNA construct, thereby producing a transfected cell;
(d) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell the genome of which contains the exogenous regulatory sequence, the construct-derived exon, and the construct-derived splice-donor site, all upstream of the endogenous transcription initiation site of the targeted gene;
and
(e) maintaining the homologously recombinant cell under conditions appropriate for transcription under the control of the exogenous regulatory sequence, to produce a transcript of the construct-derived exon, the targeted gene, and any sequence lying between the construct-derived exon and the targeted gene.

10 more elements

A cultured vertebrate cell the genome of which bears a transcription unit comprising an exogenous regulatory sequence, an exogenous exon, and a splice-donor site at the 3′ end of the exogenous exon, the splice-donor site being operatively linked to the endogenous splice-acceptor site of the second endogenous exon of an endogenous gene, wherein the genome comprises the exogenous exon in addition to all endogenous exons of the endogenous gene.

A cultured vertebrate cell the genome of which bears a transcription unit comprising an exogenous regulatory sequence, an exogenous exon and a splice-donor site at the 3′ end of the exogenous exon, all located upstream of the transcription initiation site of an endogenous gene in a chromosome of the cell, the splice-donor site being operatively linked to the endogenous splice-acceptor site of the second endogenous exon of the endogenous gene.

A DNA construct which alters the expression of an targeted gene in a cell when the DNA construct is homologously recombined with a target site within the chromosomal DNA of the cell, the DNA construct comprising:

(a) a targeting sequence homologous to the target site;
(b) an exogenous regulatory sequence;
(c) an exon;
(d) a splice-donor site;
(e) an intron;
and
(f) a splice-acceptor site;
wherein upon homologous recombination, (b)-(f) are present in the chromosomal DNA of the cell in addition to all exons of the targeted gene, and the exogenous regulatory sequence controls transcription of (c)-(f) as well as part or all of the targeted gene.

5 more elements

A method of altering the expression of a targeted gene in a cell, comprising the steps of:

(a) transfecting the cell with a DNA construct, the DNA construct comprising:
(i) a targeting sequence;
(ii) an exogenous regulatory sequence;
(iii) an exon;
(iv) a splice-donor site;
(v) an intron;
and
(vi) a splice-acceptor site,
to generate a transfected cell;
(b) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell the chromosomal DNA of which comprises the construct-derived exon and intron in addition to the endogenous coding sequence of the targeted gene;
and
(c) maintaining the homologously recombinant cell under conditions appropriate for transcription of (iii)-(vi) and the targeted gene under the control of the exogenous regulatory sequence, thereby producing a transcript.

9 more elements

A cultured vertebrate cell having incorporated therein a transcription unit comprising an exogenous regulatory sequence, an exogenous exon, a splice-donor site, an intron, and a splice-acceptor site, wherein the exogenous regulatory sequence is positioned to control transcription of the exogenous exon, splice-donor site, intron, and splice-acceptor site, in addition to all endogenous exons of an endogenous gene.

The method of making a homologously recombinant cell wherein the expression of a targeted gene is altered, comprising the steps of:

(a) transfecting a cell with a DNA construct, the construct comprising:
(i) a targeting sequence homologous to a target site in the chromosomal DNA of the cell;
(ii) an exogenous regulatory sequence;
(iii) an exon;
(iv) a splice-donor site;
(v) an intron;
and
(vi) a splice-acceptor site,
to generate a transfected cell;
and
(b) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell the chromosomal DNA of which comprises the construct-derived exon and intron in addition to the endogenous sequence of the targeted gene, wherein the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to (iii)-(vi) and part or all of the targeted gene.

8 more elements

A method of altering the expression of a targeted gene in a cell, comprising the steps of:

(a) transfecting a cell with a DNA construct, the construct comprising:
(i) a targeting sequence;
(ii) an exogenous regulatory sequence;
(iii) an exon;
(iv) a splice-donor site;
(v) an intron;
and
(vi) a splice-acceptor site,
thereby producing a transfected cell, wherein the targeting sequence directs the integration of elements (ii)-(vi) into genomic DNA of the cell such that the regulatory sequence is positioned to direct transcription of a sequence comprising (iii)-(vi) and sequence of the targeted gene;
(b) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell;
and
(c) maintaining the homologously recombinant cell under conditions appropriate for expression under the control of the regulatory sequence.

