Gene targeting technology and advances in the pathophysiology of inflammation

Pathology (2002 ) 34, pp. 109– 114

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Gene targeting technology and advances in the pathophysiology of inflammation D. SEAN RIMINTON Centenary Institute for Cancer Medicine and Cell Biology, Camperdown, and Department of Immunology, Concord Hospital, Concord, NSW, Australia

Summary Gene targeting (‘knock-out’) technology is now widely used in the basic science of all disciplines of pathology and particularly auto-immune and inflammatory disease. Gene targeting is the wilful introduction of precise mutations into the genome of an animal, usually a mouse, affecting the function of a single gene or genes. The phenotyping of knockout mice provides whole animal data on the functions of individual genes in pathophysiological settings, and frequently provides the basis for novel therapeutic strategies. ‘Knock-ins’, the Cre-LoxP system and conditional knockout s are important new advances. This paper serves as an introduction to the methodology and draws on examples of major advances in inflammation research provided by the targeting of cytokines, in particular the tumour necrosis factor family of ligands. Key words: Gene targeting, knock-out, inflammation, auto-immunity, cytokines, pathology. Received 23 November 2001, revised 2 January, accepted 7 January 2002

INFLAMMATION AND INFLAMMATORY PATHOLOGY Inflammation is the dominant feature of the immediate host response to injury. Although inflammatory physiology has evolved by providing adaptive advantage to the host, inflammatory pathologies appear to be an inevitable consequence, and for any given individual in an outbred population, the balance of protective advantage and pathology may be adversely skewed towards disease. Autoimmune and inflammatory pathologies are an important cause of morbidity and mortality in humans, estimated to affect 4–5% of the population,1 and medical management of these conditions is largely unsatisfactory. The inflammatory response is a fundamentally coordinated process. This is despite the recruitment of an apparently chaotic range of interdependent cells, mediators and their inhibitors into inflammatory lesions. Cogently designed medical therapies will depend upon the characterisation of the key mediators. However, the simple identification of any single participating cell or molecule in an inflammatory lesion fails to inform the observer of its importance, or of the balance of factors controlling its expression. Methods with a higher level of resolving power have been required. Over the last century, complex

physiological processes have often been advanced by observations of naturally occurring inborn errors in genes. With the advent of gene targeting technology, it is now possible to introduce predefined mutations to relevant genes, providing experimental systems that are ideally adapted to tackling the complexity of inflammation. This paper describes that technology and summarises some of those advances.

TARGETED DISRUPTION OF GENES Gene targeting is the wilful introduction of mutations to genes, and the stable transmission of that mutation through the germline to subsequent generations. Thus, the normal function of genes can be derived from in vivo studies in the context of whole animal physiology, providing a remarkable source of definitive experimental data in recent years ( reviewed in Ref. 2–4). Key advances enabling this technology include the development of stable pluripotent embryonic stem ( ES) cell lines,5,6 germline transmission after passage in culture,7–9 and homologous DNA recombination in mammalian cells.10,11 Some of the relevant terms are defined in Table 1. The targeted disruption of b2-microglobulin was the first knock-out of immunological significance. 12 A database of gene targeted mice and mutations is available at http://research.bmn.com /mkmd.

OVERVIEW OF GENE TARGETING TECHNIQUE To successfully disrupt a gene in vivo a number of tasks need to be completed ( Fig. 1). A targeting vector needs to be designed using a knowledge of the genomic sequence containing the genes of interest ( Fig. 2). Design is governed by three principles.3,11 First, to disrupt one or more exons of the target gene by the interruption/excision of sequence, or by the introduction of a stop codon. Second, to incorporate a selectable marker, such as neomycin phosphotransferase resistance cassette ( neor), that enables the identification of transfected ES cells in culture. Third, to provide adequate sequence homology in flanking regions enabling homologous recombination. The ES cells are transfected with the targeting vector by one of several methods, the most common being electroporation. The vector sequence may be incorporated into the host genome randomly, or by the homologous replacement of endogenous sequence. Successfully targeted ES cells are selected by their ability to grow

ISSN 0031–3025 printed/ISSN 1465– 3931 online/02/020109 – 06 © 2002 Royal College of Pathologists of Australasia DOI:10.1080/003130201201111230

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TABLE 1

Common terms

Gene targeting

Introduction of precise genetic changes into the genome of an animal, usually a mouse, affecting the function of a single gene or genes.

