ES2587344T3 - Transgenic mouse comprising a polynucleotide encoding human or humanized C5aR - Google Patents

Abstract

A transgenic mouse comprising and expressing a polynucleotide encoding a human or humanized C5aR, in which the C5aR-coding region of endogenous exon 3 of the mouse C5aR locus has been targeted replaced by a corresponding sequence encoding human or humanized C5aR. humanized and wherein said humanized C5aR differs from mouse C5aR encoded by said mouse locus in that the extracellular domains are replaced by corresponding extracellular domains of human C5aR.

Description

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(b)

 optionally, determine the structure of the candidate compound; Y

(C)

 provide the candidate compound or the structure name of the candidate compound.

Naturally, for agents that are known but have not been previously analyzed for their function using a screening provided by the present invention, the determination of the structure of the compound is implicit in step (b). This is because the person skilled in the art will know the name and / or structure of the compound at the time of screening.

As used herein, the term "provide the agent" should be understood to include any chemical or recombinant synthetic means to produce said agent or, alternatively, the arrangement of an agent that has been previously synthesized by any person or means, medium.

Compounds identified for administration to a non-human or human animal as described herein may be formulated for administration by injection through a subcutaneous, intravenous, intranasal or intraperitoneal route. Alternatively, they may be suitable for oral administration in the form of food additives, tablets, troches, etc.

The various aspects and embodiments described herein, referred to in the previous individual sections, apply, where appropriate, to other sections, changing whatever is necessary. Consequently, the aspects specified in one section can be combined with aspects specified in other sections, when appropriate.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Diagram showing the design of an artificial structure with a target for the generation of mice with an activated human C5aR gene.

Figure 2: Diagram showing a transgenic mouse C5aR locus after deletion of the PGKneo gene by Cre recombinase.

Figure 3: Diagram showing selected restriction sites and exons of the mouse C5aR (C5r1) gene.

Figure 4: Location of oligonucleotide primers used in the genotyping assay by PCR.

Figure 5: FACS analysis showing that human C5aR is expressed in neutrophils of mice that are carriers of the hC5aR gene. Blood from wild-type mice (+ / +) was incubated; heterozygous (H5Rf / +) and homozygous (H5Rf / H5Rf) with a 7F3 antibody specific for human C5aR conjugated to FITC. The area on the right of the dashed line in the neutrophil histogram is the positive staining for 7F3-FITC.

Figure 6: Progress of an AR-like inflammatory disease in mice homozygous for the activated human C5aR gene and wild-type mice. The panel on the left shows the increase in ankle thickness after injecting via i.p. K / BxN serum on days 0 and 2. The panel on the right shows the clinical score. Mice # 10 and # 25 are homozygous for the human C5aR gene, mouse # 38 is a wild-type litter mate. Ankle thickening and clinical disease developed in both wild-type and homozygous mice for the hC5aR gene in the typical manner described for this model (Lee et al. (2002) Science, 297, 1689-1692).

Figure 7: Graphs showing the progress and prevention of an inflammatory disease similar to RA in mice homozygous for the activated human C5aR gene. The panel on the left shows the increase in thickening of the ankles after injecting via i. p. K / BxN serum on days 0 and 2. The panel on the right shows the clinical score. Mice # 7, 10, 24 and 25 are homozygous for the human C5aR gene. Mice # 7 and # 24 were injected with the 7F3 antibody neutralizing human C5aR, while mice # 10 and # 25 were injected with an isotype control antibody. The wild-type litter mate mouse (mouse # 38) was also treated with 7F3. The antibody was injected on days -1 and +3. The mice to which the control antibody had been injected developed an inflammatory disease. In contrast, mice with the activated hC5aR gene, which had been injected 7F3, were protected against inflammation. Wild-type mice injected with 7F3 were not protected against inflammation.

KEY TO THE SEQUENCE LISTING

SEQ ID NO: 1: Locus sequence of the mouse C5aR (C5r1) gene and flanking DNA.

SEQ ID NO: 2: Human C5aR cDNA sequence.

SEQ ID NO: 3: Amino acid sequence of human C5aR.

SEQ ID NO: 4: F1C primer (specific for exon 3 of human C5aR):

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SEQ ID NO: 5: Primer R2a (specific for exon 3 of human C5aR):

SEQ ID NO: 6: F3C primer (specific for the mouse C5aR gene):

SEQ ID NO: 7: R4C primer (specific for the mouse C5aR gene):

DETAILED DESCRIPTION OF THE INVENTION

General techniques and definitions

Unless otherwise indicated, the recombinant DNA techniques used in the present invention are conventional methods, well known to those skilled in the art. Such techniques are described and explained through scientific publications, in sources such as: J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press (1989), TA Brown (compiler), Essential Molecular Biology: A Practical Approach, volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (compilers), DNA Cloning: A Practical Approach, volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (compilers), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date) and are incorporated herein by reference. In particular, these documents describe in detail methods for transcribing or replicating nucleic acid molecules and the conditions required for it.

Definitions

Through this specification the word "comprise" or variations, such as "comprises" or "comprising (n)", is understood to imply the inclusion of an established element, an integer or a stage or a group of elements , integers or stages, but not the exclusion of any other element, integer or stage or group of elements, integers or stages.

As used herein, a "ligand" of a C5aR protein refers to a particular class of substances that bind to a mammalian C5aR protein, including natural ligands and synthetic and / or recombinant forms of natural ligands. . In a preferred embodiment, ligand binding of a C5aR protein takes place with high affinity.

As used herein, an "antagonist" is a substance that inhibits the binding of an agonist or an inverse agonist to a C5aR protein and thus prevents at least one characteristic function of a C5aR protein, such as a binding activity (e.g., ligand binding, promoter binding, antibody binding), a signaling activity (e.g., activation of a mammalian G protein, induction of a rapid and transient increase in the concentration of free calcium in cytosol) and / or a cellular response function (eg, stimulation of chemotaxis, exocytosis or release of inflammatory mediators by leukocytes). The term "antagonist" encompasses substances that bind to the receptor (for example, an antibody, a mutant of a natural ligand, organic molecules of low molecular weight, other competitive inhibitors of ligand binding) and substances that block the function of receptors without join them.

As used herein, an "agonist" is a substance that favors (induces, causes, potentiates or increases) at least one characteristic function of a C5aR protein, such as a binding activity (eg, binding of ligands, inhibitors and / or promoters), a signaling activity (for example, activation of a mammalian G protein, induction of a rapid and transient increase in the concentration of free calcium in the cytosol) and / or a cellular response function (for example, stimulation of chemotaxis, exocytosis or release of inflammatory mediators by leukocytes). The term agonist encompasses substances that bind to the receptor (for example, an antibody, a homologue of a natural ligand of another species) and substances that favor the function of receptors without binding to them (for example, by activating an associated protein). In a preferred embodiment, the agonist is distinct from a homologue of a natural ligand.

