building a better mouse

In particular, the use of mutant mice as models of human disease, and more recently their use to explore somatic genetherapy, has been expanding.. Reviewing several common methods of man

CHAPTER 3

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 idealexperimental subject for many applications In particular, the use of mutant mice

as models of human disease, and more recently their use to explore somatic genetherapy, has been expanding Multiple genetic assets of the mouse make the devel-opment of new models of human disease relatively straightforward in the mouse ascompared to other species These include the existence of inbred strains of mice,each with a unique but uniform genetic background, an increasingly dense map ofthe murine genome, and defined experimental methods for manipulating the mousegenome

INTRODUCTION

In mice, genetic mutations may occur spontaneously or they can be induced byexperimental manipulation of the mouse genome via high-efficiency germline muta-genesis, 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 develop-ing gene therapy technology Reviewing several common methods of manipulatingthe mouse genome, addressing questions relating to genetic disease that can beasked (and answered) using mouse models, describing how mouse models can beused to evaluate somatic gene transfer, and finally speculating on what experimen-tal approaches to model development might be used in the future are the scope ofthis chapter

47

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)

PRODUCING MOUSE MODELS OF HUMAN DISEASE

Spontaneous Mutations

Spontaneous mutations occurring in existing mouse colonies are a historical andcontinuing source for models of genetic disease In the past, pet mice were selectedand propagated based on the presence of an unusual phenotype Phenotypes such

as coat color alterations or neurological disorders were chosen because of theirstriking visual impact For phenotypes with a heritable basis, subsequent mating ofaffected animals produced “lines” of mice displaying the genetic-based phenotype.More recently, with the establishment of large scientific and commercial breedingfacilities along with careful programs of animal monitoring, many additional lines

of spontaneous mutants have been established In some cases, an observed type 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 phenotypicsimilarities between the mouse and human diseases The mutated gene needs to beidentified if these models are to assist in the research or testing of somatic genetherapies Identification will require genetic mapping and positional cloning of themutated gene, made easier in mouse by the availability of well-established genemapping reagents When the gene causing or associated with the human disease hasbeen identified in the mouse, the mouse homolog of the human gene (a “candidategene”) can be screened for the presence of a mutation A partial list of prominentspontaneous genetic disease mouse models is presented in Table 3.1

pheno-High-Efficiency Germline Mutagenesis

As with selection of spontaneous mutations, high-efficiency germline mutagnesisusing ethylnitrosourea (ENU) is phenotype driven Young, sexually mature malemice are treated with the alkylating agent ENU, which introduces random basechanges (mutations) into spermatogonial stem cells Treated males are matedapproximately 100 days later following recovery from a period of ENU-inducedsterility The resulting mutations can be transmitted to progeny, which are screenedfor the disease phenotype of interest (Fig 3.1) In principle, ENU-induced muta-

TABLE 3.1 Selected Spontaneous Mouse Genetic Disease Models

Symbol

Btk xid Btk, Bruton’s tyrosine kinase X-linked agammaglobulinemia

Dmd mdx Duchenne muscular dystrophy Duchenne muscular dystrophy

protein (dystrophin)

Hfh11 nu Hfh11, HNF T-cell immunodeficiency

Lepr db Lepr, leptin receptor Diabetes mellitus

Lyst bg Lysosomal trafficking disorder Chediak–Higashi syndrome

Pdeb rd1 Pdeb, phosphodiesterase, cGMP Retinal degeneration

(rod receptor), betapolypeptide

Prkdc scid Prkdc, protein kinase, DNA- Severe combined

activated catalytic peptide immunodeficiency

Prph2 Rd2 Prph2, peripherin Retinal degeneration

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 inyoung mice Thus, large numbers of animals need not be maintained for an extendedperiod of time prior to screening Strategies for detecting phenotypes are quite variable For example, dominant mutations may be based on an obviously visiblephenotype, or altered electrophoretic mobility of a protein in a gel, or a change inbehavior Detection of recessive mutations generally requires (1) producing off-spring from mice derived from mutagenized sperm, (2) interbreeding brothers withsisters 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