Targeting vector

A DNA plasmid construct carrying the mutated target gene, normal flanking sequence homology and a selectable marker ( e.g., the neomycin resistance cassette neor ).

‘Knock-out’

Gene targeting strategy designed to inactivate the function of a gene in all tissues of a whole animal at all stages of ontogeny.

‘Knock-in’

Gene targeting strategy designed to alter the function of a gene in all of tissues of a whole animal, by the introduction of a stable mutation.

Transgenic

Enforced expression of an exogenous gene. In contrast to knock-outs, the construct is inserted randomly into the genome with variable copy number, but may have targeted tissue expression controlled by the inclusion of an exogenous cis-acting promoter.

Homologous recombination

Intrinsic function of eukaryotic cells to exchange sequences between two DNA molecules according to sequence similarity, exploited in gene targeting strategies.

Embryonic stem ( ES) cells

Cells derived from the inner cell mass of early embryos, capable of propagation and manipulation in culture without loss of pluripotency after reimplantation.

Cre-LoxP system

Cre recombinase recognises two 34-bp LoxP sites in the same orientation, and excises intervening sequence to leave a single LoxP sequence. By flanking genes with LoxP sites ( ‘floxing’ ) and transiently expressing Cre, unwanted sequences can be eliminated.

Conditional ( inducible ) knock-out

Limiting gene disruption to specific cells or organs ( site-specific), or at predetermined times during ontogeny ( temporal restriction) . These methods often employ the Cre-LoxP system and are particularly useful when mutations are lethal to embryos.

Backcrossing

Interbreeding ( ‘introgressing’) the targeted allele onto a strain other than that of the original ES cell. An important potential confounding factor when comparing gene targeting phenotypes with controls.

in aminoglycoside-containing media, and homologous recombination is identified by nested set polymerase chain reaction ( PCR) screening strategy and Southern blotting. ES cell clones are micro-injected into the 3.5-day blastocyst, and reimplanted into pseudopregnant foster mothers. Blastocysts and ES cells derived from mice of different coat colour allow identification of chimaeras in the progeny. These chimaeric mice are bred back onto the strain from which the ES cell line was derived, maintaining strain purity. Heterozygous germline transmission is then identified by PCR or Southern blotting. A homozygous colony is established by interbreeding, and phenotyping experiments are used to derive insights into the normal physiological function of the targeted gene.

PHENOTYPIC INTERPRETATION OF MUTANT MICE The interpretation of phenotype in gene targeted animals relies on the principle that the alteration of structure and function observed in the absence of a molecule represents its normal function in the intact animal. The investigator needs to be unambiguously informed about what is normal, and this depends on the availability of normal ‘wild-type’ controls. This concept has proved to be very robust but with two important qualifications. 1. Backcrossing For technical reasons, backcrossing between strains is often required. Backcrossing is the interbreeding of gene targeted mice with another strain, with positive selection of the progeny carrying the mutation in an attempt to move

( ‘introgress’) the mutation onto an alternative strain back-

ground. Backcrossing introduces random and non-random genomic heterogeneity between the targeted mice and their controls, including littermate controls. With backcrossing, it is simply impossible to study the phenotypic effect of a single gene disruption in comparison with a valid control. This is so even after 12 generations of interbreeding, which is rarely achieved in practice.13,14 2. True artefacts of gene targeting The genomic manipulations required for gene targeting may introduce ‘neighbourhood effects’ ( epistatic interactions) in vivo, altering the function of genes that are located in proximity to the mutated allele. Examples include the effects of constitutive transcription of the neomycin resistance gene, and the presence of a potent heterologous promoter within the construct. True artefacts of conventional gene targeting have been described,15 although they appear to be rare and may not be of practical importance in most cases.