"Inverse agonist" as used herein refers to a molecule that binds or otherwise interacts with C5aR to inhibit the intracellular response at the baseline, initiated by the active form of C5aR below the base level. normal activity observed in the absence of agonists.

By "transgenic mammal" is meant a mammal (eg, mouse, rat, hamster, etc.), which has a non-endogenous nucleic acid sequence (ie, heterologous) present as an extrachromosomal element in a portion of its cells or stably integrated into your germline DNA (that is, in the genomic sequence of most of your cells). The heterologous nucleic acid is introduced into the germ line of such transgenic animals by genetic manipulation, for example, of embryos or embryonic stem cells of the host animal.

The term "biologically active" or "functional", when used herein as a modifier of human C5aR, refers to a polypeptide that shows at least one of the functional characteristics attributed to natural human C5aR, such as the ability to act as a C5a receptor or to bind to one or more natural ligands of human C5aR.

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A "deactivation" of a gene ("knock-out") means an alteration in the sequence of the gene that results in a decrease in the function of the target gene, preferably so that the expression of the target gene is undetectable or insignificant. A deactivation of an endogenous gene means that the gene's function has decreased substantially, so that the expression is not detectable or only at negligible levels. "Deactivated" transgenic animals can be transgenic animals that have a heterozygous deactivation of a gene or a homozygous deactivation of a gene. "Deactivations" also include conditional deactivations, where an alteration of the target gene can occur, for example, after an exposure of the animal to a substance that favors the alteration of the target gene, the introduction of an enzyme that favors recombination in the target gene site (for example, Cre in the Cre-lox system), or another method to direct the alteration of the target gene postnatally.

An "activation" of a target gene (from English, "knock-in") means an alteration in a host cell genome that results in an altered expression (eg, increased (including ectopic)) of the target gene, by example, by the introduction of an additional copy of the target gene or by a functional insertion of a regulatory sequence that provides enhanced expression of an endogenous copy of the target gene. The "activated" transgenic animals of interest for the present invention are transgenic animals that have an activation of a human or humanized C5aR gene. Such transgenic animals may be heterozygously activated for the human or humanized C5aR gene or be homozygous for the activation of the human or humanized C5aR gene. "Activations" also cover conditional activations.

By "artificial structure" is meant a recombinant nucleic acid, generally recombinant DNA, which has been generated in order to express a specific nucleotide sequence or to be used in the construction of other recombinant nucleotide sequences.

By "functionally linked" is meant a DNA sequence and a regulatory sequence that are connected in such a way that they allow gene expression when the appropriate molecules (for example, transcription activating proteins) are bound to the regulatory sequence.

By "functionally inserted" is meant a nucleotide sequence of interest that is placed adjacent to a nucleotide sequence that directs the transcription and translation of the introduced nucleotide sequence of interest (ie, that facilitates the production, for example, of a polypeptide encoded by a human C5aR sequence).

The term "corresponds to" or "corresponding to" means homologous to, or substantially equivalent to, or functionally equivalent to the designated sequence.

The term "artificial structure of the transgenic gene" refers to a nucleic acid molecule, for example, a vector, which contains the object polynucleotide, for example, the human C5aR polynucleotide or one of its fragments, functionally linked in a way that It is capable of expressing the polynucleotide in a host cell. As used herein, the term "polynucleotide" includes deoxyribonucleic acid (DNA), and, when appropriate, ribonucleic acid (RNA). As used herein, the term also includes RNA or DNA analogs prepared from nucleotide analogs, and when applicable for the described embodiment, single stranded polynucleotides (such as sense or antisense) and double stranded.

Transgenic animals

Methods for producing transgenic animals are well known in the art. A general textbook, useful for this topic is that of Houdebine, Transgenic animals-Generation and Use (Harwood Academic, 1997) which is a comprehensive review of the techniques used to generate transgenic animals from fish to mice and cows. Transgenic mice are of particular interest in the context of the present invention.

Advances in technologies for micromanipulation of embryos now allow the introduction of heterologous DNA, for example, into fertilized mammalian eggs. For example, totipotent or pluripotent stem cells can be transformed by microinjection, calcium phosphate mediated precipitation, liposome fusion, retroviral infection or other means, then the transformed cells are introduced into the embryo, and the embryo then develops into a transgenic animal In a preferred method, developing embryos are transfected with the desired DNA by electroporation, and transgenic animals are produced from the infected embryo. In another preferred method, however, the appropriate DNAs are co-injected into the pronucleus or cytoplasm of embryos, preferably at the stage of a single cell, and embryos are allowed to develop to mature transgenic animals. These techniques are well known. See reviews of conventional laboratory methods for microinjection of heterologous DNAs in fertilized mammalian eggs, including Hogan et al., Manipulating the Mouse Embryo, (Cold Spring Harbor Press 1986); Krimpenfort et al., Bio / Technology 9: 844 (1991); Palmiter et al., Cell, 41: 343 (1985); Kraemer et al., Genetic manipulation of the Mammalian Embryo, (Cold Spring Harbor Laboratory Press 1985); Hammer et al., Nature, 315: 680 (1985); Wagner et al., US Pat. No. 5,175,385; Krimpenfort et al., US Pat. No. 5,175,384.

Another method used to produce a transgenic animal involves microinjecting a nucleic acid into eggs in the pronuclear stage by conventional methods. Injected eggs are grown after transferring them to

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the oviducts of the pseudo-pregnant recipients.

Transgenic animals can also be produced by nuclear transfer technology, as described in Schnieke, A.E. et al., 1997, Science, 278: 2130 and Cibelli, J.B. et al., 1998, Science, 280: 1256. Using this method, donor animal fibroblasts are stably transfected with a plasmid that incorporates the coding sequences of a binding domain or a binding partner of interest, under the control of regulatory elements Stable transfectants are then fused with enucleated oocytes, cultured and transferred to recipient females.

The analysis of animals that may contain transgenic sequences would normally be performed by PCR or Southern blot analysis, following conventional methods.

By way of specific example for the construction of transgenic mammals, such as cows, artificial nucleotide structures comprising a sequence encoding a binding domain fused with GFP are microinjected using, for example, the technique described in US Pat. .UU. No. 4,873,191, in oocytes that are obtained from ovaries recently removed from the mammal. The oocytes are aspirated from the follicles and allowed to settle before fertilization with frozen sperm, trained with heparin and previously fractionated by Percoll gradient to isolate the mobile fraction.

The fertilized oocytes are centrifuged, for example, for eight minutes at 15,000 g to visualize the pronuclei for injection and then cultured from the zygote stage to the morula or blastocyst stage in conditioned medium of oviduct tissue. This medium is prepared using scratched luminous tissues of the oviducts and diluted in culture medium. Zygotes should be placed in the culture medium two hours after microinjection.