9 more elements

A method for expressing an erythropoietin gene in the genome of a mammalian cell, comprising transfecting the cell with a construct comprising

(a) a targeting sequence,
(b) an exogenous regulatory sequence,
(c) an exon;
and
(d) an unpaired splice-donor site at the 3′ end of the exon,
wherein the targeting sequence directs the integration of (b)-(d) into the chromosomal DNA of the cell upstream and within 30 kb of the endogenous ATG initiation codon of the erythropoietin gene such that the exogenous regulatory sequence directs expression of a transcript comprising sequence corresponding to the construct-derived exon and all endogenous exons of the erythropoietin gene.

3 more elements

A cultured vertebrate cell which expresses erythropoietin, the genome of said cell having incorporated therein a transcription unit comprising an exogenous regulatory region, an exogenous exon, and an exogenous splice-donor site, wherein transcription under the control of the exogenous regulatory region produces a transcript comprising RNA corresponding to the exogenous exon, the exogenous splice-donor site, and all endogenous exons of an endogenous erythropoietin gene, wherein the RNA corresponding to the splice-donor site directs splicing to a splice-acceptor site of the transcript which corresponds to a site within the endogenous erythropoietin gene.

A cultured vertebrate cell comprising the dhfr gene, the neo gene, the CMV immediate early promoter, intron 1 of the CMV immediate early gene, hGH exon 1 and an unpaired splice-donor site inserted at a position upstream of the ATG initiation codon of an endogenous erythropoietin gene, such that after transcription driven by the CMV promoter, the hGH exon 1 portion of the transcript is spliced to the portion of the transcript transcribed from exon 2 of the erythropoietin gene.

A DNA construct which alters expression of an endogenous GM-CSF (colony stimulating factor-granulocyte/macrophage) gene in a cell when the DNA construct is homologously recombined with a target site within or upstream of a GM-CSF gene in the chromosomal DNA of the cell, the construct comprising:

(a) a targeting sequence homologous with the target site;
(b) an exogenous regulatory sequence;
(c) an exon;
and
(d) an unpaired splice-donor site at the 3′ end of the exon;
wherein following homologous recombination of the targeting sequence with the target site, the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to the construct-derived exon in addition to all endogenous exons of the GM-CSF gene.

3 more elements

A method of producing a homologously recombinant cell wherein the expression of a GM-CSF gene is altered, comprising the steps of:

(a) providing a cell, the genome of which contains an endogenous GM-CSF gene;
(b) transfecting the cell with a DNA construct, the construct comprising
(i) a targeting sequence homologous with a target site within or upstream of the endogenous GM-CSF gene;
(ii) an exogenous regulatory sequence;
(iii) an exon;
and
(iv) an unpaired splice-donor site at the 3′ end of the exon,
thereby producing a transfected cell;
and
(c) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell in which the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to the construct-derived exon, the construct-derived splice-donor site, and all endogenous exons of the GM-CSF gene.

7 more elements

A method for producing GM-CSF, comprising the steps of:

(a) transfecting a cell, the chromosomal DNA of which contains an endogenous GM-CSF gene, with a DNA construct comprising
(i) a targeting sequence homologous with a target site within or upstream of the endogenous GM-CSF gene;
(ii) an exogenous regulatory sequence;
(iii) an exon;
and
(iv) an unpaired splice-donor site at the 3′ end of the exon,
thereby producing a transfected cell;
(b) maintaining the transfected cell under conditions appropriate for homologous recombination to occur, thereby producing a homologously recombinant cell in the genomic DNA of which (a)(ii)-(iv) are positioned upstream of the endogenous transcription initiation site of the GM-CSF gene, and the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to (a)(iii)-(iv) and the GM-CSF gene;
and
(c) maintaining the homologously recombinant cell produced in step (b) under conditions appropriate for the production of GM-CSF under the control of the exogenous regulatory sequence.

7 more elements

A DNA construct which alters the expression of an endogenous G-CSF (colony stimulating factor-granulocyte) gene in a cell when the DNA construct is homologously recombined with a target site within or upstream of the coding region of the G-CSF gene in the chromosomal DNA of the cell, said construct comprising:

(a) a targeting sequence homologous with the target site;
(b) an exogenous regulatory sequence;
(c) an exon;
and
(d) an unpaired splice-donor site at the 3′ end of the exon;
wherein following homologous recombination of the targeting sequence with the target site, the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to the construct-derived exon in addition to all endogenous exons of the G-CSF gene.

