Claims for Patent: 6,201,168
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Summary for Patent: 6,201,168
Title: | Pathogenesis of cardiomyopathy |
Abstract: | Disclosed is a mouse, cells derived therefrom, and methods for using the mouse, the mouse being homozygous for a disrupted .delta.-sarcoglycan gene, the disruption in the gene having been introduced into the mouse or an ancestor of the mouse at an embryonic stage. The disruption prevents the synthesis of functional .delta.-sarcoglycan in cells of the mouse and results in the mouse having a reduced amount of .beta.- and .epsilon.-sarcoglycan and sarcospan, and a disruption of the sarcoglycan-sarcospan complex in smooth muscle of the mouse. Also disclosed is a mouse, cells derived therefrom, and methods for using the mouse, the mouse being homozygous for a disrupted .beta.-sarcoglycan gene, the disruption in the gene having been introduced into the mouse or an ancestor of the mouse at an embryonic stage. The disruption prevents the synthesis of functional .beta.-sarcoglycan in cells of the mouse and results in the mouse having a reduced amount of .delta.-and .epsilon.-sarcoglycan and sarcospan and .alpha.-dystroglycan in smooth muscle of the mouse. |
Inventor(s): | Campbell; Kevin P. (Iowa City, IA), Coral; Ramon (Iowa City, IA), Cohn; Ronald (Iowa City, IA), Williamson; Roger (Iowa City, IA), Durbeej; Madeleine (Iowa City, IA) |
Assignee: | University of Iowa Research Foundation (Iowa City, IA) |
Application Number: | 09/378,418 |
Patent Claims: | 1. A transgenic knockout mouse whose genome comprises a homozygous disruption in its endogenous .beta.-sarcoglycan gene, wherein said homozygous disruption prevents the
expression of a functional .beta.-sarcoglycan protein in cells of the mouse, and wherein said homozygous disruption results in said transgenic knockout mouse exhibiting conditions of: (i) a reduced amount of .delta.-and .epsilon.-sarcoglycan, sarcospan
and .alpha.-dystroglycan in a smooth muscle, (ii) a disruption of the sarcoglycan-sarcospan complex in a smooth muscle, and (iii) a reduced amount of sarcospan and .alpha.-, .gamma.-, -.delta., and .epsilon.-sarcoglycan in the sarcolemma of a skeletal
and a cardiac muscle, compared to the amounts of .delta.-and .epsilon.-sarcoglycan, sarcospan and .alpha.-dystroglycan in the smooth muscles, the sarcoglycan-sarcospan complex in the smooth muscles, and sarcospan and .alpha.-, .gamma.-, -.delta., and
.epsilon.-sarcoglycan in the sarcolemma of the skeletal and cardiac muscles of a wild type mouse, and wherein said homozygous disruption results in ischemia in said transgenic knockout mouse.
2. The transgenic knockout mouse of claim 1, wherein said homozygous disruption consists of a deletion of a 1606 nucleotides of intron 2 upstream of exon 3, entirety of exons 3-6 and introns 3-5, and 498 nucleotides immediately downstream of exon 6 of the .beta.-sarcoglycan gene, and wherein the deleted portion of the endogenous .beta.-sarcoglycan gene is replaced with a PGK-neomycin resistance cassette. 3. The transgenic knockout mouse of claim 1, wherein the method of making said transgenic knockout mouse comprises: introducing a DNA construct into a mouse embryonic stem cell, and wherein said DNA construct comprises in order: the first 1800 nucleotides of intron 2 of the .beta.-sarcoglycan gene starting at the 5' end of the intron 2, a DNA sequence encoding a neomycin resistance gene, and 6500 nucleotides starting at 498 nucleotides immediately downstream of exon 6 of the .beta.-sarcoglycan gene, wherein said DNA construct lacks exons 3-6 and introns 3-5 of the .beta.-sarcoglycan gene, wherein the introduction of said DNA construct into said mouse embryonic stem cell results in the disruption of the endogenous .beta.-sarcoglycan gene; introducing said mouse embryonic stem cell into a mouse blastocyst and transplanting said blastocyst into a pseudopregnant mouse; allowing said blastocyst to develop into a chimeric mouse whose genome contains the DNA construct; breeding said chimeric mouse to produce heterozygous offspring; producing progeny from said heterozygous offspring; and screening said progeny to identify a homozygous transgenic knockout mouse whose genome comprises disruption of the endogenous .beta.-sarcoglycan gene. 4. A smooth muscle, skeletal muscle, or cardiac muscle cell isolated from the transgenic knockout mouse of claim 1, wherein the genome of said smooth muscle, skeletal muscle, or cardiac muscle cell comprises a homozygous disruption in its endogenous .beta.-sarcoglycan gene, wherein said homozygous disruption consists of a deletion of a 1606 nucleotides of intron 2 upstream of exon 3, entirety of exons 3-6 and introns 3-5, and 498 nucleotides immediately downstream of exon 6 of the .beta.-sarcoglycan gene, and wherein the deleted portion of the endogenous .beta.-sarcoglycan gene is replaced with a PGK-neomycin resistance cassette, and wherein said homozygous disruption prevents the expression of a functional .beta.