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 ENU- induced infertility, treated males with mutagenized sperm are mated with normal females Offspring bearing the mutation are analyzed for the phenotype of interest.

carriers A mating between two carrier offspring would produce progeny with a 25%chance of carrying two mutant alleles, thereby displaying a recessive phenotye Astrength of ENU mutagenesis is that disease models can be generated even thoughmutations are at unidentified loci These new mutations can be mapped in the mouse genome and perhaps the human gene location inferred through synteny ho-mologies 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 aregenotype driven Here a known genetic alteration is introduced into the germlineand the phenotypic consequences are observed As noted earlier, classical mutage-nesis 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 facili-tate 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 tion or microinjection of ES cells, as well as by microinjection of fertilized mouseeggs (see Chapter 2)

infec-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 containthree basic components: the gene regulatory elements (enhancer/promoter), mes-senger ribonucleic acid (mRNA) encoding sequence, and polyadenylation signal(Fig 3.2) The enhancer/promoter regulates transgene expression in an either/ordevelopmental and tissue-specific manner For example, gene regulatory elementsfrom the albumin gene will be expressed in fetal hepatocytes beginning shortly aftermidgestation Expression will reach a maximal (and steady state) level in youngadult hepatocytes The coding sequence may be in the form of genomic DNA or a

TABLE 3.2 Selected ENU-Induced Mouse Genetic Disease Models

Apc Adenomatous polyposis coli (APC) Adenomatous intestinal polyposis

protein

Car2 Carbonic anhydrase II (CAII) CA-II deficiency syndrome

Dma mdx Dystrophin dehydrogenase (G6PD) Muscular dystrophy syndrome

GTP-cyclohydrolase I Tetrahydrobiopterin-deficient

hyperphenylalaninemia

Pah Phenylalanine hydroxylase Phenylketonuria

Tpi Triosephosphate isomerase (TPI) TPI deficiency

cDNA and generally can be transcribed into an mRNA capable of being translatedinto a protein Genomic DNA is preferred for transgene construction since it is morereliably expressed, possibly because of the presence of gene expression regulatoryelements within introns In practice, complementary DNA (cDNA) are commonlyused because of their smaller size and ready availability The use of cDNA’s neces-sitates 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 endogenouspolyadenylation sequence with a heterologous polyadenylation sequence takenfrom a gene that produces a very stable message, such as the human growthhormone gene or the simian virus 40 (SV40) T antigens gene The end result of thejoining 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 injec-tion into fertilized mouse eggs More recently, transgenes have been created usinglarge DNA fragments, including yeast and bacterial artificial chromosomes and P1 phage

Once microinjected into the pronucleus of fertilized mouse eggs, as shown inFigure 3.3, transgenes can become integrated into chromosomal DNA in an appar-ently random manner and through an unknown mechanism However, integrationmay be favored at sites of DNA double-strand breaks In most instances, multiplecopies of the DNA fragment will integrate in a head-to-tail tandem array at a singlegenomic locus Microinjected eggs are then surgically transferred into the oviducts

of pseudopregnant recipients and develop to term Pseudopregnant females havebeen bred by vasectomized males, so that a state of “physiological pregnancy” is

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.

