An Introduction to Molecular Medicine and Gene Therapy Edited by Thomas F Kresina, PhD Copyright © 2001 by Wiley-Liss, Inc ISBNs: 0-471-39188-3 (Hardback); 0-471-22387-5 (Electronic) CHAPTER Building a Better Mouse: Genetically Altered Mice as Models for Gene Therapy William C Kisseberth, D.V.M., M.S and ERIC SANDGREN, V.M.D., PH.D BACKGROUND Mice have been used in biomedical research for many years: their small body size, efficient reproductive characteristics, and well-defined genetics make mice an ideal experimental subject for many applications In particular, the use of mutant mice as models of human disease, and more recently their use to explore somatic gene therapy, has been expanding Multiple genetic assets of the mouse make the development of new models of human disease relatively straightforward in the mouse as compared to other species These include the existence of inbred strains of mice, each with a unique but uniform genetic background, an increasingly dense map of the murine genome, and defined experimental methods for manipulating the mouse genome INTRODUCTION In mice, genetic mutations may occur spontaneously or they can be induced by experimental manipulation of the mouse genome via high-efficiency germline mutagenesis, via transgenesis, or via targeted gene replacement in embryonic stem (ES) cells Although each of these methods has potential advantages and disadvantages, all have been successful in generating models of human disease for use in developing gene therapy technology Reviewing several common methods of manipulating the mouse genome, addressing questions relating to genetic disease that can be asked (and answered) using mouse models, describing how mouse models can be used to evaluate somatic gene transfer, and finally speculating on what experimental approaches to model development might be used in the future are the scope of this chapter 47 48 BUILDING A BETTER MOUSE: GENETICALLY ALTERED MICE AS MODELS FOR GENE THERAPY PRODUCING MOUSE MODELS OF HUMAN DISEASE Spontaneous Mutations Spontaneous mutations occurring in existing mouse colonies are a historical and continuing source for models of genetic disease In the past, pet mice were selected and propagated based on the presence of an unusual phenotype Phenotypes such as coat color alterations or neurological disorders were chosen because of their striking visual impact For phenotypes with a heritable basis, subsequent mating of affected animals produced “lines” of mice displaying the genetic-based phenotype More recently, with the establishment of large scientific and commercial breeding facilities along with careful programs of animal monitoring, many additional lines of spontaneous mutants have been established In some cases, an observed phenotype may be caused by mutation of a gene that is responsible, in humans, for a specific genetic disease These models are usually identified based on phenotypic similarities between the mouse and human diseases The mutated gene needs to be identified if these models are to assist in the research or testing of somatic gene therapies Identification will require genetic mapping and positional cloning of the mutated gene, made easier in mouse by the availability of well-established gene mapping reagents When the gene causing or associated with the human disease has been identified in the mouse, the mouse homolog of the human gene (a “candidate gene”) can be screened for the presence of a mutation A partial list of prominent spontaneous genetic disease mouse models is presented in Table 3.1 High-Efficiency Germline Mutagenesis As with selection of spontaneous mutations, high-efficiency germline mutagnesis using ethylnitrosourea (ENU) is phenotype driven Young, sexually mature male mice are treated with the alkylating agent ENU, which introduces random base changes (mutations) into spermatogonial stem cells Treated males are mated approximately 100 days later following recovery from a period of ENU-induced sterility The resulting mutations can be transmitted to progeny, which are screened for the disease phenotype of interest (Fig 3.1) In principle, ENU-induced mutaTABLE 3.1 Mouse Gene Symbol Btkxid Dmdmdx Hfh11nu Leprdb Lystbg NOD Pdebrd1 Prkdcscid Prph2Rd2 Selected Spontaneous Mouse Genetic Disease Models Deficiency Produced Disease Modeled Btk, Bruton’s tyrosine kinase Duchenne muscular dystrophy protein (dystrophin) Hfh11, HNF Lepr, leptin receptor Lysosomal trafficking disorder Polygenic Pdeb, phosphodiesterase, cGMP (rod receptor), betapolypeptide Prkdc, protein kinase, DNAactivated catalytic peptide Prph2, peripherin X-linked agammaglobulinemia Duchenne muscular dystrophy T-cell immunodeficiency Diabetes mellitus Chediak–Higashi syndrome Diabetes mellitus Retinal degeneration Severe combined immunodeficiency Retinal degeneration PRODUCING MOUSE MODELS OF HUMAN DISEASE 49 FIGURE 3.1 High-efficiency