-sarcoglycan protein in said smooth muscle, skeletal muscle, or cardiac muscle cell. 5. A smooth muscle, skeletal muscle, or cardiac muscle cell isolated from the transgenic knockout mouse of claim 2, wherein the genome of said smooth muscle, skeletal muscle, or cardiac muscle cell comprises a homozygous disruption in its endogenous .beta.-sarcoglycan gene, wherein said homozygous disruption consists of a deletion of a 1606 nucleotides of intron 2 upstream of exon 3, entirety of exons 3-6 and introns 3-5, and 498 nucleotides immediately downstream of exon 6 of the .beta.-sarcoglycan gene, and wherein the deleted portion of the endogenous .beta.-sarcoglycan gene is replaced with a PGK-neomycin resistance cassette, and wherein said homozygous disruption prevents the expression of a functional .beta.-sarcoglycan protein in said smooth muscle, skeletal muscle, or cardiac muscle cell. 6. A smooth muscle, skeletal muscle, or cardiac muscle cell isolated from the transgenic knockout mouse of claim 3, wherein the genome of said smooth muscle, skeletal muscle, or cardiac muscle cell comprises a homozygous disruption in its endogenous .beta.-sarcoglycan gene, wherein said homozygous disruption consists of a deletion of a 1606 nucleotides of intron 2 upstream of exon 3, entirety of exons 3-6 and introns 3-5, and 498 nucleotides immediately downstream of exon 6 of the .beta.-sarcoglycan gene, and wherein the deleted portion of the endogenous .beta.-sarcoglycan gene is replaced with a DNA construct wherein said DNA comprises in order: the first 1800 nucleotides of intron 2 of the .beta.-sarcoglycan gene starting at the 5' end of the intron 2, a DNA sequence encoding a neomycin resistance gene, and 6500 nucleotides starting at 498 nucleotides immediately downstream of exon 6 of the .beta.-sarcoglycan gene, wherein said DNA construct lacks exons 3-6 and introns 3-5 of the .beta.-sarcoglycan gene, and wherein said homozygous disruption prevents the expression of a functional .beta.-sarcoglycan protein in said smooth muscle, skeletal muscle, or cardiac muscle cell. 7. A method for identifying a candidate therapeutic compound for the treatment of an individual diagnosed with .beta.-sarcoglycan-deficient limb-girdle muscular dystrophy, comprising: a) providing a transgenic knockout mouse whose genome comprises a homozygous disruption in its endogenous .beta.-sarcoglycan gene, wherein said homozygous disruption prevents the expression of a functional .beta.-sarcoglycan protein in cells of the transgenic knockout mouse, and wherein said homozygous disruption results in said transgenic knockout mouse exhibiting conditions of: (i) a reduced amount of .delta.-and .epsilon.-sarcoglycan, sarcospan and .alpha.-dystroglycan in a smooth muscle, (ii) a disruption of the sarcoglycan-sarcospan complex in a smooth muscle, and (iii) a reduced amount of sarcospan and .alpha.-, .gamma.-, .delta., and .epsilon.-sarcoglycan in the sarcolemma of a skeletal and a cardiac muscle, compared to the amounts of .delta.-and .epsilon.-sarcoglycan, sarcospan and .alpha.-dystroglycan in the smooth muscles, the sarcoglycan-sarcospan complex in the smooth muscles, and sarcospan and .alpha.-, .gamma.-, .delta., and .epsilon.-sarcoglycan in the sarcolemma of the skeletal and cardiac muscles of a wild type mouse, and wherein said homozygous disruption results in ischemia in said transgenic knockout mouse; b) administering the candidate therapeutic compound to the transgenic knockout mouse of step a); and c) assaying the therapeutic effects of the candidate therapeutic compound by comparing the parameters of: (i) a reduced amount of .delta.-and .epsilon.-sarcoglycan, sarcospan and .alpha.-dystroglycan in smooth muscles, (ii) a disruption of the sarcoglycan-sarcospan complex in smooth muscles, (iii) a reduced amount of sarcospan and .alpha.-, .gamma.-, .delta., and .epsilon.-sarcoglycan in the sarcolemma of skeletal and cardiac muscles, and (iv) ischemia, in the transgenic knockout mouse which has received the candidate compound as in step b) with the same parameters of a transgenic knockout mouse of step a) which has not received the candidate therapeutic compound, wherein a difference in one or more of the measured parameters of the transgenic knockout mouse of step b) is an indication of a potential therapeutic effect of the candidate therapeutic compound on .beta.-sarcoglycan-deficient limb-girdle muscular dystrophy. 8. The method of claim 7, wherein the administration of said candidate therapeutic compound results in the delivery of the candidate compound to the smooth muscle cells of the transgenic knockout mouse. 9. The method of claim 7, wherein the administration of said candidate therapeutic compound results in the delivery of the candidate compound to the skeletal muscle cells of the transgenic knockout mouse. 10. The method of claim 7, wherein the administration of said candidate therapeutic compound results in the delivery of the candidate compound to the cardiac muscle cells of the transgenic knockout mouse. 