FIGURE 3.3 Transgenic mouse production A dilute DNA solution containing the gene construct is microinjected into the pronucleus of fertilized mouse eggs The microin- jected 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.

trans-induced by cervical stimulation during copulation However, these females have nonaturally 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 ofthe 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 asspecified by its gene regulatory elements In practice, transgene expression often ishighly variable and dependent on the genomic site of integration Thus, in a “typical”injection experiment in which nine lineages are generated that carry a particulartransgene, 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 tissue-

or development-specific pattern This outcome may be a consequence of transgeneintegration near powerful endogenous enhancer or promoter elements These wouldovertly influence expression of the integrated DNA Finally, mice in the final threelineages will express the transgene as expected based upon the transgene’s regula-tory elements However, the level of expression may vary among lineages Oneimportant aspect of transgenic animals is that transgenes permit assessment of thephenotypic consequences only of dominant acting genes The transgenic mouseretains normal copies of all endogenous genes Selected models created by the trans-genic approach are listed in Table 3.3

PRODUCING MOUSE MODELS OF HUMAN DISEASE 53

TABLE 3.3 Representative Transgenic Mice as Models of Human Disease

Bone morphogenic protein Inherited photoreceptor degeneration (Retina) Protein-4

Epidermal growth factor receptor Glioblastoma multiforme

Connexins -CX43 or Cx40 Arrhythmias/sudden cardiac death

Neurotrophins and receptors Nociceptive or analgesic pain

Targeted Mutagenesis

The ideal model for the study of somatic gene therapy should exhibit the samegenetic deficiency as the human disease In general, the greater the similaritybetween the mouse mutation and the mutation as it occurs in humans, the greaterthe likelihood that the mouse will produce a reliable model of the human disease,that is, mimic human pathogenesis Through the selective replacement of normalmouse genes with mutated genes, one can attempt to reproduce the molecular basis

of human genetic diseases A powerful method to accomplish this, developed in the1990s involves inserting a mutant copy of the desired gene into a targeting vectorand then introducing this vector into ES cells ES cells are derived from cells of theinner cell mass of a blastocyst (see Chapter 2) They have retained an ability to dif-ferentiate 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 therapypurposes Most DNA targeting vectors that integrate into ES cell chromosomes do

so randomly However, with a low frequency, the construct will be “targeted “ to thegene of interest in some cells and replaced by homologous recombination (Fig 3.4;also see Chapter 5) ES cell colonies that have undergone homologous recombina-tion carry the mutation in one allele of the targeted gene They can be identified byPCR or Southern blotting Individual cells from these colonies are microinjectedinto mouse embryos at the blastocyst stage of development (Fig 3.5) Injected blas-tocysts develop into chimeric animals, whose tissues comprise a mixture of mutant

ES cell-derived and blastocyst-derived (normal) cells If mutant cells are rated into the germline, the mutation can be passed on to progeny heterozygous forthe mutant allele Matings between heterozygotes produce offspring, one-fourth ofwhich carry two mutant alleles (homozygotes) This approach of targeted mutage-nesis can identify the phenotypic consequences of deleting or modifying endoge-nous mouse DNA Several models generated via targeted mutagenesis are listed inTable 3.4

incorpo-Analysis of Phenotype

Techniques for altering the mouse genome to create models of human diseasedepend upon a systematic and thorough evaluation of phenotype Without a carefulanalysis 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 musttake into account that the genetic change is expressed within a complex context:the living organism Thus, the phenotype will be determined by specific molecularconsequences of the mutation such as loss of gene expression, increased geneexpression, 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 presup-poses an understanding of normal anatomy and complex processes of physiology.However, for studies of gene therapy, these requirements represent an advantagebecause human disease does not exist in a test tube but within an environmentalconstruct 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 3 kb

of neo insertion splitting exon C (Top) Arrows indicate transcriptional orientation of

pro-moters and dotted lines indicate regions of homology where recombination may occur.

(Middle) Wild-type locus (Bottom) Predicted structure of locus after undergoing gous recombination (b) Homologous recombination using an insertional targeting vector (Top) Plasmid with sequence insertion vector containing recombinant DNA homologous to

homolo-the endogenous wild-type locus presented in homolo-the middle frame Prior to electroporation homolo-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.