3 more elements

A method of producing a homologously recombinant cell wherein the expression of a G-CSF gene is altered, comprising the steps of:

(a) providing a cell, the genome of which contains an endogenous G-CSF gene;
(b) transfecting the cell with a DNA construct comprising
(i) a targeting sequence homologous with a target site within or upstream of the endogenous G-CSF gene;
(ii) an exogenous regulatory sequence;
(iii) an exon;
and
(iv) an unpaired splice-donor site at the 3′ end of the exon,
thereby producing a transfected cell;
and
(c) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell in which the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to the construct-derived exon, construct-derived splice-donor site, and all endogenous exons of the G-CSF gene.

7 more elements

A cultured vertebrate cell which expresses G-CSF, the genome of the cell having incorporated therein a transcription unit comprising an exogenous regulatory region, an exogenous exon, and an exogenous splice-donor site at the 3′ end of the exogenous exon, wherein transcription under the control of the exogenous regulatory region produces a transcript comprising RNA corresponding to the exogenous exon, the exogenous splice-donor site, and all of the endogenous exons of an endogenous G-CSF gene, wherein the RNA corresponding to the splice-donor site directs splicing to a splice-acceptor site of the transcript which corresponds to a site within the endogenous G-CSF gene.

A method for producing G-CSF, comprising the steps of:

(a) transfecting a cell, the chromosomal DNA of which contains an endogenous G-CSF gene, with a DNA construct comprising,
(i) a targeting sequence homologous with a target site within or upstream of the endogenous G-CSF gene;
(ii) an exogenous regulatory sequence;
(iii) an exon;
and
(iv) an unpaired splice-donor site at the 3′ end of the exon,
thereby producing a transfected cell;
(b) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell in the genomic DNA of which (a)(ii)-(iv) are positioned upstream of the endogenous transcription initiation site of the G-CSF gene, and the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to (a)(iii)-(iv) and the G-CSF gene;
and
(c) maintaining the homologously recombinant cell produced in step (b) under conditions appropriate for the production of G-CSF under the control of the exogenous regulatory sequence.

7 more elements

A DNA construct which alters the expression of an FSHβ (follicle stimulating hormone-β) gene in a cell when the DNA construct is homologously recombined with a target site within or upstream of an FSHβ gene in the chromosomal DNA of the cell, the construct comprising:

(a) a targeting sequence homologous with the target site;
(b) an exogenous regulatory sequence;
(c) an exon;
and
(d) an unpaired splice-donor site at the 3′ end of the exon,
wherein following homologous recombination of the targeting sequence with the target site, the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to the construct-derived exon in addition to all endogenous exons of the FSHβ gene.

3 more elements

A DNA construct which alters the expression of an FSHβ (follicle stimulating hormone-β) gene in a cell when the DNA construct is homologously recombined with a target site within or upstream of the first intron of an FSHβ gene in the chromosomal DNA of the cell, the construct comprising:

(a) a targeting sequence homologous with the target site;
(b) an exogenous regulatory sequence;
(c) an exon;
and
(d) an unpaired splice-donor site at the 3′ end of the exon,
wherein following homologous recombination of the targeting sequence with the target site, the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to the construct-derived exon in addition to all endogenous exons of the FSHβ gene.

3 more elements

A method of producing a homologously recombinant cell wherein the expression of an FSHβ gene is altered, comprising the steps of:

(a) providing a cell, the genome of which contains an endogenous FSHβ gene;
(b) transfecting the cell with a DNA construct comprising,
(i) a targeting sequence homologous with a target site within or upstream of the endogenous FSHβ gene;
(ii) an exogenous regulatory sequence;
(iii) an exon;
and
(iv) an unpaired splice-donor site at the 3′ end of the exon,
thereby producing a transfected cell;
and
(c) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell in which the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to the construct-derived exon, the construct-derived splice-donor site, and all of the endogenous exons of the FSHβ gene.