ENU-induced germline mutagenesis Young, sexually mature male mice are treated with the mutagen ethylnitrosourea (ENU) After recovery from ENUinduced infertility, treated males with mutagenized sperm are mated with normal females Offspring bearing the mutation are analyzed for the phenotype of interest tions are sufficiently frequent so that only 500–1000 offspring of treated males need to be screened to recover one animal with a mutation at a given genetic locus Because of the number of animals to be screened, it is important for the phenotype to be well defined, easily and inexpensively identifiable, as well as expressed in young mice Thus, large numbers of animals need not be maintained for an extended period of time prior to screening Strategies for detecting phenotypes are quite variable For example, dominant mutations may be based on an obviously visible phenotype, or altered electrophoretic mobility of a protein in a gel, or a change in behavior Detection of recessive mutations generally requires (1) producing offspring from mice derived from mutagenized sperm, (2) interbreeding brothers with sisters from these litters, and (3) determining the phenotype of resulting offspring If the original parent carried one mutant allele, half of its offspring also should be 50 BUILDING A BETTER MOUSE: GENETICALLY ALTERED MICE AS MODELS FOR GENE THERAPY TABLE 3.2 Selected ENU-Induced Mouse Genetic Disease Models Gene Symbol Apc Car2 Dmamdx G6pt Hba Hbb Pah Sar Tpi Deficiency Produced Disease Modeled Adenomatous polyposis coli (APC) protein Carbonic anhydrase II (CAII) Dystrophin dehydrogenase (G6PD) Glucose-6-phosphate GTP-cyclohydrolase I Adenomatous intestinal polyposis Hemoglobin, a-chain Hemoglobin, b-chain Phenylalanine hydroxylase Sarcosine dehydrogenase Triosephosphate isomerase (TPI) CA-II deficiency syndrome Muscular dystrophy syndrome G6PD deficiency syndrome Tetrahydrobiopterin-deficient hyperphenylalaninemia a-Thalassemia b-Thalassemia Phenylketonuria Hypersarkosinemia TPI deficiency carriers A mating between two carrier offspring would produce progeny with a 25% chance of carrying two mutant alleles, thereby displaying a recessive phenotye A strength of ENU mutagenesis is that disease models can be generated even though mutations are at unidentified loci These new mutations can be mapped in the mouse genome and perhaps the human gene location inferred through synteny homologies A partial list of ENU-induced animal models is presented in Table 3.2 Transgenic Mice Whereas the previous methods are phenotype driven, the following methods are genotype driven Here a known genetic alteration is introduced into the germline and the phenotypic consequences are observed As noted earlier, classical mutagenesis does have inherent limitations Because mutations are produced randomly, extensive screening may be necessary to identify carriers of a mutation at the locus of interest Second, induced mutations are not “tagged” in any way to facilitate identification of the mutant gene: ENU-induced deoxyribonucleic acid (DNA) lesions are typically single nucleotide changes Transgenic animals circumvent some of these problems by allowing introduction of a precisely designed genetic locus of known sequence into the genome Foreign DNA, or transgenes, can be introduced into the mammalian genome by several different methods, including retroviral infection or microinjection of ES cells, as well as by microinjection of fertilized mouse eggs (see Chapter 2) The first step in the creation of transgenic mice is construction of the transgene (trans refers to the fact that, historically, the introduced DNA was not from the mouse; thus DNA was being transferred trans species) Most transgenes contain three basic components: the gene regulatory elements (enhancer/promoter), messenger ribonucleic acid (mRNA) encoding sequence, and polyadenylation signal (Fig 3.2) The enhancer/promoter regulates transgene expression in an either/or developmental and tissue-specific manner For example, gene regulatory elements from the albumin gene will be expressed in fetal hepatocytes beginning shortly after midgestation Expression will reach a maximal (and steady state) level in young adult hepatocytes The coding sequence may be in the form of genomic DNA or a PRODUCING MOUSE MODELS OF HUMAN DISEASE 51 FIGURE 3.2 Transgene construction Constituent parts of a simple transgene may come for one or more sources Gene regulatory elements (promoters/enhancers) from gene A may be fused to the mRNA coding sequence from gene B and the polyadenylation signal of gene C Transgene expression is directed in a developmental- and tissue-specific pattern specified by regulatory elements from gene A The stability of transgene mRNA is modified by the polyadenylation signal from gene C cDNA and generally can be transcribed into an mRNA capable of being translated into a protein Genomic DNA is