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Chapter 14Chapter 14Gene Cloning inGene Cloning in

MedicineMedicine14.1 Production of recombinant

pharmaceuticals14.2 Identification of genes

responsible for human disease14.3 Gene therapy

14.1 Production ofrecombinant pharmaceuticalsTreatment of disorders absence or

malfunction proteinSupply by human protein

Need large amountSupply by animal protein

Side effects: an allergenic response

How are these techniques being applied tothe production of proteins for use aspharmaceuticals

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14.1.1 Recombinant Insulin Insulin controls level of glucose in blood

by β-cells of islets of Langerhans in pancreas

Disease of insulin deficiency: diabetes mellitus Treatment of DM: pig or cow insulin

Slight differences between animal & human proteins Potentially dangerous contaminants

Two features facilitate production of insulin byrecombinant DNA techniques1. Human protein is not modified after translation

Synthesized by a bacterium should therefore be active

2. Relative small protein (A: 21, B: 30 amino acids)

Fig 14.1 Structure ofinsulin molecule & a

summary of itssynthesis by processing

from preproinsulin

One of the firstprojects Synthesis of artificial

gene for A & Bchains

Production of fusionproteins in E. coli

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Synthesis & expression ofartificial insulin genes (1978)

(a) Artificial gene synthesisAmino acid sequences trinucleotides codonsTwo recombinant plasmids: each carry artificial

gene for A & BpBR322-type vector

提供E.coli 轉錄及轉譯所需訊息1. lac P For RNA polymerase2. lacZ’ For ribosome binding

Fig 14.2 Synthesisof recombinant

insulin fromartificial A & Bchain genes

p.233

The final step is actually rather inefficient

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Synthesis & expression ofartificial insulin genes

A subsequent improvementArtificial gene synthesis contain B-C-A

proinsulin chainA more daunting proposition in DNA synthesisThe prohormone: folding spontaneously into

correct disulphide-bonded structureC-chain: excised relatively easily by

proteolytic cleavage在此章中要注意的重點是，用何種載體、何種宿主以及如何表現？一開始如何進行？碰到何種困難？如何改進？

14.1.2 Synthesis of humangrowth hormones in E. coli

Somatostatin & SomatotropinControl growth processes in human bodyMalfunction: painful & disabling disorders such as

acromegaly (uncontrolled bone growth) & dwarfism Somatostatin or Growth hormone-inhibiting

hormone (GHIH) : acromegalyFirst human protein synthesized by E. coliA very short protein: only 14 amino acidsIdeally suited for artificial gene synthesisUse the same strategy for recombinant insulin

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Fig 14.3 Production ofrecombinant somatostatin

Insertion of artificialgene into a lacZ’vector (pBR322 type)

Synthesis of a fusionprotein

Cleavage withcyanogen bromide

14.1.2 Synthesis of HumanGrowth Hormones in E. coli

Somatotropin or Growth Hormone dwarfism; 191 amino acids, almost 600 bpOut of DNA synthesis capabilities (late 1970s)

Artificial gene synthesis + cDNA cloningObtain mRNA from pituitary RT-PCR cDNACut by restriction endonuclease (Hae III)Longer segment: condons 24-191, retainSmaller segment replaced by artificial DNA

provided correct signals for translation in E. coliInsert into an expression vector carrying lac promoter

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Fig 14.4 Production ofrecombinant somatotropin

Smaller segment replaced byartificial DNA

mRNA cDNA

14.1.3 Recombinant factor VIII Human factor VIII: role in blood clotting

An inability to synthesize factor VIII commonestform of hemophilia

Treat: injection of purified factor VIII protein (donors)? Hepatitis & AIDS

Factor VIII gene: very large (>186 kb)26 exons & 25 introns 2351 AA

A complex series of post-translationalprocessing eventsDimeric protein17 disulphide bonds & a number of glycosylated sites

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Fig 14.5 Factor VIII gene & itstranslation product