The estrus is then synchronized in the intended recipient mammals, such as cattle, administering coprostanol. Estrus occurs within the next two days and embryos are transferred to recipients 57 days after estrus. Successful transfer can be assessed in the offspring by Southern transfer.

Alternatively, the desired artificial structures can be introduced into embryonic stem cells (abbreviated ES cells, by the English expression Embryonic Stem) and the cells are cultured to ensure modification through the transgene. The modified cells are then injected into the embryonic stage of the blastula and the blastulas are replaced in the pseudopregnant hosts. The resulting offspring is chimeric with respect to ES cells and host cells, and non-chimeric strains comprising exclusively the ES progeny can be obtained, using conventional cross reproduction. This technique is described, for example, in WO91 / 10741.

Transgenic animals comprise an exogenous nucleic acid sequence, present as an extrachromosomal element or stably integrated into all or a portion of their cells, especially germ cells. It is preferred that a transgenic animal comprises stable changes for the germline sequence. A stable change is generally achieved by introducing the DNA into the genome of the cell. Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACs and the like. During the initial construction of the animal "chimeras" or "chimeric animals" are generated, in which only a subset of cells has the altered genome. Chimeras are mainly used for breeding purposes, in order to generate the desired transgenic animal. Animals that have a heterozygous disorder are generated by chimera reproduction. Normally male and female heterozygous are raised to generate homozygous animals.

In a preferred embodiment, the transgenic mice of the invention are produced by introducing a human C5aR transgene into the mouse germ line. Embryonic stem cells (ES) are the primary type of target cell for the introduction of the human C5aR transgene into the mouse, to achieve homologous recombination. ES cells can be obtained from preimplantation embryos grown in vitro and fused with embryos (Evans, MJ, et al., (1981) Nature 292, 154-156; Bradley, MO, et al., (1984) Nature 309, 255-258; Gossler, et al., (1986) Proc. Natl. Acad. Sci. USA 83, 9065-9069; and Robertson, et al., (1986) Nature 322, 445-448). Transgenes can be efficiently introduced into ES cells by DNA transfection or by retrovirus mediated transduction. Such transformed ES cells can then be combined with the blastocysts of a non-human animal. ES cells then colonize the embryo and contribute to the germ line of the resulting chimeric animal. For a review see Jaenisch, R. (1988) Science 240, 1468-1474. Transfected embryonic cells can be incubated in vitro for variable times or reimplanted in the rental host

or both.

The transgenic offspring of the rental host can be screened to analyze the presence and / or expression of the transgene by any suitable method. Screening is frequently performed by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene. As an alternative or additional method, Western blot analysis using an antibody against the protein encoded by the transgene can be used to screen for the presence of the transgene product. In transgenic mice, DNA is normally prepared from tail tissue and analyzed by

Southern analysis or PCR for the transgene. Alternatively, tissues or cells believed to express the transgene are analyzed at a higher level to determine the presence and expression of the transgene using Southern or PCR analysis, although some tissues or cell types can be used for this analysis. The progeny of transgenic animals can be obtained by pairing the transgenic animal with a suitable partner or by fertilizing

5 in vitro expression of ovules and / or sperm obtained from the transgenic animal.

The methods for producing transgenic mice through homologous recombination between the endogenous gene and an artificial transgene structure are described by Hanks, M et al. (Science 269: 679-682, 1995). Artificial structures of human and humanized C5aR The artificial polynucleotide structure introduced can encode a wild-type human C5aR sequence

10 as shown in SEQ ID NO: 3 or an allelic variant. Non-limiting examples of allelic variants of SEQ ID NO: 3 comprise one or more of the following substitutions

tes: Amino Acid No. 2: Asp> Asn Amino Acid No. 279: Asn> Lys.

The artificial polynucleotide structure may comprise a sequence such as that shown in SEQ ID NO: 2, or a

allelic variant of it. Non-limiting examples of allelic variants of SEQ ID NO: 2 comprise one or more of the following base changes:

Base No. 28: g> a

20 Base No. 474: c> t Base No. 861: t> g Base No. 1313-1314: insertion of a Base No. 1447-1448: insertion of a.

In a preferred aspect, the sequence encoding C5aR is functionally linked to a promoter, which may be an endogenous or heterologous, constitutive or inducible regulatory sequence and other necessary regulatory sequences.

for expression in the host mouse. Alternatively, the artificial structure introduced can encode humanized C5aR comprising intracellular domains of endogenous C5aR and extracellular domains of human C5aR,

The various domains of human C5aR that can be introduced into humanized C5aR are listed in the Table

30 1. Table 1:

amino acids 1 -37 extracellular domain -extremo N-terminal amino acids 38 -60 transmembrane domain amino acids 61 -71 intracellular domain amino acids 72 -94 transmembrane domain amino acids 95 -110 extracellular domain -bucle extracellular 1 amino acids 111 -132 transmembrane domain amino acids 133 -153 domain intracellular amino acids 154-174 transmembrane domain amino acids 175 -200 extracellular domain-extracellular loop 2 amino acids 201-222 transmembrane domain amino acids 227-242 intracellular domain amino acids 243-265 transmembrane domain amino acids 266-282 extracellular domain extracellular loop 3 amino acids 283-303 domain transmembrane

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amino acids 304-350 intracellular domain -extremo C-terminal.

Artificial structures of interest may contain cDNA, genomic sequences or both. Methods for isolating and cloning a desired sequence, as well as artificial structures suitable for the expression of a selected sequence in a host animal, are well known in the art. The artificial structure may include sequences other than the sequences encoding C5aR. For example, a marker gene, such as lac Z, may be included in the artificial structure, where increased regulation of the expression of the encoded sequence will result in a change in the phenotype, easily detected.

The term "C5aR gene" is generally used with the meaning of a human C5aR gene and isoforms, alternative forms, splicing variants, mutated variants, etc. of this human gene. This expression is also understood to mean the open reading frame that encodes specific polypeptides, introns and non-coding nucleotide sequences adjacent to 5 'and 3', involved in the regulation of expression, up to about 1 kb beyond the coding region , but possibly further in any direction. The DNA sequence encoding C5aR can be cDNA or genomic DNA or one of its fragments. The gene can be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host.

Genomic sequences of particular interest comprise the nucleic acid present between the initiation codon and the stop codon, including all introns that are normally present in the natural chromosome. These may also include regions not translated into 3 'and 5', found in mature mRNA. Such sequences may further include specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc., including approximately 1 kb, but possibly more of the genomic DNA flanking at the 5 'or 3' end of the transcribed region. Genomic DNA can be isolated as a fragment of 100 kb or smaller; and is substantially free of flanking chromosomal sequences.