7 more elements

An isolated DNA molecule comprising a sequence selected from the group consisting of SEQ ID NO:3 and a fragment of SEQ ID NO:3 which selectively promotes homologous recombination with genomic DNA upstream of a thrombopoietin gene.

A method for producing FSHβ, comprising the steps of:

(a) transfecting a cell, the chromosomal DNA of which contains an endogenous FSHβ gene, with a DNA construct comprising
(i) a targeting sequence homologous with a target site within or upstream of the endogenous FSHβ gene;
(ii) an exogenous regulatory sequence;
(iii) an exon;
and
(iv) an unpaired splice-donor site at the 3′ end of the exon,
thereby producing a transfected cell;
(b) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell in the genomic DNA of which (a)(ii)-(iv) are positioned upstream of the endogenous transcription initiation site of the FSHβ gene, and the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to (a)(iii)-(iv) and the FSHβ gene;
and
(c) maintaining the homologously recombinant cell produced in step (b) under conditions appropriate for the production of FSHβ under the control of the exogenous regulatory sequence.

7 more elements

A DNA construct which 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 homologous with a target site upstream of the endogenous thrombopoietin gene;
(b) a regulatory sequence;
(c) a non-coding exon;
and
(d) an unpaired splice-donor site at the 3′ end of the exon,
wherein, following homologous recombination of the targeting sequence with the target site, exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to the construct-derived exon in addition to all endogenous exons of the thrombopoietin gene.

3 more elements

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

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

(a) a targeting sequence homologous with a target site within or upstream of the endogenous thrombopoietin coding region;
(b) a regulatory sequence;
(c) a non-coding exon;
and
(d) an unpaired splice-donor site at the 3′ end of the exon,
wherein, following homologous recombination of the targeting sequence with the target site, the regulatory sequence of (b), the non-coding exon of (c), and the unpaired splice-donor site of (d) are integrated upstream of the first exon of the thrombopoietin gene and upon expression under the control of the regulatory sequence, a transcript is produced in which sequence corresponding to the construct-derived splice-donor site is spliced to sequence corresponding to the splice-acceptor site of the second endogenous exon of the thrombopoietin gene.

3 more elements

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

(a) transfecting a cell containing the thrombopoietin gene with a DNA construct comprising
(i) a targeting sequence homologous with a target site within or upstream of the thrombopoietin gene;
(ii) an exogenous regulatory sequence;
(iii) a non-coding exon;
and
(iv) an unpaired splice-donor site at the 3′ end of the exon,
thereby producing a transfected cell, and
(b) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell in which the exogenous regulatory sequence controls expression of a transcript comprising the construct-derived exon, the construct-derived splice-donor site, and all endogenous exons of the thrombopoietin gene.

5 more elements

A cultured vertebrate cell which expresses thrombopoietin, said cell having incorporated therein a transcription unit which comprises an exogenous regulatory sequence, an exogenous non-coding exon, and an exogenous splice-donor site, the splice-donor site being operatively linked to the endogenous splice-acceptor site of the second endogenous exon of he thrombopoietin gene, wherein the cell comprises the exogenous non-coding exon in addition to all endogenous exons of the thrombopoietin gene.

A DNA construct which alters the expression of a gene encoding DNase I when inserted by homologous recombination into chromosomal DNA of the cell, said construct comprising:

(a) a targeting sequence homologous with a target site within or upstream of the DNase I gene;
(b) an exogenous regulatory sequence;
(c) an exon;
and
(d) an unpaired splice-donor site at the 3′ end of the exon,
wherein following homologous recombination of the targeting sequence with the target site, the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to the construct-derived exon in addition to all endogenous exons of the DNase I gene.

3 more elements

An isolated DNA molecule comprising a sequence selected from the group consisting of SEQ ID NO:17 and a fragment of SEQ ID NO:17 which selectively promotes homologous recombination with genomic DNA upstream of a DNase I gene.

An isolated DNA molecule of at least about 20 nucleotides which selectively promotes homologous recombination with genomic DNA within or upstream of the DNase I gene, the DNA molecule comprising a sequence selected from the group consisting of a sequence between about nucleotides −328 to −2 of FIG. 11 (SEQ ID NO:18) and a sequence which hybridizes to a sequence between about nucleotides −328 to −2 of FIG. 11 (SEQ ID NO:18).