Impossible to synthesize an activeversion in E. coli Mammalian cells

14.1.3 Recombinant factor VIII Initial attempts

Entire cDNA was cloned in hamster cellBut yields of protein were disappointingly lowProbably because post-translational events

Did not convert all initial product into an active form

An alternativeTwo separated segments from cDNA were usedEach fragment: ligated into an expression vector

Downstream of Ag promoter & upstream of SV40polyadenylation signal

Introduced into a hamster cell line & recombinantprotein obtained

> 10X yields; function as native form

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Fig 14.6 Expression signals used inproduction of recombinant factor VIII

A & C separated: downstream of Ag promoter &upstream of SV40 polyadenylation signalAg promoter: an artificial hybrid of chicken β-actin &

rabbit β-globin sequencesPolyadenylation signal (needed for correct

processing of mRNA before translation into protein)is obtained from SV40 virus

14.1.3 Recombinant factor VIII Most recent

technology: pharmingComplete human

cDNA: promoter forwhey (乳漿) acidicprotein gene of pigLeading to synthesis of

human factor VIII in pigmammary tissue

Subsequent secretionof protein in the milk

Exactly same as nativeprotein & fully functional inblood clotting assays

2002 台灣首例第九凝血因子及乳鐵蛋白霜轉殖基因猪「酷比」: #1~4

Fig 13.20

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14.1.4 Synthesis of otherrecombinant human proteins Human proteins synthesized by recombinant

technology continues to grow (Table 14.1)

Proteins used to treatdisordersReplacement or

supplementation ofdysfunction

Potential uses in cancertherapySome growth factors

(interferons & interleukins)

Proteins are verylimited amount in bodyInterferons &

interleukins Proteins need very

large quantitiesSerum albumin

Table 14.1 Some human proteins that have beensynthesized from genes cloned in bacteria &/or

eukaryotic cells or by pharming

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14.1.5 Recombinant Vaccines Vaccine

An antigenic preparation injection into bloodstream stimulate immune system to synthesize antibodies protect body against infection

Two problems have hindered preparation ofattenuated viral vaccines (減毒疫苗)Inactivation must be 100% efficient

Just one live virus particle could result in infection口服沙賓疫苗, cattle disease foot-&-mouth

Need large amounts of virus particlesSome virus do not grow in tissue culture (HBV)

whole virusparticle

Producing vaccines asrecombinant proteins

Isolate viral components also can induce virus-specific antibody (Fig 14.7)Advantages: free of intact virus particle & could be

obtain in large quantities Greatest success: hepatitis B virus (42 nm)

Hepatitis B surface antigen (HBsAg, 22 nm )Synthesized in both Saccharomyces cerevisiae(vector based on 2 µm plasmid) & Chinese hamsterovary (CHO) cells

Both have been approved for use in humansWHO is promoting their use

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recombinant proteins

• Gene cloning its gene• Expression & purification

Fig 14.7 Use ofa preparation of

isolated viruscoat proteins as

a vaccine

Recombinant vaccines intransgenic plants

Immunity could be acquired simply by eating part or all ofthe transgenic plant: simpler, cheaper

HBsAg & coat proteins of measles virus & Respiratorysyncytial virus

pharmingOral administration

Tobacco, tomato, & rice plants

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牛痘

Live recombinant virusvaccines

Use live vaccinia virus as a vaccine forsmallpox1796, Edward Jenner 1980, eradication

smallpoxUse recombinant vaccinia virus as live

vaccine against other diseases (Fig 14.8)A gene coding for HBsAg is ligated into

vaccinia genome under control of a vacciniapromoter the gene will be expressed

Immunity against both smallpox & hepatitis B

天花

Fig 14.8 Potential useof a recombinant

vaccina virus

Immunity against bothsmallpox & hepatitis B

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Table 14.2 Some foreign genes that have beenexpressed in recombinant vaccinia viruses