The sequences of the 5 'regions of the human C5aR gene, and the additional sequences of regions upstream of the 5' end and sequences downstream of the 3 'end, can be used for promoter elements, including enhancer binding sites, which provide a expression in tissues where C5aR is normally expressed. Specific expression in tissues is useful for providing promoters that mimic the natural model of expression. Polymorphisms that occur naturally in the promoter region are useful for determining natural variations in expression, particularly those associated with a disease. Alternatively, mutations can be introduced in the promoter region to determine the effect of altering expression in experimentally defined systems. Methods for the identification of specific DNA motifs, involved in the binding of transcriptional factors are known in the art, for example, similarity of sequences with known binding motifs, gel delay studies, etc. For example, see Blackwell et al., (1995) Mol. Med. 1: 194205; Mortlock et al. (1996) Genome Res. 6: 327-33; and Joulin and Richard-Foy (1995) Eur. J. Biochem. 232: 620-626.

In one aspect, vectors suitable for use in the present invention may comprise at least one expression control element functionally linked to the nucleic acid sequence encoding human C5aR. The expression control elements can be inserted into the vector to control and regulate the expression of the nucleic acid sequence. Examples of expression control elements include, but are not limited to, the lac system, lambda phage operator and promoter regions, yeast promoters and promoters obtained from polyoma, adenovirus, retrovirus or SV40. The vector may further comprise functional elements that include, but are not limited to, leader sequences, termination codons, polyadenylation signals and any other sequence necessary or preferred for the appropriate transcription and / or translation of the nucleic acid sequence encoding human C5aR .

In a further preferred aspect, the artificial structure comprises regions homologous to the sequences of the 3 'and 5' ends flanking the endogenous C5aR coding sequence of the non-human mammal. It will be understood that these homology regions are useful for the target-directed integration of the artificial structure in the endogenous C5aR locus of the transgenic mammal.

Preferably, the artificial polynucleotide structure comprises regions homologous at least 2 kb, more preferably at about 3 kb, located upstream and downstream of exon 3 of the endogenous C5aR gene. For example, the downstream and upstream sequences may come from the sequence between nucleotides 7377-15045 of SEQ ID NO: 1.

Those skilled in the art will understand that vectors of this type are constructed using a conventional methodology (See for example Sambrook et al., (Compilers) (1989) "Molecular Cloning, A Laboratory Manual" Cold Spring Harbor Press, Plainview, NY; Ausubel et al., (Compilers) (1987) "Current Protocols in Molecular Biology" John Wiley and Sons, New York, NY) or are commercially available.

In some aspects, it may be preferable to express human C5aR in tissues that mimic the natural model of expression in humans. A specific expression model can be achieved by placing the nucleic acid encoding human C5aR under the control of an inducible or developmentally regulated promoter or under the control of a tissue-specific or cell-specific promoter. As an example, specific models of

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Candidate compounds are also among the biomolecules that include, but are not limited to, saccharides, fatty acids, steroids, purines, pyrimidines and their derivatives, structural analogues or combinations thereof.

Candidate compounds can be obtained from a wide variety of sources including collections of natural or synthetic compounds. For example, numerous means are available for random and direct synthesis of a wide variety of organic compounds and biomolecules, including the expression of randomized oligonucleotides and oligopeptides. Alternatively, collections of natural compounds are available in the form of bacterial, fungal, vegetable and animal extracts, or are easily produced. Alternatively, collections and compounds produced naturally or synthetically are easily modified by conventional chemical, physical and biochemical means and can be used to produce combinatorial collections. Known pharmacological agents can be subjected to direct or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc., to produce structural analogues.

The screening can also be directed to pharmacologically active compounds and their analogous chemicals.

The candidate agent may be administered to the transgenic mouse of the invention (or to cells from the transgenic mouse) in any desired manner and / or appropriate for the administration of the agent, to achieve the desired result. For example, the candidate agent can be administered by injection (for example, intravenous, intramuscular, subcutaneous injection or directly into the tissue in which the desired effect is desired), orally or by any other desirable means. Preferably, in vivo screening will involve a number of animals that receive varying amounts and concentrations of the candidate compound (from absence of compound to an amount of compound that approaches the upper limit of the amount that can be satisfactorily administered to the mouse), and may include the administration of the agent in a different formulation. The compounds can be administered alone or in combinations of two or more, especially when administration of a combination of agents can result in a synergistic effect. The effect of agent administration on the transgenic mouse can be monitored with a conventional methodology.

The range of candidate compounds contemplated herein includes C5aR inhibitor compounds.

or antagonists of a biological function of C5aR. In one embodiment, the compound is selected from the group consisting of: a peptide, including a peptide obtained from C5aR or C5a or other peptides that are not C5aR and are capable of inhibiting, reducing or suppressing a C5aR function, a dominant dominant mutant of C5aR; a non-peptide inhibitor of C5aR; an antibody or an antibody fragment that binds to C5aR and inhibits a C5aR function; a small organic molecule and nucleic acid, including nucleic acid encoding said peptide obtained from C5aR or C5a or another peptide inhibitor that is not C5aR, an antisense nucleic acid directed against mRNA encoding C5aR or an anti-C5aR ribozyme or a small interference RNA (RNAi) that targets the expression of the C5aR gene. A certain number of these types of compounds are discussed below.

Small molecules

The candidate compounds include numerous chemical classes, although preferably they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 Daltons. Candidate compounds comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding and usually include at least one amino, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. Candidate compounds frequently comprise cyclic or heterocyclic carbon structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate compounds are also among biomolecules that include peptides, saccharides, fatty acids, steroids, purines, pyrimidines and derivatives, structural analogues or combinations thereof. In fact, virtually any small organic molecule that is potentially capable of binding to a biological target molecule can find use in the present invention as long as it is sufficiently soluble and stable in aqueous solutions to be analyzed for its ability to bind to the target molecule. biological

Candidate compounds are obtained from a wide variety of sources, including collections of natural or synthetic compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including the expression of randomized oligonucleotides. Alternatively, collections of natural compounds in the form of bacterial, fungal, vegetable and animal extracts are readily available or produced. Additionally, collections and compounds produced naturally or synthetically, are easily modified by conventional chemical, physical and biochemical means. Known pharmacological compounds may be subjected to random or directed chemical modifications, such as acylation, alkylation, esterification, amidification, etc., to produce structural analogues.

Peptide or protein inhibitors

In another aspect, the candidate compounds are proteins. By "protein" in this context is meant at least two covalently fixed amino acids, which includes proteins, polypeptides, oligopeptides and peptides. The protein

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It may consist of naturally occurring amino acids and peptide bonds or synthetic peptidomimetic structures. Thus "amino acid" or "peptide residue", as used herein, means both natural and synthetic amino acids. For example, homophenylalanine, citrulline and norleucine are considered amino acids for the purposes of the invention. "Amino acid" also includes imino acid residues, such as proline and hydroxyproline. The side chains can have the configuration (R) or (S). In the preferred embodiment, the amino acids have the configuration (S) or (L). If non-natural side chains are used, substituents other than amino acids can be used, for example, to prevent or delay degradations in vivo.

