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C1 inhibitor produced in the milk of transgenic mammals

a technology of c1 inhibitor and transgenic mammals, which is applied in the field of recombinant genetics and medicine, can solve the problems of ineffectiveness, life-threatening situation of laryngeal edema, and painful swelling of intestinal mucous, and achieve the effect of promoting the expression of dna segments

Inactive Publication Date: 2005-10-06
PHARMING INTPROP BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008] In one aspect, the invention provides transgenic non-human mammals expressing C1 inhibitor in their milk. Such mammals have a transgene comprising a recombinant DNA segment encoding a C1 inhibitor operably linked to at least one regulatory sequence effective to promote expression of the DNA segment in mammary gland cells of the transgenic nonhuman mammal and a segment encoding signal peptide functional in mammary secretory cells of th

Problems solved by technology

Swelling of the intestinal mucous can be very painful and laryngeal edema is a life-threatening situation.
Prophylactic treatment using androgens or fibrinolytic agents is used to reduce the number and severity of attacks but they are not efficient against acute crises and besides, they induce side effects that are incompatible with long-term therapy (Zurlo J. et al 1990, Fertility and sterility 54: 64)
However, product isolated from plasma poses substantial risk of contamination.
Although taking the precautions for virus removal / inactivation there is still a risk for transmission of viruses such as HIV and hepatitis (De Filippi F. et al.
In addition to the safety problem the lack of availability of purified plasma C1 inhibitor as well as the high costs involved are disadvantages.
However, the reported yield in the jig / ml range is too low for therapeutic application.
Expression of C1 inhibitor in microorganisms would not be expected to result in correct posttranslational modification for functional inhibitor, and as far as we are aware, has not been attempted.

Method used

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  • C1 inhibitor produced in the milk of transgenic mammals
  • C1 inhibitor produced in the milk of transgenic mammals
  • C1 inhibitor produced in the milk of transgenic mammals

Examples

Experimental program
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Effect test

example 1

Construction of Transgenes

a. Overlapping Genomic Constructs (CINH1)

[0065] A set of two expression vectors containing overlapping parts of the genomic sequence of the human C1 inhibitor gene was constructed. Together these plasmids contain the bovine αS1-casein promoter and the complete human C1 inhibitor genomic sequence. All C1 inhibitor fragments used were derived from P1 clone DMDC-HFF#1-1112-69, obtained from Genome Systems Inc. (8620 Pennell Drive, St. Louis, Mo. 63114), which was isolated from a P1 human genomic library by PCR with two C1 inhibitor specific primers (Appendix 1A).

[0066] Plasmid pαS1 / 5′C1, which includes 6.3 kb of bovine αS1-casein regulatory sequences fused to the 5′-part of the C1 inhibitor gene, was constructed as follows. First, pKUN1 [Konings, 1986 Gene 46,269-76] was digested with EcoRI and SalI and ligated to linker 1 (Appendix 1B), followed by removal of the ClaI site by filling in with Klenow and ligating. From the resulting plasmid pKUN2ΔC, pKUN2ΔC...

example 2

Transgenesis

A. Overlapping Constructs (CINH1) in Mice.

[0069] Transgenic mice were produced by pronuclear injection of fertilized oocytes, essentially as described by Hogan et al., 1986, “Manipulating the Mouse Embryo”, Cold Spring Harbour Press NY. pαS1-5′C1 (see FIG. 1A) was digested with NotI, yielding an 11.3 kb fragment which was isolated by gelpurification and electroelution. Similarly, a 15.8 kb ScaI-EcoRV fragment from pIC20R / 3′-C1 , extending from intron 3 to 2 kb beyond the last exon, was prepared. Both fragments were combined (at a concentration of 3 ng / μl) and injected into the pronucleus of fertilized mouse oocytes, which were implanted in the uterus of pseudopregnant mouse foster mothers. The offspring was analyzed for the insertion of the human C1 inhibitor genomic gene construct by Southern blotting of DNA isolated from clipped tails. Whether correct homologous recombination between the overlapping fragments had occurred, was checked on Southern blots and by PCR. 3...

example 3

Analysis Of C1 Inhibitor in the Milk of Transgenic Animals

A, B. Overlapping and Single Genomic Constructs in Mice (CINH1 and CINH2).

[0074] Milk from transgenic mice and non-transgenic controls was analyzed by an enzyme-linked-radio-immuno-assay (for a description of the ELISA, see Appendix 2; for the expression data see Tables 1, 2 and 3). The ELISA measures the total amounts of C1 inhibitor (both active and inactive). The average levels of C1 inhibitor obtained in the transgenic mice made with the overlapping fragments ranged from 0.04-5 mg / ml. Some individual samples of the highest producing lines contained more than 20 mg / ml. The average levels of C1 inhibitor obtained in the transgenic mice made with the single fragment ranged from 0.1 μg / ml-10 mg / ml. Some individual samples of the highest producing line (5903) contained more than 20 mg / ml.

TABLE 1C1 inhibitor expression data of rH-C1INH1 mouse linesC1 inhibitor# integrationexpression F1 μg / ml1Line nositessublineAverage2Max3...

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Abstract

The invention provides transgenic nonhuman mammals expressing C1 inhibitor in their milk. The C1 inhibitor is useful in treating patients with hereditary angioedema or patients requiring immunosuppression.

Description

TECHNICAL FIELD [0001] The present invention resides in the fields of recombinant genetics, and medicine, and is directed to transgenic production of C1 inhibitor and its use as a therapeutic molecule, e.g. in replacement therapy in patients with hereditary angioedema or patients requiring immunosuppression. BACKGROUND OF THE INVENTION [0002] Human C1 inhibitor, also known as C1 esterase inhibitor, is a well-known and identified substance. C1 inhibitor belongs to the superfamily of serine proteinase inhibitors and is the only inhibitor of C1r and C1s of the complement system and is the major inhibitor of factor XIIa and kallikrein of the contact system. In addition C1 inhibitor inhibits also other serine proteases of the coagulation and fibrinolytic systems like factor XI, tissue type plasminogen activator and plasmin (Schapira M. et al. 1985, Complement 2:111 / Davis A. E. 1988, Ann. Rev. Immunol. 6:595). [0003] C1 inhibitor is encoded by a single gene on chromosome 11 and consists o...

Claims

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Application Information

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IPC IPC(8): A01K67/027A23L1/305A61K38/00A61K38/55A61P9/00A61P9/10A61P17/02A61P29/00A61P31/04A61P37/06C07K14/81C12N15/09C12N15/85
CPCA01K67/0278A01K2207/15A01K2217/00A01K2217/05A01K2227/105A01K2227/107A01K2267/01A23C2230/05A61K38/00C07K14/8121C12N15/8509A61P1/18A61P11/00A61P15/06A61P17/02A61P19/02A61P25/08A61P29/00A61P31/04A61P37/06A61P43/00A61P9/00A61P9/10
Inventor NUIJENS, JOHANNESVAN VEEN, HENRICUSPIEPER, FRANKHEUS, JORIS
Owner PHARMING INTPROP BV
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