preferred for transgene construction since it is more reliably expressed, possibly because of the presence of gene expression regulatory elements within introns In practice, complementary DNA (cDNA) are commonly used because of their smaller size and ready availability The use of cDNA’s necessitates the use of special transgene construction techniques to enhance expression Finally, for many applications transgene mRNA stability is an important issue Message stability often can be improved by replacement of the gene’s endogenous polyadenylation sequence with a heterologous polyadenylation sequence taken from a gene that produces a very stable message, such as the human growth hormone gene or the simian virus 40 (SV40) T antigens gene The end result of the joining of these pieces of DNA is a transgene that will target stable expression of a selected coding sequence to specific tissue(s) during selected stage(s) of life Most commonly, these elements of the transgene are assembled in plasmid vectors Transgenes are then excised from the vector, isolated, and purified prior to injection into fertilized mouse eggs More recently, transgenes have been created using large DNA fragments, including yeast and bacterial artificial chromosomes and P1 phage Once microinjected into the pronucleus of fertilized mouse eggs, as shown in Figure 3.3, transgenes can become integrated into chromosomal DNA in an apparently random manner and through an unknown mechanism However, integration may be favored at sites of DNA double-strand breaks In most instances, multiple copies of the DNA fragment will integrate in a head-to-tail tandem array at a single genomic locus Microinjected eggs are then surgically transferred into the oviducts of pseudopregnant recipients and develop to term Pseudopregnant females have been bred by vasectomized males, so that a state of “physiological pregnancy” is 52 BUILDING A BETTER MOUSE: GENETICALLY ALTERED MICE AS MODELS FOR GENE THERAPY FIGURE 3.3 Transgenic mouse production A dilute DNA solution containing the transgene construct is microinjected into the pronucleus of fertilized mouse eggs The microinjected embryos are transferred to the oviduct of pseudopregnant foster mothers in which they develop until birth Tissue samples (generally tail) are analyzed by Southern blotting or PCR for presence of the transgene Mice that have incorporated the transgene into their genome and pass the transgene to their offspring are referred to as “founders” of a lineage PRODUCING MOUSE MODELS OF HUMAN DISEASE 53 induced by cervical stimulation during copulation However, these females have no naturally occurring fertilized eggs Offspring of implanted eggs with incorporated transgene in genomic DNA (termed founder mice) can be identified by PCR or Southern blotting of tissues using transgene-specific DNA as a probe Presence of the transgene in the germline results in passage to progeny A single founder mouse and its transgene-bearing offspring constitute a lineage Although present in every cell of the body, transgene expression is regulated as specified by its gene regulatory elements In practice, transgene expression often is highly variable and dependent on the genomic site of integration.Thus, in a “typical” injection experiment in which nine lineages are generated that carry a particular transgene, mice in three lineages will not express the transgene This may be a result of transgene integration into untranscribed or silent regions of the genome Mice in another three lineages will express the transgene but in an unexpected tissueor development-specific pattern This outcome may be a consequence of transgene integration near powerful endogenous enhancer or promoter elements These would overtly influence expression of the integrated DNA Finally, mice in the final three lineages will express the transgene as expected based upon the transgene’s regulatory elements However, the level of expression may vary among lineages One important aspect of transgenic animals is that transgenes permit assessment of the phenotypic consequences only of dominant acting genes The transgenic mouse retains normal copies of all endogenous genes Selected models created by the transgenic approach are listed in Table 3.3 TABLE 3.3 Representative Transgenic Mice as Models of Human Disease Transgene Fc g-RIII Bone morphogenic protein Protein-4 Epidermal growth factor receptor (EGFR) Stromelysin-1 Matrix metalloproteinase (MMP-3) SAD mouse Db/Db Ob/Ob EL/EL Juvenile cystic kidney jck mutation Troponin I (TNI 1-193) Amyloid precursor protein or Presenilin-1 Connexins -CX43 or Cx40 Neurotrophins and receptors Lipoprotein Human Disease Autoimmune hemolytic anemia (AIHA) Inherited photoreceptor degeneration (Retina) Glioblastoma multiforme Atheroma Sickle cell disease Type diabetes Obesity Epilepsy Polycystic kidney disease (PKD) Reversible contractile heart