In a further preferred aspect, the candidate compounds are natural proteins or fragments of natural proteins. Thus, for example, cell extracts containing randomly or directed digested proteins or products of protein cell extracts can be used. In this way, collections of prokaryotic and eukaryotic proteins can be prepared. In this embodiment, bacterial, fungal, viral and mammalian protein collections are particularly preferred, the latter being preferred and human proteins being especially preferred.

In another preferred aspect, the candidate compounds are peptides with a length of about 5 to about 30 amino acids, peptides of about 5 to about 20 amino acids being preferred, and those of about 7 to about 15 amino acids being particularly preferred. The peptides can be digested materials of natural proteins, random peptides or "biased" random peptides. By "randomized" or grammatical equivalents, it is understood herein that each peptide consists essentially of random amino acids. Since these random peptides are generally chemically synthesized, they can incorporate any amino acid in any position. The synthetic process can be designed to generate randomized proteins to allow the formation of all or most of the possible combinations along the length of the sequence, thereby forming a collection of randomized candidate bioactive protein compounds.

The preparation and scrutiny of combinatorial chemical collections are well known to those skilled in the art. Such combinatorial collections include, but are not limited to, peptide collections (see, for example, US Patent No. 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37: 487 -493, Houghton et al. (1991) Nature, 354: 84-88). Peptide synthesis is by no means the only approach intended and intended for use in the present invention. Other chemical methods can also be used to generate a variety of chemical collections. Such chemical methods include but are not limited to: peptoids (PCT Publication No. WO 91/19735, Dec. 26, 1991), encoded peptides (PCT Publication Document WO 93/20242, Oct. 14, 1993), random bio-oligomers ( PCT Publication Document WO 92/00091, January 9, 1992), benzodiazepines (US Patent No. 5,288,514), diversomers, such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., (1993) Proc. Nat. Acad. Sci. USA 90: 6909-6913), vinistic polypeptides (Hagihara et al. (1992) J. Amer. Chem. Soc. 114: 6568), non-peptide peptidomimetics with a major beta-D-glucose structure (Hirschmann et al., (1992) J. Amer. Chem. Soc. 114: 9217-9218), analogous organic syntheses of small compound collections (Chen et al. (1994) J. Amer. Chem. Soc. 116: 2661), oligocarbamates (Cho, et al., (1993) Science 261: 1303) and / or peptidyl phosphonates (Campbell et al., (1994) J. Org. Chem. 59: 658). See, in general, Gordon et al., (1994) J. Med. Chem. 37: 1385, nucleic acid collections, peptide and nucleic acid collections (see, for example, U.S. Patent No. 5,539,083), antibody collections (see, for example, Vaughn et al. (1996) Nature Biotechnology, 14 (3): 309-314), and PCT / US96 / 10287), carbohydrate collections (see, for example, Liang et al. (1996) Science, 274: 1520-1522, and US Patent No. 5,593,853), and collections of small organic molecules (see, for example, benzodiazepines, Baum ( 1993) C&EN, Jan 18, page 33, isoprenoids U.S. Patent No. 5,569,588, thiazolidinones and methathiazanones U.S. Patent No. 5,549,974, pyrrolidines U.S. Patent No.

5,525,735 and 5,519,134, morpholino compounds US Pat. No. 5,506,337, benzodiazepines US Pat. No. 5,288,514 and the like).

Devices for the preparation of combinatorial collections are commercially available (see, for example, 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.).

In one aspect, C5aR peptide inhibitors are chemically or recombinantly synthesized as oligopeptides (usually 10-25 amino acids in length) obtained from a C5aR or C5a sequence. Alternatively, the C5aR or C5a fragments are produced by digestion of natural or recombinantly produced C5aR or C5a, for example, using a protease, for example, trypsin, thermolysin, chymotrypsin or pepsin. Computer analysis (using commercially available software, for example, MacVector, Omega, PCGene, Molecular Simulation, Inc.) is used to identify proteolytic cleavage sites. Proteolytic or synthetic fragments may comprise all amino acid residues that are necessary to partially or completely inhibit the function of C5aR. Preferred fragments will be at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids.

Protein or peptide inhibitors can also be dominant dominant mutants of C5aR. The term "negative dominant mutant" refers to a C5aR polypeptide that has mutated from its natural state and that interacts with a protein with which C5aR normally interacts, thereby preventing endogenous C5aR

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Anti-C5aR antibodies

The term "antibody" as used herein includes intact molecules, as well as fragments thereof, such as Fab, F (ab ') 2 and Fv that are capable of binding to an epitope determinant of C5aR. These antibody fragments retain some ability to selectively bind with their antigen and are defined as follows:

(one)

 Fab, the fragment containing a monovalent fragment that binds antigen of an antibody molecule, can be produced by digestion of a complete antibody with the papain enzyme to provide an intact light chain and a portion of a heavy chain;

(2)

 Fab ', the fragment of an antibody molecule can be obtained by treating a complete antibody with pepsin, followed by reduction, to provide an intact light chain and a portion of the heavy chain; two Fab 'fragments are obtained for each antibody molecule;

(3)

 F (ab ') 2, the antibody fragment that can be obtained by treating a complete antibody with the enzyme pepsin without further reduction; F (ab ') 2 is a dimer of two Fab' fragments that are held together through two disulfide bridges;

(4)

Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain, expressed as two chains; Y

(5)

Single chain antibody ("SCA"), defined as a genetically engineered molecule that contains the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule .

The methods for preparing these fragments are known in the art. (See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988)).

Antibodies can be prepared using intact C5aR or fragments thereof, as an immunizing antigen. A peptide used to immunize an animal can be derived from translated cDNA or chemical synthesis and is purified and conjugated with a carrier protein, if desired. Such commonly used vehicles that chemically couple to the peptide, include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA) and tetanus toxoid. The coupled peptide can then be used to immunize the animal (for example, a mouse or a rabbit).

If desired, polyclonal antibodies can be further purified, for example, by binding them to a matrix and eluting them from the matrix to which the peptide binds, against which the antibodies were produced. Those skilled in the art will know several common methods in immunological techniques for the purification and / or concentration of polyclonal antibodies, as well as monoclonal antibodies (See for example, Coligan, et al. Unit 9, Current Protocols in Immunology, Wiley Interscience, 1991).

Monoclonal antibodies can be prepared using any technique that provides for the production of antibody molecules through continuous cell lines in culture, such as, for example, the hybridoma technique, the human B lymphocyte hybridoma technique and the EBV technique. -hybridoma (Kohler et al., Nature 256, 495-497, 1975; Kozbor et al., J. Immunol. Methods 81, 31-42, 1985; Cote et al., Proc. Natl. Acad. Sci. USA 80 , 2026-2030, 1983; Cole et al., Mol. Cell Biol. 62, 109-120, 1984).