A method of producing a homologously recombinant cell wherein the expression of a DNase I gene is altered, comprising the steps of:

(a) transfecting a cell containing the DNase I gene with a DNA construct comprising
(i) a targeting sequence homologous with a target site within or upstream of the DNase I gene;
(ii) an exogenous regulatory sequence;
(iii) an exon;
and
(iv) an unpaired splice-donor site at the 3′ end of the exon;
and
(b) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell in which the exogenous regulatory sequence controls expression of a transcript comprising the construct-derived exon, the construct-derived splice-donor site, and all of the endogenous exons of the DNase I gene.

5 more elements

A cultured vertebrate cell which expresses DNase I, the genome of the cell having incorporated therein a transcription unit comprising an exogenous regulatory region, an exogenous exon, and an exogenous splice-donor site at the 3′ end of the exogenous exon, wherein transcription under the control of the exogenous regulatory region produces a transcript comprising RNA corresponding to the exogenous exon, the exogenous splice-donor site, and all of the endogenous exons of an endogenous DNase I gene, wherein the RNA corresponding to the splice-donor site directs splicing to a splice-acceptor site of the transcript which corresponds to a site within the endogenous DNase I gene.

A method for producing DNase I comprising maintaining the cell of claim 289 under conditions appropriate for the production of DNase I under the control of the exogenous regulatory region.

A method for producing DNase I, comprising the steps of:

(a) transfecting a cell, the chromosomal DNA of which contains an endogenous DNase I gene, with a DNA construct comprising
(i) a targeting sequence homologous with a target site within or upstream of the DNase I gene;
(ii) an exogenous regulatory sequence;
(iii) an exon;
and
(iv) an unpaired splice-donor site at the 3′ end of the exon,
thereby creating a transfected cell;
(b) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell in the genomic DNA of which (a)(ii)-(iv) are positioned upstream of the endogenous transcription initiation site of the DNase I gene, and the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to (a)(iii)-(iv) and all of the endogenous exons of the DNase I gene;
and
(c) maintaining the homologously recombinant cell produced in step (b) under conditions appropriate for the production of DNase I under the control of the exogenous regulatory sequence.

7 more elements

A DNA construct which alters the expression of a β-interferon gene in a cell when the DNA construct is homologously recombined with a target site within the chromosomal DNA of the cell, the construct comprising:

(a) a targeting sequence homologous with a target site within or upstream of the β-interferon coding region;
(b) an exogenous regulatory sequence;
(c) an exon;
(d) a splice-donor site;
(e) an intron;
and
(f) a splice-acceptor site,
wherein, upon integration of the construct into chromosomal DNA by homologous recombination at the target site, the regulatory sequence of (b) controls expression of a transcript comprising sequence corresponding to the exon of (c), the splice-donor site of (d), the intron of (e), the splice-acceptor site of (f), and part or all of the β-interferon coding region.

5 more elements

An isolated DNA molecule of at least about 20 nucleotides having a sequence selected from the group consisting of SEQ ID NO:23, a fragment of SEQ ID NO:23, and a sequence which hybridizes to the complement of SEQ ID NO:23 and which selectively promotes homologous recombination with a target site upstream of a β-interferon coding sequence.

A method of producing a homologously recombinant cell wherein the expression of a β-interferon gene is altered, comprising the steps of:

(a) transfecting a cell containing the β-interferon gene with a DNA construct comprising
(i) a targeting sequence homologous to a target site within or upstream of the β-interferon coding sequence;
(ii) an exogenous regulatory sequence;
(iii) an exon;
(iv) a splice-donor site;
(v) an intron;
and
(vi) a splice-acceptor site,
thereby producing a transfected cell;
and
(b) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell in which the regulatory sequence controls expression of a transcript comprising sequence corresponding to (a)(iii)-(vi) and part or all of the β-interferon gene.

8 more elements

A cultured vertebrate cell which expresses β-interferon, the genomic DNA of the cell comprising an exogenous regulatory region positioned to control expression of an exogenous exon, an exogenous splice-donor site, an exogenous intron, an exogenous splice-acceptor site, and part or all of an endogenous β-interferon gene.