failure (stunned myocardium) Early-onset familiar Alzheimer’s disease Arrhythmias/sudden cardiac death Nociceptive or analgesic pain Atherosclerosis 54 BUILDING A BETTER MOUSE: GENETICALLY ALTERED MICE AS MODELS FOR GENE THERAPY Targeted Mutagenesis The ideal model for the study of somatic gene therapy should exhibit the same genetic deficiency as the human disease In general, the greater the similarity between the mouse mutation and the mutation as it occurs in humans, the greater the likelihood that the mouse will produce a reliable model of the human disease, that is, mimic human pathogenesis Through the selective replacement of normal mouse genes with mutated genes, one can attempt to reproduce the molecular basis of human genetic diseases A powerful method to accomplish this, developed in the 1990s involves inserting a mutant copy of the desired gene into a targeting vector and then introducing this vector into ES cells ES cells are derived from cells of the inner cell mass of a blastocyst (see Chapter 2) They have retained an ability to differentiate into all cell types in the body Thus, ES cells are “totipotent” and now can be maintained and manipulated in cell culture for animal model and gene therapy purposes Most DNA targeting vectors that integrate into ES cell chromosomes so randomly However, with a low frequency, the construct will be “targeted “ to the gene of interest in some cells and replaced by homologous recombination (Fig 3.4; also see Chapter 5) ES cell colonies that have undergone homologous recombination carry the mutation in one allele of the targeted gene They can be identified by PCR or Southern blotting Individual cells from these colonies are microinjected into mouse embryos at the blastocyst stage of development (Fig 3.5) Injected blastocysts develop into chimeric animals, whose tissues comprise a mixture of mutant ES cell-derived and blastocyst-derived (normal) cells If mutant cells are incorporated into the germline, the mutation can be passed on to progeny heterozygous for the mutant allele Matings between heterozygotes produce offspring, one-fourth of which carry two mutant alleles (homozygotes) This approach of targeted mutagenesis can identify the phenotypic consequences of deleting or modifying endogenous mouse DNA Several models generated via targeted mutagenesis are listed in Table 3.4 Analysis of Phenotype Techniques for altering the mouse genome to create models of human disease depend upon a systematic and thorough evaluation of phenotype Without a careful analysis of the consequences to the host of altered gene expression, the relevance of the model to the study of human disease is limited Analysis of phenotype must take into account that the genetic change is expressed within a complex context: the living organism Thus, the phenotype will be determined by specific molecular consequences of the mutation such as loss of gene expression, increased gene expression, and production of a mutant protein In addition phenotypic expression is influenced by cellular biochemistry, tissue- and organ-specific physiology, as well as the environment, the organism-wide homeostatic mechanisms that regulate adaptation of an individual to its surroundings The analysis of phenotype presupposes an understanding of normal anatomy and complex processes of physiology However, for studies of gene therapy, these requirements represent an advantage because human disease does not exist in a test tube but within an environmental construct Thus, it is within this organismal context that any therapy must be effective PRODUCING MOUSE MODELS OF HUMAN DISEASE 55 (a) (b) FIGURE 3.4 Homologous recombination in the generation of gene-targeted animals (a) Use of a replacement vector having a 10-kb homology with the endogenous locus and kb of neo insertion splitting exon C (Top) Arrows indicate transcriptional orientation of promoters and dotted lines indicate regions of homology where recombination may occur (Middle) Wild-type locus (Bottom) Predicted structure of locus after undergoing homologous recombination (b) Homologous recombination using an insertional targeting vector (Top) Plasmid with sequence insertion vector containing recombinant DNA homologous to the endogenous wild-type locus presented in the middle frame Prior to electroporation the vector is linearized within the region of homology (5¢ and 3¢ ends lie adjacent to each other) (Bottom) Structure of altered gene locus based on homologous recombination 56 BUILDING A BETTER MOUSE: GENETICALLY ALTERED MICE AS MODELS FOR GENE THERAPY FIGURE 3.5 Microinjection of blastocysts, embryo-derived totipotent stem cells to generate germline chimeric animals (From Jacenko, 1997.) HR, homologous recombination; PNS, positive–negative selection Phenotypic analysis usually involves examination of animal behavior, longevity, and cause of death, as well as gross and microscopic