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 todetermine the cause of the observed abnormalities or because the induced muta-tion failed to alter the desired biological processes In general, the analysis of phenotype focuses on detecting abnormalities that are expected from the specificmutation produced However, these abnormalities would be correlative to thosedetermined by the assessment of physiological and behavioral changes of humandisease Unanticipated phenotypic consequences should not be ignored Results of

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.

the model analysis should be compared to relevant observations of the disease inhumans, to assess how closely the animal and human conditions resemble oneanother at the biochemical, anatomical, and physiological levels Additional studiesshould proceed only after phenotypic analysis has been appropriately performed.

MOUSE MODELS FOR GENE THERAPY: WHAT MAKES A GOOD MODEL

OF HUMAN DISEASE?

The ideal mouse model of a human genetic disease should recapitulate exactly thegenotypic and phenotypic characteristics of the human disease This, in reality,rarely occurs All animal models of human disease have limitations The limitations,however, should not preclude the use of the model It is important to recognize thestrengths and weaknesses of any model, as well as to use the model to addresstestable hypotheses and answer questions For example, spontaneous, ENU-induced

or targeted mutants have gene deficiencies possibly correctable by somatic genereplacement Animal models of monogenic disorders created by these techniques,such as cystic fibrosis, Duchenne’s muscular dystrophy, and hemophilia, can be used

in gene therapy experiments Specifically, studies should investigate the addition of

MOUSE MODELS FOR GENE THERAPY: WHAT MAKES A GOOD MODEL OF HUMAN DISEASE? 57

TABLE 3.4 Selected Animal Models Produced by Targeted Mutagenesis

Human Gene Characteristic Magel 2–7C region Prader–Willi MAGEL-2 MAGE proteins

(neurodegenerative expressed disorder)

Murine ceruloplasmin Aceruloplasminemia Ceruloplasmin Autosomal

b-adducin null mice Hereditary b-Adducin delete Loss of protein

OA-1 knock-out Ocular albinism Ocular albinism Expressed intracell

(loss of pigment type 1 gene in melanosome glycoprotein)

Lymphotoxin (LT-a) Enhanced tumor Lymphotoxin Knock-out lacks

Mouse ApC gene Colorectal tumors Adenomatous An early mutation

(ApC 1638 T)]

Nkx2.1 locus Tracheoesophageal NKX 2.1 Expressed in Targeted disruption fistula (transcription thyroid, lung,

Col 6 a1 gene Bethlem myopathy Type VI collagen Connective tissue

in muscle

a normally functioning gene(s) into somatic cells relevant to the inherited gene ciency Less straightforward is the modeling and evaluation of gene therapy for poly-genic diseases (see Chapter 1) such as cancer, diabetes mellitus, and cardiovasculardisease These diseases, by definition, do not have a single genetic cause However,hypotheses regarding pathogenesis and treatment can be addressed using mice thathave been genetically manipulated such that they exhibit altered susceptibility tothat disease.

defi-For many monogenic disorders, the inherited mutant allele has decreased orabsent function (hypo- or nullimorph) compared to the normal allele Such diseaseshold the greatest promise for use of somatic gene therapy Here, a normal copy ofthe gene could be introduced into affected cells to correct the genetic basis ofdisease For monogenetic diseases, either targeted mutagenesis or high-efficiencygermline mutagenesis are generally the most efficient methods for creating appro-priate models Other genetic diseases result from increased or novel function of amutant allele (hyper- or neomorph) For these diseases, a transgenic approach mayproduce a phenotypic model of the disease However, both normal alleles need to

be present

Transgenic animal models also are useful for introducing highly expressed didate target genes into a mouse for testing of gene therapy strategies These micemay not model a particular disease but produce a particular protein that can serve

can-as a gene therapy target For example, transgenic mice overexpressing transforminggrowth factor alpha (TGF-a) in mammary epithelium provide a uniform popula-tion of experimental animals with a defined genetic lesion that efficiently causescancer These animals can be used to evaluate the effectiveness of gene therapystrategies that interfere, directly or indirectly, with increased TGF-a growth signal-ing and thereby potentially inhibit mammary carcinogenesis