A method for producing β-interferon comprising the steps of:

(a) transfecting a cell containing a β-interferon gene with a DNA construct comprising
(i) a targeting sequence homologous to a target site within or upstream of the β-interferon gene;
(ii) an exogenous regulatory sequence;
(iii) an exon;
(iv) a splice-donor site;
(v) an intron;
and
(vi) a splice-acceptor site,
thereby producing a transfected cell;
(b) maintaining the transfected cell under conditions appropriate for homologous recombination in which the exogenous regulatory sequence is positioned to control expression of a transcript comprising RNA corresponding to (a)(iii)-(vi) and part or all of the β interferon gene, thereby producing a homologously recombinant cell;
and
(c) maintaining the homologously recombinant cell produced in step (b) under conditions appropriate for the expression of β-interferon under the control of the exogenous regulatory sequence.

9 more elements

A method for altering expression of a targeted gene, comprising the steps of:

(a) providing a cell, the genome of which comprises
(i) a targeted gene;
and
(ii) a target site within or upstream of the targeted gene;
(b) providing a DNA construct comprising:
(i) a targeting sequence homologous to the target site, and
(ii) an actin promoter from a mammalian gene;
(c) transfecting the cell with the DNA construct, thereby producing a transfected cell;
and
(d) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell the genome of which contains the construct-derived actin promoter positioned to regulate transcription of the targeted gene, the targeted gene encoding a protein other than actin;
and
(e) maintaining the homologously recombinant cell under conditions appropriate for transcription of the targeted gene under the control of the actin promoter.

9 more elements

A cultured vertebrate cell having incorporated therein a transcription unit comprising an exogenous actin promoter positioned to control transcription of an endogenous gene other than an actin gene in the chromosomal DNA of the cell.

A method of altering expression of a targeted gene, comprising the steps of:

(a) providing a cell, the genome of which comprises
(i) a targeted endogenous gene;
and
(ii) a target site within or upstream of the targeted gene;
(b) providing a DNA construct comprising:
(i) a targeting sequence homologous to the target site, and
(ii) a collagen promoter from a mammalian gene;
(c) transfecting the cell with the DNA construct, thereby producing a transfected cell;
and
(d) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell the genome of which contains the collagen promoter positioned to control transcription of the targeted gene, the targeted gene encoding a protein other than a collagen;
and
(e) maintaining the homologously recombinant cell under conditions appropriate for transcription of the targeted gene under the control of the collagen promoter.

9 more elements

A cultured vertebrate cell having incorporated therein a transcription unit, wherein the transcription unit comprising an exogenous collagen promoter positioned to control transcription of an endogenous gene other than a collagen gene in the chromosomal DNA of the cell.

A method of altering the expression of a targeted gene in the chromosomal DNA of a cell, comprising the steps of:

(a) transfecting the cell with a DNA construct comprising:
(i) a targeting sequence homologous with genomic DNA within or upstream of the coding region of the targeted gene;
(ii) a non-viral promoter;
(iii) an exon;
and
(iv) an unpaired splice-donor site at the 3′ end of the exon,
thereby producing a transfected cell;
(b) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell the genome of which comprises the non-viral promoter, the construct-derived exon, and the construct-derived splice-donor site, in addition to all endogenous exons of the targeted gene;
and
(c) maintaining the homologously recombinant cell under conditions appropriate for expression of the targeted gene under the control of the non-viral promoter.

7 more elements

A method of making a protein by altering the expression of a targeted gene that encodes the protein in a cell, comprising the steps of:

(a) transfecting the cell with a DNA construct, the DNA construct comprising:
(i) a targeting sequence homologous with genomic DNA within or upstream of the coding region of the targeted gene;
and
(ii) a non-viral promoter which is not homologous to the endogenous promoter of the targeted gene,
thereby producing a transfected cell;
(b) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell in which the non-viral promoter is positioned to control expression of the targeted gene;
and
(c) maintaining the homologously recombinant cell under conditions appropriate for expression of the protein under the control of the non-viral promoter.

5 more elements

An HT1080 cell into the genome of which is incorporated a transcription unit comprising an exogenous regulatory sequence positioned to control expression of a sequence comprising an endogenous gene, wherein the expression of the endogenous gene is altered compared to expression of the endogenous gene in an HT1080 cell lacking the transcription unit.

An RPMI 8226 cell into the genome of which is incorporated a transcription unit comprising an exogenous regulatory sequence positioned to control expression of a sequence comprising an endogenous gene, wherein the expression of the endogenous gene is altered compared to expression of the endogenous gene in an RPMI 8226 cell lacking the transcription unit.