ADVANCES IN GENE TARGETING TECHNIQUES Recent advances in gene targeting technology, enabling the specific inactivation of genes restricted both in time and anatomic location, may ultimately supersede currently established techniques. Conditional, cell type-specific and inducible gene targeting techniques have been developed that include the use of the recombinase protein Cre, and the Crespecific recognition sequence LoxP.16 The targeting construct is designed with the targeted allele flanked by two LoxP sites ( ‘floxed’ ), with site-specific excision of the intervening sequences achieved by Cre expression. Cre

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TUMOUR NECROSIS FACTOR AS AN EXAMPLE OF THE GENE TARGETING APPROACH Tumour necrosis factor ( TNF), the ubiquitously expressed and most studied of cytokines, provides an ideal illustration of the effectiveness of gene targeting experiments in the investigation of inflammatory biology. TNF is expressed in cell surface and secreted forms, binds two separate receptors, and is frequently co-expressed in inflammatory lesions with lymphotoxin ( LT) a3, which also shares TNF-R1 and TNF-R2 binding. These complexities halted definitive progress in TNF biology.18 Gene targeting enabled precise inactivation of TNF and/ or the LTa or b molecules, or their receptors, and the definition of their roles. TNF-deficient mice displayed resistance to endotoxin-induced shock, greater susceptibility to intracellular pathogens such as Mycobacterium tuberculosis,19 marked alteration in chemokine-dependent inflammatory leukocyte traffic within tissues,18 and defects in germinal centre formation in lymphoid tissue.20 Mice lacking TNF were markedly less susceptible to auto-immune pathology in the central nervous system.21 Undoubtedly, the most surprising outcome from studies of this type was the discovery of the role of the membrane-bound form of LT ( LTa1b2) in the histiogenesis of lymph nodes and Peyer’s patches, as well as the structural organisation of the spleen.22,23 Secreted LTa3 is independently involved in defence against M. tuberculosis.24 Findings from TNF-/- mice remind us that, although this molecule has an enormous capacity for self-harm, imprudent inhibition may well compromise vital host defence roles and lymphoid integrity. A case series of active tuberculosis precipitated by the use of a TNF-inhibitor in humans is an example,25 and confirms the animal data in gene targeted mice.19 However, some reassurance may be taken from the fact that TNF-/- mice exhibited normal longevity and no excess morbidity when studied in the absence of specific infectious disease challenge (data not shown), consolidating its suitability as a therapeutic target in the appropriate setting.

Fig. 1 Overview of tasks for successful gene targeting in vivo.

expression may be achieved either as a transient transfection of ES cells, or by the crossing of mice homozygous for the floxed allele with transgenic mice carrying the Cre recombinase gene under ( a) general ( ‘Cre-deleter’), ( b) cell type-specific, or ( c) inducible promoters. These techniques have been proved in principle.17 The Cre-LoxP system is used in ‘knock-ins’ ( the targeted replacement of a gene, most often carrying a specific mutation) where the neomycin resistance cassette is floxed and deleted from the replaced gene.

SINGLE GENES AND INFLAMMATION The study of the pathophysiology of inflammation is now aided by a collection of single gene-deficient strains similar to the TNF-/- mice, some of which are detailed in Table 2. These are described briefly in view of the scope of this paper and references are provided for further reading. Although inflammation is clearly controlled by multiple genes – most typically cytokines, cytokine inhibitors and intracellular molecules – single genes can be almost uniquely responsible for individual components of inflammatory pathology ( e.g., airway esosinophilia and IL-5,26 and acute phase response and IL-627). Gene targeting has uncovered entirely unsuspected roles for genes, such as lymphoid tissue histiogenesis and LTa/b. Some knock-outs have indicated that the importance of individual mediators may have been overestimated, by the observation of minimal phenotypes in the absence of expression (e.g., IL-1, COX-1, Fractalkine). Others form excellent models of human single gene defect diseases ( e.g., CD18-/- and human leukocyte adhesion deficiency).

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Fig. 2 Diagrammatic representation of the gene targeting construct used to inactivate both the tumour necrosis factor ( TNF) and lymphotoxin ( LT) a genes in the mouse ( not to scale). The construct is designed to remove or disrupt exons of the native genes, insert the selectable marker ( neor ), and provide adequate flanking sequence for homologous recombination .