In many instances, mutation of a locus in the mouse does not produce the samephenotype observed in patients with mutation of the corresponding human locus.The reasons are varied The precise character of the murine and human mutationsmay differ That is, different sites in the gene may have been mutated in the mouseand human Thus, the level of residual mutant protein function varies There may

be different patterns of expression of the target gene or modifier genes betweenspecies Finally, there may exist biochemical or physiological differences betweenspecies that affect the resulting phenotype Although in principle, one desiresmodels that closely mimic the disease in humans, models that fall short of this idealare still useful For example, humans carrying germline mutations in the tumor

suppressor gene retinoblastoma (rb) develop the ocular tumor retinoblastoma.

However, mice deficient for the same gene develop tumors of the intermediate lobe

of the pituitary In spite of this difference, rb-null mice can provide general mation about mechanisms of rb-mediated tumor genesis Such studies allow an eval- uation of gene therapy protocols designed to restore rb function to deficient cells

infor-regardless of specific tumor

MODELS OF MONOGENIC DISORDERS

Modeling monogenic disorders is conceptually straightforward Nonetheless, opment of a model to evaluate gene-based treatment may be difficult Two genetic

devel-diseases that are promising candidates for molecular therapeutics are Duchenne’smuscular dystrophy and cystic fibrosis The following sections discuss the geneticbases of these diseases and evaluate the strengths and shortcomings of currentmodels being used to study both disease pathogenesis and treatment.

Duchenne’s Muscular Dystrophy

Duchenne’s muscular dystrophy (DMD) is an X-linked recessive disorder with aworldwide incidence of 1 in 3500 male births DMD is characterized clinically bysevere, progressive weakness and fibrosis of muscle tissue that eventually leads torespiratory or cardiac failure DMD is caused by mutations within a 2.3-Mb genecomprised of 79 exons located on the short arm of the X chromosome Because it

is present on the X chromosome, all carrier males (with only one X chromosome)are affected The transcribed mRNA encodes a large subsarcolemma cytoskeletonprotein, dystrophin Dystrophin is tightly associated with a large oligomeric com-plex of membrane glycoproteins, the dystrophin–glycoprotein complex (DGC) TheDGC spans the sarcolemma of skeletal and cardiac muscle, linking the actincytoskeleton and the extracellular matrix Structurally, the dystrophin protein iscomposed of four polypeptide domains They are (1) an a-actinin-like actin bindingdomain at the amino terminus, (2) the rod domain, composed of a series of 24 spec-trinlike a-helical repeats, (3) a cysteine-rich region, and (4) a variable C-terminaldomain that is subject to alternative transcript splicing At the molecular level,dystophin deficiency results in loss of the DGC and weakening of the muscle cellmembrane The exact function of the protein is poorly understood, but it is pre-sumed to serve a structural function in force transmission or stabilization of the sarcolemma The characteristic lesions of DMD patients include muscle cell necrosis and regeneration and elevated serum levels of muscle creatinine kinase (anindicator of muscle damage) As the disease progresses, muscle fibers are replaced

by fat and connective tissue

Mutations resulting in DMD and the clinically milder Becker’s muscular phy can cause complete or partial loss of dystrophin or production of a truncated,nonfunctional dystrophin protein Resulting phenotypes are variable and depend

dystro-on the precise mutatidystro-on involved The mdx mouse, a spdystro-ontaneous mutant, displays many of the biochemical and pathological features of DMD The mdx mice have a

stop codon mutation in the mRNA transcript of the dystrophin gene The

bio-chemical and histopathological defects observed in mdx mice are similar to those

present in DMD patients Histologically, these mice display muscle necrosis, sis and phagocytic infiltration within muscle tissue, variation in myofiber size,

fibro-an increased proportion of myofibers with centrally located nuclei (fibro-an indicator ofregeneration), and elevated serum levels of muscle creatinine kinase However, mice

do not display severe progressive myopathy In mice the only muscle to undergoprogressive myopathy is the diaphragm Clinically, these animals do not exhibitvisible signs of muscle weakness or impaired movement