examination of animal tissues Specialized physiological and behavioral tests also may be performed as a means to determine the cause of the observed abnormalities or because the induced mutation failed to alter the desired biological processes In general, the analysis of phenotype focuses on detecting abnormalities that are expected from the specific mutation produced However, these abnormalities would be correlative to those determined by the assessment of physiological and behavioral changes of human disease Unanticipated phenotypic consequences should not be ignored Results of MODELS OF MONOGENIC DISORDERS 61 in regulation of an outwardly rectified chloride channel, sodium reabsorption, and sulfation Little is known about these other functions or their relevance to CF The CF gene is large, spanning approximately 230 kb and consisting of 28 exons The most common human mutation results in a single amino acid deletion, phenylalanine 508 (DF508) The DF508 accounts for 70% of the disease alleles in the human population Hundreds of additional mutant alleles have been identified, each occurring at a much lower frequency The DF508 mutant protein is mislocalized in epithelial cells, presumably because of improper folding Other mutations prevent proper synthesis of full-length normal protein because of either nonsense, frameshift mutations, aberrant mRNA splicing, or they alter protein function thereby affecting chloride channel regulation, conductance, or gating The initial animal models of this disease were created using ES cells to target disruption of either exon 10 or exon of the murine cftr gene Disruption of exon 10 gave rise to a truncated protein similar to that seen with several types of human CF mutations For all four of the initial CF mouse models, affected animals displayed defective cAMP-mediated chloride transport, consistent with CFTR dysfunction However, despite producing an apparent phenocopy of the biochemical and electrophysiological defect, the histopathological features of the human disease were only partially reproduced in these models The most striking phenotypic abnormality in mouse homozygotes in three of the four mutant lineages was a high incidence of death between birth and weaning The causative lesion resembles meconium ileus (an intestinal obstruction caused by failure to pass a thick, viscous meconium), also found in 10 to 15% of CF patients Additional human pathology was not seen in the mouse model Approximately 85% of human CF patients have pancreatic insufficiency from birth However, histologically, none of these CF mutants reported severe pancreatic pathology It was also unclear whether the relatively mild pancreatic lesions observed in some mutant mice were primarily effects of the cftr mutations or secondary to intestinal disease Also in humans, lung disease accounts for virtually all of the morbidity and mortality in CF patients But no histological abnormalities are observed at birth Over time CF patients develop progressive inflammatory lung disease None of the mouse mutants developed lung lesions when housed under standard conditions One cftr mutant lineage displayed a low level (90% of affected animals surviving to adulthood Interestingly, mice in this mutant lineage, when repeatedly exposed to the common bacterial pathogens Staphylococcus aureus and Pseudomonas aeruginosa, displayed a significant incidence of lung disease, consistent with findings in human CF patients In view of the major differences outlined above between the human and mouse diseases, how good are the animal models of CF? As is often true for a disease with a complex of clinical features and associated lesions, some characteristics are faithfully reproduced, others less so, some not at all Furthermore, other new and apparently unique phenotypes may appear in the mouse All four lineages of mutant mice display the expected alterations in electrophysiology, specifically cAMP-mediated chloride conductance, as predicted based on the proposed role of the CFTR in chloride conductance.Thus, at the level of electrophysiology pathogenesis is reproduced With respect to reproducing the clinical and histopathological manifestations of the human disease, however, the models are less satisfying The differences in repro- 62 BUILDING A BETTER MOUSE: GENETICALLY ALTERED MICE AS MODELS FOR GENE THERAPY duction of clinical features of the disease may be related to species differences in cftr gene expression, protein function, or organ-specific differences in physiology There may also be differences in the specific mutation introduced However, the models are providing useful information An important finding relevant to gene therapy is that a small amount of CFTR can have profound phenotypic consequences When nullimorph cftr mutants are crossed with the lineage displaying slight residual cftr expression, the double heterozygotes express