狂犬病

皰疹

口腔炎

Live recombinant virusvaccines

Possibility of broad spectrum vaccines israisedA single recombinant vaccinia virus

expresses influenza virus HA, HBsAg, HSVglycoprotein

Immunity against each diseases in monkeysVaccinia viruses expressing rabies

glycoproteinDeletion of vaccinia gene for thymidine kinasePrevent virus from replicatingNow being used in Europe & north America

狂犬病

避免接觸此活疫苗的動物得到牛痘

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14.2 Identification of GenesResponsible for Human Disease A genetic or inherited disease

Cause by a defect in a specific gene Table 14.3

Individuals carry the defective gene Predisposed toward developing the disease X-linked: hemophilia, male Autosomal recessive: most Autosomal dominant: a few disease,

Huntignton’s chorea

Table 14.3 Some commonest geneticdiseases in the UK

1 in 200 000Blindness, loss of motor controlTay-Sachs disease1 in 25 000 malesBlood disorderHaemophilia B1 in 20 000Cancer of the eyeRetinoblastoma1 in 20 000Blood disorderβ-thalassaemia1 in 12 000Mental retardationPhenylketonuria1 in 10 000Blood disorderSickle cell anaemia1 in 4000 malesBlood disorderHaemophilia A

1 in 3000 malesProgressive muscle weaknessDuchenne musculardystrophy

1 in 2000NeurodegenerationHuntington’s chorea1 in 2000Lung diseaseCystic fibrosis

1 in 300 femalesCancerInherited breast cancerFrequencySymptomsDisease

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Why identifying the gene responsiblefor a genetic disease is important?• Provide an indication of biochemical basis

enable therapies to be designed• Be used to devise a screening program

Mutant gene can be identified in individualsCarrier or who have not yet developed the disease

Counseling for carrierEarly identification in individuals: precautions

• A prerequisite for gene therapy

14.2.1 How to identify a genefor a genetic disease

No single strategyBest approach

Depend on the available information aboutthe disease

Breast cancerMost common & most difficult scenario 方案To understand its principle

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Locating approximate position ofgene in human genome

If: no information about the desired geneHow can it be located in human genome?

Genetic mapping: by linkage AnalysisTarget gene; genetic loci whose map

positions are already knownComparing their inheritance pattern (Fig 14.9)

Key to understand its chromosomepositionDemonstration of linkage with one or more

mapping genetic loci

Fig 14.9 Inheritance patterns forlinked & unlinked genes

(c) Two genes that are far aparton a single chromosome areoften inherited together, butrecombination may unlinkthem.

(a) Two closely linked genes arealmost always inherited together.

(b) Two genes on differentchromosomes display randomsegregation.

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Locating approximate position ofgene in human genome

Human: impossible to carry out directedbreeding programs

Mapping of disease gene: data frompedigree analysisInheritance of gene is examined in families

with a high incidence of disease being studiesObtain DNA samples from at least 3

generations of each familiesMore family members: betterLinkage to DNA markers is more usually

testedHow linkage analysis is used?

This approach to locate a gene: positional cloning

One of the susceptible genes tohuman breast cancer was mapped

1990, U. C. Berkeley: first breakthroughA result of restriction fragment length polymorphism

(RFLP) Linkage analysisFamilies w/ a high incidence same version of an

RFLP (Fig 14.10)

D17S74, long arm of chromosome 17 It was far from the end of the story

>1000 genes in this 20 Mb stretchCarry out more linkage studies: short tandem

repeated (STRs) just 600 kb

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Fig 14.10 Mapping breastcancer gene

Initial: gene was mapped toa 20 Mb segment ofchromosome 17

Additional mappingexperiments narrowed thisdown to a 600 kb regionflanked by two previouslymapped loci, D17S1321 &D17S1325

After examination ofexpressed sequences, astrong candidate for BRCA1was eventually identified

Identification of candidatesfor disease gene

In breast cancer projectFortunate: just 600 kb >60 genesAny one of which could have been BRCA1

Other projectsOften 10 Mb or more of DNA sequences has

be examined

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Approaches can be used toidentify the disease gene