Methods known in the art allow antibodies that show C5aR binding to be identified and isolated from antibody expression collections. For example, a method for the identification and isolation of an antibody-binding domain that shows binding to C5aR is a bacteriophage vector system. This vector system has been used to express a combinatorial collection of Fab fragments from the mouse antibody repertoire in Escherichia coli (Huse, et al., Science, 246: 1275-1281, 1989) and the human antibody repertoire (Mullinax , et al., Proc. Nat. Acad. Sci., 87: 8095-8099, 1990). This methodology can also be applied to hybridoma cell lines that express monoclonal antibodies that bind to a preselected ligand. Hybridomas that secrete a desired monoclonal antibody can be produced in various ways using techniques well known to those of ordinary skill in the art and will not be repeated herein. Details of these techniques are described in references of the Monoclonal Antibodies-Hybridomas type: A New Dimension in Biological Analysis, Edited by Roger H. Kennett, et al., Plenum Press, 1980; and US Pat. No. 4,172,124.

In addition, methods for producing "humanized" or chimeric antibodies are known in the art and include combining murine variable regions with human constant regions (Cabili, et al. Proc. Natl. Acad. Sci. USA, 81: 3273, 1984), or graft the complementarity determining regions (CDRs) of murine antibodies in the human framework (Riechmann, et al., Nature 332: 323, 1988).

Antisense compounds

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163 (2): 985-94; Arumugam et al., (2003) Kidney Int 63 (1): 134-42; Arumugam et al., (2002) J. Surg. Res. 103 (2): 2607). In a model of myocardial IR injury in pigs, a C5aR antagonist markedly reduced the size of the infarction (Riley et al., (2000) J. Thorac. Cardiovasc. Surg. 120 (2): 350-8).

Studies with antibodies against C5 also show similar reductions in IR lesions (Fitch et al., (1999) Circulation 100 (25): 2499-506; de Vries et al., (2003) Transplantation 75 (3): 375-82 ).

Clinical and experimental studies have shown that the IR of organs, such as kidneys, liver, lungs and heart induces a rapid release of various cytokines. Although after reperfusion the level in many of these molecules is reduced, IL-8 increases significantly. IL-8 is a potent chemoattractant of neutrophils. IL-8 levels correlate negatively with lung function and elevated levels are associated with an increased fatal risk in lung transplantation. Intravenous administration of anti-IL8 antibodies at the beginning of reperfusion markedly reduces lung injury and neutrophil infiltration (from Perrot et al., (2003) Am. J. Respir. Crit. Care Med. 167 (4) : 490-511).

There is evidence to show that IR injury occurs with a biphasic pattern. The macrophages activated during ischemia mediate in the early phase of the lesion, while neutrophils and lymphocytes are mainly involved in the second, delayed phase. Neutrophil and lymphocyte recruitment results in the release of cytokines before and after reperfusion.

Since C5a is a potent chemoattractant of various myeloid cells, including macrophages and neutrophils and also induces the production of cytokines, it is considered that it may be beneficial to block C5aR with specific antibodies preventing IL-8 release and neutrophil migration and reducing in this way the IR injury.

Acute respiratory distress syndrome (ARDS) and acute lung injury (APL)

ARDS and APL are syndromes that are the result of pulmonary edema and inflammation. The development of ARDS / APL is associated with several serious clinical disorders including lung injury caused by pneumonia infection, aspiration of gastric contents, trauma, septicemia, acute pancreatitis, drug overdose and cardiopulmonary bypass. Septicemia is the main cause of ARDS / APL. As with IR, the inflammatory response in LPA is associated with the recruitment of large amounts of neutrophils and monocytes into the distal air spaces of the lungs. Proinflammatory molecules, such as cytokines, oxygenated radicals and proteases have a function and excessive inflammation can worsen the ARDS / LPA (Ware and Matthay (2000) N. Engl. J. Med. 342 (18): 1334-49; Brower et al., (2001) Chest 120 (4): 1347-67).

It has been suggested that anti-inflammatory strategies may be beneficial to reduce the number of neutrophils that migrate to the extravascular spaces of the lungs, to reduce neutrophil adhesion to the pulmonary endothelium and to reduce the release of chemotactic factors (Brower et al. (2001 ) Chest 120 (4): 1347-67).

Studies of patients with ARDS reveal that in the early stages of the disease, elevated levels of IL-8 are present in the BAL fluid or pulmonary edema fluid. In addition, antibodies that neutralize IL-8 reduced lung injury in a rabbit model (Brower et al., (2001) Chest 120 (4): 1347-67).

For ARDS and APL, blocking C5aR can also be a therapeutically viable approach. C5a is a potent chemotactic molecule and cytokine release stimulator. The studies described above that employed molecules that antagonized C5a or C5aR, showed a reduction of the lesion in various inflammation models.

Production of compounds identified in the screening methods of the invention

The object method described herein further comprises producing or synthesizing the compound that is analyzed in the genetically modified mouse.

Peptidyl compounds are conveniently prepared by conventional peptide synthesis, such as the Merrifield synthesis method (Merrifield, J. Am. Chem. Soc. 85: 2149-2154, 1963) and the thousands of improvements available for that technology ( see, for example, Synthetic Peptides: A User's Guide, Grant, compiler (1992), WH Freeman & Co., New York, pp. 382; Jones (1994) The Chemical Synthesis of Peptides, Clarendon Press, Oxford, pp. 230 ); Barany, G. and Merrifield, R. B. (1979) in The Peptides (Gross, E. and Meienhofer, J. compilers), vol. 2, p. 1-284, Academic Press, New York; Wünsch, E., compiler (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Müller, E., compiler), vol. 15, 4th ed., Parts 1 and 2, Thieme, Stuttgart; Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474.

Preferably, the peptide is synthesized on a solid phase support, such as, for example, a polystyrene gel bead comprising polystyrene crosslinked with divinylbenzene, preferably 1% divinylbenzene (w / w), which is further swollen using lipophilic solvent , such as, for example, dichloromethane or dimethylformamide (DMF). The polystyrene can be functionalized by the addition of chloromethane or aminomethyl groups.

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Alternatively, the crosslinked and functionalized polydimethyl acrylamide gel can be used once swollen and solvated, using DMF or a dipolar aprotic solvent. Other solid phase supports known to those skilled in the art can also be used for peptide synthesis, such as, for example, beads obtained from polyethylene glycol, produced by grafting polyethylene glycol on the surface of inert polystyrene beads. . Commercially available preferred solid supports include PAL-PEG-PS, PAC-PEG-PS, KA, KR or TGR (Applied Biosystems, CA 94404, USA).

For the synthesis of solid phase peptides, blocking groups that are stable against repeated treatments, necessary for the removal of the blocking amino group from the growing peptide chain and for repeated amino acid couplings, are used to protect the side chains of amino acids during synthesis and to mask the unwanted reactivity of the α-amino, carboxyl or side chain functional groups. Blocking groups (also called protecting groups or masking groups) therefore protect the amino group of the amino acid that has an activated carboxyl group that is involved in the coupling reaction or protect the carboxyl group of the amino acid that has an acylated amino group that is involved in the coupling reaction.