TABLE 2

Examples of insights into inflammatory pathology from the targeting of single genes in mice

Targeted gene

Major phenotyp e

Reference

Interleukin ( IL)-1b

Minimal phenotype, resistance to fever induction

36

IL-1 receptor antagonist

Chronic inflammatory arthropathy resembling rheumatoid arthritis, with DNA antibodies

37

IL-2

Hyper-proliferation of lymphocytes T cell-dependent inflammatory bowel disease

38

IL-5

Loss of eosinophilic airway inflammatory response and bronchial hyper-reactivity

26

IL-6

Marked reduction in antiviral antibody and acute phase response

27

IL-10

Segmental enterocolitis, ameliorated by pathogen-free housing

31

TGF-b1

Systemic lethal inflammation within 3 weeks of birth

39

Tumour necrosis factor

Absent germinal centres, resistance to LPS shock, increased susceptibility to intracellular bacteria, reduced severity of auto-immune pathology, defects in leukocyte migration

18,20,21,40

Lymphotoxin-a

Absent lymph nodes, susceptibility to tuberculosis after immune reconstitution, no observable role in CNS auto-immune pathology

24,41,42

A20

Severe inflammation and cachexia due to failure to regulate TNF-induced NF-kB and cell death responses

29

CD18

Severe inflammatory cell recruitment defect, fatal defect when combined with E-selectin deficiency, mouse model of human leukocyte adhesion deficiency ( LAD)

43

COX-1

Prostaglandin production reduced by 99% but minimal phenotype observed

44

COX-2

Reduced viability, renal dysplasia, cardiac fibrosis, reduced LPS-mediated liver toxicity

45

Complement C5a receptor

Impaired inflammatory responses and Arthus reaction

46

CCR1

Impaired host defence, haematopoiesis and granulomatous inflammation

47

Fractalkine

No observable phenotyp e

48

DNase-1

Lupus-like disorder, ANA-positive, glomerulonephriti s

49

ICAM-1

Impaired neutrophil chemotaxis, decreased mixed lymphocyte reaction

50

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Inflammation is not only attenuated in gene targeted mice. Complex dysregulated inflammatory syndromes have developed from the disruption of single genes indicating vital roles in regulation ( TGF-b,28 A20 29) and in some cases resembling human inflammatory illnesses ( enterocolitis in IL-2-/- and IL-10 -/- resembling ulcerative colitis30,31). These findings, strengthened by observations in CTLA4-/- and CD25-/- mice, are establishing the importance of nonantigen-specific control mechanisms, as distinct from auto-immune sensitisation itself, as key determinants of health and disease in auto-immune pathology.32

GENE TARGETING AND POLYGENIC DISEASE: THE CASE OF SYSTEMIC LUPUS ERYTHEMATOSUS One of the outstanding examples of the contribution of gene targeting to this field has been the description of lupus-like disorders in a series of gene targeted mice rendered deficient of DNase-1, C1q and serum amyloid P component: molecules that share roles in the clearance of nuclear debris at sites of cell death. It is surprising that these molecules, which are not directly connected with specific immunity, have essential roles in protecting mice against the prototypic auto-immune inflammatory disorder. In the case of DNase-1 and C1q the genotypic disturbance has been confirmed in a limited number of human subjects33 and has triggered a new surge of interest in the pathogenesis of systemic lupus erythematosus ( SLE).34 While individual defects lead to some of the features of SLE, the dose of mutations requiring the full expression of disease is less clear. However, seminal work with congenic mice mouse models has begun to define a gene threshold liability concept for the clinical manifestations of SLE.35 Once the genes within the relevant congenic segments have been defined, gene targeting will be employed to dissect the details.

CONCLUSIONS Gene targeting is now established as a key experimental technology supporting the advances that flow from the human genome project and other resources for the identification of participating genes in the complex disease processes such as inflammatory pathology. Gene targeting techniques are becoming more sophisticated, but backcrossing of mutated alleles between mouse strains remains an important confounder in experimental design. Gene targeting in models of auto-immune pathology illustrate that molecules that control cellular activation and regulate inflammation in a non-antigen-specific manner may be as important as the mechanisms that control tolerance itself. Gene targeting not only enables the identification of those factors, but frequently provides the platform for the evaluation of the efficacy and safety of the novel therapeutic interventions that arise from their discovery. Address for correspondence: Dr S. Riminton, Centenary Institute, Building 93, Royal Prince Alfred Hospital, Missenden Rd, Camperdown, NSW 2050, Australia. E-mail: [email protected] v.au

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