Expression profiles of candidate genesBy hybridization analysis or RT-PCR of RNA from

different tissuesBRCA1: hybridize to RNA from breast & ovary tissues

Southern hybridization analysis for differentspecies DNA (zoo blots)human gene has homologs in other mammals

Compare sequences between cancer & non-cancer women: mutations

Confirm identify of a candidate genePrepare a knockout mouse display disease

symptoms

Identification of a candidateBRCA1 gene

BRCA1: a ~100 kb gene, 22 exons, codingfor a 1863 amino acids proteinIts transcripts were detectable in breast &

ovary tissuesHomologs were present in mice, rats, rabbits,

sheep & pigs, but not chickensMost important: the genes from five

susceptible families contained mutationsEvidence was sufficiently overwhelming

回應上頁的四點

Subsequent research: BRCA2, transcription regulation & DNA repair

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14.3 Gene Therapy Original name

Aim to cure an inheriteddisease by providingpatient with a correctcopy of defective gene

Now extend toAttempts to cure any

disease by introduction ofcloned gene into patient

Germline therapyA fertilized egg is

provided with a copy ofcorrect version ofrelevant gene &reimplanted into mother

Resulting individual: thegene is present &expressed in all cells

14.3.1 Gene therapy forinherited diseases

Usual: microinjection of asomatic cell nuclear transferinto an oocyte (桃利羊)

Could be used to treat any inherited disease在道德倫理宗教上，尚具爭議性；理論上可行，但是科學家們約定不做

Fig 13.19

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14.3.1 Gene therapy forinherited diseases

Somatic cell therapyCells remove cell from organism, transfected, then

place back in bodyCells transfected in situ without removal

目前容許之治療方法

Somatic cell therapy

Fig 14.11 Differentiation of atransfected stem cell leadsto new gene being present inall mature blood cells

血液方面的疾病比較可行

Most promise for inheritedblood diseasehemophilia & thalassemiaGenes are introduced into

stem cells from BMWhich give rise to all

specialized cell types in bloodRetrovirus-based vector

High transfectionfrequency

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Somatic cell therapy

Potential in treatment of cystic fibrosisDNA cloned in adenovirus vectors or

contained in liposomesIntroduction into respiratory tract via inhalerTake up by epithelial cells in lungsGene expression occurs for only a few weeksHas not been developed into an effective

means

14.3.2 Gene therapy & cancer Most intensive area of current research Inactivation of a tumor suppress gene

Introduction of a correct version gene Activation of an oncogene

Prevent expression of oncogeneNot to replace it with a non-defective copy

One possible way: introduce an antisense version ofits mRNA (Fig 14.12)

An alternative: suicide gene therapy Another approach: improve natural killing of

cancer cells by patients immune system

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Fig 14.12 Antisense RNA can beused to silence a cellular mRNA

Prevent protein synthesis The target is therefore

inactivated

DS

Suicide gene therapy An effective general approach

Introduce a gene: selectively kills cancer cells orpromotes their destruction by drugsMany genes code for toxic proteinsEnzymes convert non-toxic prodrugs into toxic

Under control of human telomerase promoterActive only in cancerous tissues

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14.3.3 The ethical issuesraised by gene therapy

Should gene therapy be used to cure humandisease? There is no simple answer

No justifiable objectionRoutine application via a respiratory inhaler of correct

versions of CF gene If bone marrow transplants are acceptable It is difficult to argue:

gene therapies aimed at correction of blood disordersvia stem cell transfection

癌症是如此恐怖的疾病若是以道德的立場反對基因治療這種有效的治療，那它本身是否可以被批評為不道德

Somatic cell therapy

14.3.3 The ethical issuesraised by gene therapy

Germline therapy is a more difficult issue Aims being to ‘improve’ farm animals

Make genetic changes result in lower fat contentGermline manipulation of inherited characteristicsDevelopment of this technique with animals has not

prompted by any desire to cure genetic diseaseExactly same techniques

Genetic constitution of an organism is changedin a directed, heritable fashionThis type of manipulation is clearly unacceptable with

humans