During the synthesis, the coupling takes place after the removal of a blocking group without a breakage of a peptide bond or of any protective group attached to another part of the peptide. Additionally, it is required that the peptide-resin anchor that protects the C-terminal end of the peptide be protected during the synthetic process until the resin is cleaved. Accordingly, by a successful selection of orthogonally protected α-amino acids, amino acids are added at the desired positions to a growing peptide while it is still anchored to the resin.

Preferred groups that block amino can be easily removed, but are stable enough to survive under the conditions of the coupling reaction and other manipulations, such as, for example, modifications in the groups of the side chains. In one embodiment, the amino blocking groups are selected from the group comprising: (i) a benzyloxycarbonyl (Z or carbobenzoxy) group that is readily removed by catalytic hydrogenation at room temperature and ordinary pressure or using sodium in liquid and acid ammonia hydrobromic in acetic acid; (ii) a urethane derivative; (iii) a t-butoxycarbonyl (Boc) group that is introduced using t-butoxycarbonyl azide or di-tert-butyl dicarbonate and removed using a mild acid, such as, for example, 50% trifluoroacetic acid (TFA). dichloromethane) or HCl in acetic acid / dioxane / ethyl acetate; (iv) a 9-fluorenylmethyloxycarbonyl (Fmoc) group that is cleaved under mildly basic, non-hydrolytic conditions, such as, for example, using a primary or secondary amine (for example, 20% piperidine in dimethyl formamide); (v) a 2- (4-biphenylyl) propyl (2) oxycarbonyl (Bpoc) group; (vi) a 2-nitro-phenylsulfenyl group (Nps); and (vii) a dithia-succinyl group (Dts). Boc is widely used to protect the N-terminal end in Fmoc chemistry, or Fmoc is widely used to protect the N-terminal end in Boc chemistry.

The side chain protecting groups will vary for the functional side chains of the amino acids that form the peptide that is synthesized. The side chain protecting groups are generally based on the Bzl group or the tBu group. Amino acids that have alcohols or carboxylic acids in the side chain are protected as Bzl ethers, Bzl esters, cHex esters, tBu ethers or tBu esters. The side chain protection of amino acids with Fmoc requires blocking groups that are ideally stable against bases and labile against weak acids (TFA). Many different protecting groups for peptide synthesis have been described (see The Peptides, Gross et al. Compilers, vol. 3, Academic Press, New York, 1981). For example, the 4-methoxy-2,3,6-trimethylphenylsulfonyl (Nd-Mtr) group is useful for the protection of the arginine side chain, however, the deprotection of Arg (Mtr) requires prolonged treatment with TFA. To protect cystine, a certain number of labile groups are used against weak acids (TFA, thallium trifluoroacetate / TFA) or stable labile groups against TFA, but not against thallium trifluoroacetate (III) / TFA or stable groups against weak acids.

The two most widely used protection strategies are the Boc / Bzl and Fmoc / tBu strategies. In Boc / Bzl, Boc is used for amino protection and the side chains of the various amino acids are protected using Bzl or cHex based protecting groups. A Boc group is stable under catalytic hydrogenation conditions and is used orthogonally together with a Z group for the protection of many groups of side chains. In Fmoc / tBu, Fmoc is used for amino protection and the side chains are protected with tBu based protecting groups.

Alternatively, the peptidyl compound is produced by recombinant expression of a nucleic acid encoding the amino acid sequence of said peptide. Collections encoding random peptides are particularly preferred for these purposes, because they provide a wide range of different compounds to analyze. Alternatively, nucleic acids of natural origin can be screened. In accordance with this embodiment, the nucleic acid encoding the peptidyl compound is produced by conventional oligonucleotide synthesis or is derived from a natural source and is cloned into a suitable expression vector, functionally connected with a promoter or other regulatory sequence capable of regulating expression in a cell-free system or in a cellular system.

The oligonucleotides are preferably synthesized with linker or adapter sequences at the 5 'and 3' ends to facilitate subsequent cloning in a suitable vector system, using conventional techniques.

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14.

Fraction the digested DNA on a 1% agarose gel. Transfer to a nylon membrane and hybridize by Southern blotting with a hybridization probe of the appropriate target gene, to distinguish the unchanged target gene from a target gene that has undergone homologous recombination.

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 Select colonies of ES cells that show two hybridization fragments of approximately equal intensity - a fragment of the size predicted for the unchanged target gene and a fragment of the size predicted for a target gene that has undergone homologous recombination. If the two fragments have an uneven hybridization intensity, the cell population may not be clonal. Freeze the cells and keep them in liquid nitrogen.

A total of 672 colonies of ES cells transfected with the artificial structure that targets an hC5aR target were screened for homologous recombination between the artificial structure that targets a target and the mouse genome.

The DNA extracted from the colonies of ES cells was digested with EcoRV or XbaI and subjected to electrophoresis through an agarose gel. The DNA was transferred to a filter (by the Southern method) and hybridized with a DNA from a 32P labeled probe in 5 'or 3' using conventional conditions. After washing, the filter was exposed to an X-ray film. This screening provided three colonies containing the human C5aR gene. In two of these colonies (5H1 and 6C8) it was identified that they had a human C5aR gene correctly integrated into the locus of the mouse C5aR gene (data not shown).

Example 3: Implantation of ES cells, chimera generation and germline transmission of the human C5aR gene

The conventional method for generating chimeras with ES cells employs 3.5-day-old mouse embryos (blastocysts). Embryos at this stage have a large fluid-filled cavity in which ES cells can be placed by injection. The blastocysts have already left the oviducts and are found in the uterine tubes. For injections, they must be collected before they hatch (loss of the zona pellucida) and are fixed to the uterine wall. Various strains of mice are used to generate blastocysts for injections of ES cells. In this example, the strain of Balb / c mice was used to generate blastocysts. This strain provides a reasonable number of embryos. It tends to produce high quality chimeras that can be easily distinguished by coat color when used with ES cells from a variety of C57BL / 6 sub-strains.

ES cells from colonies, 5H1 and 6C8 were injected into ~ 200 blastocysts. The blastocysts were implanted in pseudopregnant female mice. About 17 chimeras were born that had between 5 and 50% the color of black fur. The 13 male chimeras mated with C57BL / 6 females and produced about 300 offspring. Germline transmission of the human C5aR gene was observed to 19 offspring from 5 pairs of chimera mating x C57BL / 6.

The presence of the human C5aR locus was confirmed by Southern blotting. Genomic DNA extracted from the mouse tail was digested with EcoRV, subjected to electrophoresis and hybridized with the 3 'probe. The filter was disassembled and re-analyzed with the Neo probe. A certain number of mice showed the C5aR gene. The genotype of these mice was named hC5aRfloxed (neoR) / wt (abbreviated H5Rf / +).