DMD is a condition that appears well suited for treatment by gene therapy It is

a monogenic disorder, and the distinctive properties of skeletal muscle favor ery of gene targeting vectors Myofibers are formed as a syncytium of embryonicmyoblasts The nuclei migrate to the periphery of the plasma membrane, and eachcontributes mRNA transcripts to the entire myofiber Furthermore, muscles also

deliv-MODELS OF MONOGENIC DISORDERS 59

contain satellite cells, which are myoblast precursors They lie at intervals along the

outside surface of the myofibers If these cells could be genetically manipulated, theycould serve as a future and potentially unlimited source of targeting vector expres-sion in regenerating muscle For this disease, gene therapy has been attempted usingvirtually every gene transfer technique developed These include retroviral and ade-noviral vector infection, direct gene transfer, receptor-mediated gene transfer, andsurgical transfer of genetically manipulated muscle cells

The general feasibility of gene therapy for DMD was demonstrated using thetransgenic mouse approach Full-length human or murine dystrophin cDNAs were

expressed in mdx mice under the control of skeletal muscle-specific gene

promot-ers Expression of as little as 5% of the normal level of dystrophin was able to tially reverse the histopathological lesions Expression of approximately 20 to 30%

par-of the normal level prevented essentially all dystrophic histopathology and restoreddiaphragmatic muscle function Unfortunately, the full-length 14-kb cDNA exceedsthe cloning capacity of current viral delivery vectors Therefore, a truncated genewith an in-frame deletion (exons 17 to 48) in the rod domain (which produces avery mild phenotype in humans with the corresponding mutation) was expressed

as a transgene in mdx mice Truncated human and murine dystrophin cDNAs were

capable of restoring most of the normal muscle phenotype and function, although

a higher level of expression may be required compared to the full-length cDNA.When an adenovirus capable of expressing a recombinant truncated dystrophin was

injected into muscles of newborn mdx mice, reduction in the histological evidence

of muscle degeneration was noted Also, protection from stretch-induced cal damage in these mice as adults were seen More recently, it was found that trun-cated utrophin, a structurally similar protein present in skeletal muscle, couldsubstitute for dystrophin as a therapeutic molecule when expressed in transgenic

mechani-mdx mice This finding is notewortlhy because DMD patients have a functional

utrophin gene Thus, it may be possible to reverse or prevent muscle damage by regulating utrophin expression

up-Cystic Fibrosis

Cystic fibrosis is a common recessive disorder in the Caucasian population thataffects about 1 in 2500 live births in populations of northern European ancestry.Clinical manifestations of this devastating disease include chronic pulmonaryobstruction, bacterial colonization of the airways, pancreatic enzyme insufficiency,meconium ileus, elevated sweat electrolytes, and reduced fertility in males The gene causing cystic fibrosis is the cystic fibrosis transmembrane conductance regu-

lator (cftr) gene, a transmembrane protein that functions as a cyclic adenosine

5¢-monophosphate (cAMP)-regulated chloride channel in the apical membrane ofrespiratory and intestinal epithelial cells Elevation of cAMP within normal cellsresults in opening of the chloride channel and subsequent chloride secretion ontothe mucosal surface Water follows by osmosis This flushing process is thought to

be important in maintaining proper mucociliary clearance in the airways Mutations

in the cftr gene result in reduced or absent cAMP-mediated chloride secretion

because the protein is either mislocalized or functions with reduced efficiency Cysticfibrosis (CF) mutations have other primary effects in addition to chloride conduc-tance dysfunction The CF transmembrane regulator (CFTR) also may be involved