A U-937 cell into the genome of which is incorporated a transcription unit comprising an exogenous regulatory sequence positioned to control expression of a sequence comprising an endogenous gene, wherein the expression of the endogenous gene is altered compared to expression of the endogenous gene in an RPMI 8226 cell lacking the transcription unit.

A WI-38VA13 subline 2R4 cell into the genome of which is incorporated a transcription unit comprising an exogenous regulatory sequence positioned to control expression of a sequence comprising an endogenous gene, wherein the expression of the endogenous gene is altered compared to expression of the endogenous gene in an WI-38VA13 subline 2R4 cell lacking the transcription unit.

A heterohybridoma cell into the genome of which is incorporated a transcription unit comprising an exogenous regulatory sequence positioned to control expression of a sequence comprising an endogenous gene, wherein the expression of the endogenous gene is altered compared to expression of the endogenous gene in a heterohybridoma cell lacking the transcription unit.

A linear DNA construct which alters the expression of a targeted gene in a cell when the DNA construct is homologously recombined with a target site within the chromosomal DNA of the cell, the DNA construct comprising:

(a) a targeting sequence homologous to the target site, and
(b) an exogenous regulatory sequence,
wherein each end of the DNA construct has an exonuclease-generated, single-stranded overhang.

A method of producing a protein comprising:

(a) providing an HT1080 cell containing DNA comprising an exogenous regulatory region which controls expression of a sequence encoding the protein,
(b) culturing the cell under conditions appropriate for production of the protein, thereby producing the protein, and
(c) confirming that the protein was produced.

A purified preparation of glucocerebrosidase produced by a method comprising:

(a) providing a genetically engineered human cell into the genome of which is incorporated a DNA comprising an exogenous regulatory sequence positioned to control expression of a glucocerebrosidase gene, wherein said expression under the control of the exogenous regulatory sequence is altered compared to expression of glucocerebrosidase in a human cell lacking the DNA;
(b) culturing the genetically engineered cell under conditions appropriate for production of glucocerebrosidase under the control of the exogenous regulatory sequence;
and
(c) isolating the glucocerebrosidase from the genetically engineered cell, thereby producing a purified preparation of glucocerebrosidase.

1 more element

A fusion protein comprising amino acids 1-3 of human growth hormone signal peptide and amino acids 5-165 of human erythropoietin.

A fusion protein consisting of a first sequence consisting of amino acids 1-3 of human growth hormone signal peptide fused to a second sequence which is not part of human growth hormone signal peptide.

A cultured vertebrate cell having incorporated therein a transcription unit, wherein the transcription unit comprises an exogenous non-viral promoter positioned to control expression of an endogenous gene in the chromosomal DNA of the cell, provided that the exogenous non-viral promoter alters expression of the endogenous gene compared to expression under the control of the endogenous promoter of the endogenous gene.

A cultured vertebrate cell which expresses a GM-CSF, the genome of said cell having incorporated therein a transcription unit comprising an exogenous regulatory sequence, an exogenous exon, and an exogenous splice-donor site, wherein transcription under the control of the exogenous regulatory sequence produces a transcript comprising RNA corresponding to the exogenous exon, the exogenous splice-donor site, and all of the endogenous exons of an endogenous GM-CSF gene, wherein the RNA corresponding to the splice-donor site directs splicing to a splice-acceptor site of the transcript which corresponds to a site within the endogenous GM-CSF gene.

A cultured vertebrate cell which expresses FSHβ, the genome of the cell having incorporated therein a transcription unit comprising an exogenous regulatory region, an exogenous exon, and an exogenous splice-donor site at the 3′ end of the exogenous exon, wherein transcription under the control of the exogenous regulatory region produces a transcript comprising RNA corresponding to the exogenous exon, the exogenous splice-donor site, and all of the endogenous exons of an endogenous FSHβ gene, wherein the RNA corresponding to the splice-donor site directs splicing to a splice-acceptor site of the transcript which corresponds to a site within the endogenous FSHβ gene.

Dependencies are inferred from the claim text.

Protein production and delivery

Life of this patent

Protein production and delivery

Life of this patent

Abstract

Claims356