Example 4: Reproduction and identification of homozygous mice for the human C5aR gene

A generation of H5Rf / + mice was paired with C57BL / 6 Cre suppressor mice to eliminate the neoR marker gene, thereby generating H5R / wt / Cre mice. H5R / wt / Cre mice were paired with C57BL / 6 mice to eliminate the Cre gene, thereby generating heterozygous H5R / wt mice (H5R / +). Mating between H5R / + mice produced homozygous mice with activated H5R / H5R genes. Some H5Rf / + x H5Rf / + pairings were also established and generated H5Rf / H5Rf homozygous.

Confirmation that mice are carriers of the human C5aR gene was performed using a PCR-based genotyping assay. The Southern blot genotype assay previously used to identify mice bearing the human C5aR gene was based on hybridization with a mouse specific DNA probe (not a specific probe of the human C5aR gene). The PCR assay described below used primers that specifically amplify the sequences of the human or mouse C5aR gene.

Preparation of the tail DNA

3 mm tail tips were placed in sterile 1.5 ml Eppendorf tubes and incubated overnight at 65 ° C in 200 ul of DNA isolation buffer (67 mM Tris-Cl, pH 8.0 (Calbiochem), 16.6 mM ammonium sulfate (Sigma), 6.5 mM magnesium chloride (Promega), 1% β-mercaptoethanol (Biomedical ICN) and 0.05% Triton X100 (Sigma). The supernatant liquid was transferred to a new 1.5 ml tube containing 500 ul of 100% ethanol (UNIVAR), mixed and incubated for 2 hours at -20 ° C, then centrifuged for 15 minutes at 4 ° C and 13,000 rpm (Labofuge 400R, Heraeus Instruments) to precipitate purified genomic DNA. The DNA pellet was washed in 70% ethanol and allowed to dry before re-suspending it in 50 ul of distilled water. A 1 ul aliquot was used as the template DNA in the following PCR assay.

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PCR to amplify human and mouse C5aR genes

Each PCR reaction contained 5 ul of 10x magnesium-free PCR buffer, 5 ul of 25 mM magnesium chloride, 0.5 ul of Taq DNA polymerase (2.5 units / ul), 1 ul of 10 mM dNTPs (Promega ), 1 ul of each 10 mM oligonucleotide primer (F1C, R2a, F3C and R4C) and 1 ul of template DNA. The total volume was completed up to 50 ul with distilled water. The oligonucleotide primers used were:

5'-TGGACTACAGCCACGACAAACG-3 '(F1C) (SEQ ID NO: 4) and 5'-AGGAAGGACATCATTATCCCCG-3' (R2a) (SEQ ID NO: 5), both specific to exon 3 of human C5aR; Y,

5'-CACCAGCCCCGAGATTTTTTC-3 '(F3C) (SEQ ID NO: 6) and 5'-TCAGAAACCAGATGGCGT-3' (R4C) (SEQ ID NO: 7), both specific to the mouse C5aR gene.

The target sequences were then amplified using a System 2700 PCR thermal cycler (Applied Biosystems). The PCR began with a 5-minute denaturation stage at 95 ° C, followed by 25 three-stage cycles: 95 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for 60 seconds. A final extension step: 5 minutes at 72 ° C was followed by storage at 4 ° C.

Gel electrophoresis

The genotype of each sample was visualized using gel electrophoresis with 10 ul of PCR product and 6x loading dye (Promega). The gel consisted of 80 ml of quality agarose for 1.5% DNA (Progen) in 1x TBE and 2 ul of ethidium bromide (MP Biomedicals). The samples were run at 100 volts for 30 minutes along with a 1 kb DNA chain (Promega). It was expected that the DNA of homozygous H5Rf / H5Rf mice would have a single band of 237 bp, the DNA of heterozygous H5Rf / + mice would have bands of 237 bp and 565 bp and the DNA of wild-type mice would have a single band of 565 bp . Gel analysis showed that mice # 10, 24 and 25 were homozygous (H5Rf / H5Rf) (data not shown). In some samples, an irrelevant transcript with a length of 1139 bp (amplified with primers F3C and R2a) was observed.

The relative location in the mouse / human C5aR locus of the PCR primers, F1C, R2a, F3C and R4C, used in the genotyping assay is shown in Figure 4.

Example 5: Human C5aR is expressed in isolated mouse cells with the activated gene

To confirm that the human C5a receptor is expressed in mice that are carriers of the human C5aR gene, neutrophils were isolated from the blood of a wild-type mouse (+ / +), a heterozygous (H5Rf / +) and a homozygous (H5Rf / H5Rf).

Direct immunofluorescence staining of C5aR in whole mouse blood

Approximately 30 ul of peripheral venous blood were collected from homozygous (H5Rf / H5Rf), heterozygous (H5Rf / +) and wild type (+ / +) mice in a tube containing EDTA (the final EDTA concentration was 5 mM). For the detection of the human C5aR receptor, 30 ul of blood was stained with 5 ul of 7F3 labeled with FITC (fluorescein isothiocyanate, Sigma), an antibody specific for human C5aR (7F3 has no cross-reaction with the mouse C5aR). Blood and antibody were incubated in a 12 x 75 mm test tube for 15 minutes at room temperature. To lyse the erythrocytes and fix the cells, 500 ul of lysis solution BD (Becton Dickinson) was added to the blood / antibody solution. After 15 minutes, the cells were centrifuged at 1,200 rpm for 3 minutes at room temperature and the cell pellet was resuspended in 1 ml of wash buffer (1 x PBS, 0.1% BSA, 0% sodium azide , 02%). The cells were analyzed on a FACS Calibur flow cytometer (Becton Dickinson) using the Cell Quest software (Becton Dickinson). Approximately 5,000 cells were counted for each sample.

Figure 5 shows the results of this analysis. Neutrophils of both homozygous and heterozygous KI mice bound 7F3, an antibody specific for human C5aR. In contrast, the neutrophils of wild-type mice were not stained with 7F3. Lymphocytes of all mouse genotypes were not stained with AcMo 7F3. This was expected, since C5aR is not expressed in lymphocytes. Monocytes from homozygous and heterozygous mice with the activated gene were partially stained with 7F3, indicating that a small percentage of the cells expressed human C5aR. None of the monocytes from the wild-type mouse tested positive for staining through 7F3.

In summary, mice that are carriers of the human C5aR gene express human C5aR on the surface of the expected cell types. This indicates that the mouse cell is capable of expressing and processing the human C5a receptor.

Example 6: Mice with the activated human C5aR gene can be used to screen anti-inflammatory compounds

To demonstrate the utility of mice with the activated human C5aR gene, we subjected homozygous mice (H5Rf / H5Rf) to an inflammatory disease model, specifically the K / BxN model of rheumatoid arthritis.

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