11. The method of claim 7, wherein said candidate therapeutic compound is a DNA construct encoding a protein and administration of said DNA construct results in the expression of the protein in the cells of the transgenic knockout mouse. 12. A method of identifying a candidate therapeutic compound for the treatment of ischemic heart disease in an individual caused by a reduced expression of .beta.-sarcoglycan in the vascular smooth muscles of the individual, comprising: a) providing a transgenic knockout mouse whose genome comprises a homozygous disruption in its endogenous .beta.-sarcoglycan gene, wherein said homozygous disruption prevents the expression of a functional .beta.-sarcoglycan protein in cells of the transgenic knockout mouse, and wherein said homozygous disruption results in said transgenic knockout mouse exhibiting conditions of: (i) a reduced amount of .delta.-and .epsilon.-sarcoglycan, sarcospan and .alpha.-dystroglycan in a smooth muscle, (ii) a disruption of the sarcoglycan-sarcospan complex in a smooth muscle, and (iii) a reduced amount of sarcospan and .alpha.-, .gamma.-, .delta., and .epsilon.-sarcoglycan in the sarcolemma of a skeletal and a cardiac muscle, compared to the amounts of .delta.-and .epsilon.-sarcoglycan, sarcospan and .alpha.-dystroglycan in the smooth muscles, the sarcoglycan-sarcospan complex in the smooth muscles, and sarcospan and .alpha.-, .gamma.-, .delta., and .epsilon.-sarcoglycan in the sarcolemma of the skeletal and cardiac muscles of a wild type mouse, and wherein said homozygous disruption results in ischemia in said transgenic knockout mouse; b) administering the candidate therapeutic compound to the transgenic knockout mouse of step a) whereby the candidate compound is delivered to the vascular smooth muscle cells of the mouse; and c) assaying the therapeutic effects of the candidate therapeutic compound by comparing ischemia in the transgenic knockout mouse which has received the candidate compound as in step b) with the ischemia in a transgenic knockout mouse of step a) which has not received the candidate therapeutic compound, and wherein a reduction or a reversal of ischemia in the transgenic knockout mouse of step b) is an indication of a potential therapeutic effect of the candidate therapeutic compound on ischemic heart disease. 13. A method of identifying a candidate therapeutic compound for the prevention of ischemic injury in an individual caused by a reduced expression of .beta.-sarcoglycan in the vascular smooth muscles of the individual, comprising: a) providing a transgenic knockout mouse whose genome comprises a homozygous disruption in its endogenous .beta.-sarcoglycan gene, wherein said homozygous disruption prevents the expression of a functional .beta.-sarcoglycan protein in cells of the transgenic knockout mouse, and wherein said homozygous disruption results in said transgenic knockout mouse exhibiting conditions of: (i) a reduced amount of .delta.-and .epsilon.-sarcoglycan, sarcospan and .alpha.-dystroglycan in a smooth muscle, (ii) a disruption of the sarcoglycan-sarcospan complex in a smooth muscle, and (iii) a reduced amount of sarcospan and .alpha.-, .gamma.-, .delta., and .epsilon.-sarcoglycan in the sarcolemma of skeletal and cardiac muscles, compared to the amounts of .delta.- and .epsilon.-sarcoglycan, sarcospan and .alpha.-dystroglycan in the smooth muscles, the sarcoglycan-sarcospan complex in the smooth muscles, and sarcospan and .alpha.-, .gamma.-, .delta., and .epsilon.-sarcoglycan in the sarcolemma of the skeletal and cardiac muscles of a wild type mouse, and wherein said homozygous disruption results in ischemia in said transgenic knockout mouse; b) administering the candidate therapeutic compound to the transgenic knockout mouse of step a) whereby the candidate compound is delivered to the vascular smooth muscle cells of the mouse; and c) assaying the therapeutic effects of the candidate therapeutic compound by comparing ischemia in the transgenic knockout mouse which has received the candidate compound as in step b) with the ischemia in a transgenic knockout mouse of step a) which has not received the candidate therapeutic compound, and wherein a reduction or a reversal of ischemia in the transgenic knockout mouse of step b) is an indication of a potential preventive effect of the candidate therapeutic compound on ischemic injury. |
Details for Patent 6,201,168
Applicant | Tradename | Biologic Ingredient | Dosage Form | BLA | Approval Date | Patent No. | Expiredate |
---|---|---|---|---|---|---|---|
Merck Sharp & Dohme Corp. | INTRON A | interferon alfa-2b | For Injection | 103132 | 06/04/1986 | ⤷ Try a Trial | 2039-02-26 |
Merck Sharp & Dohme Corp. | INTRON A | interferon alfa-2b | For Injection | 103132 | ⤷ Try a Trial | 2039-02-26 | |
Merck Sharp & Dohme Corp. | INTRON A | interferon alfa-2b | Injection | 103132 | ⤷ Try a Trial | 2039-02-26 | |
>Applicant | >Tradename | >Biologic Ingredient | >Dosage Form | >BLA | >Approval Date | >Patent No. | >Expiredate |
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