BIOTECHNOLOGY & YOUBIOTECHNOLOGY &...

Your World /Our World 1

BIOTECHNOLOGY & YOUBIOTECHNOLOGY & YOU

Volume 8, Issue No. 1

a magazine of biotechnology application in healthcare, agriculture, the environment, and industry

Genes andMedicineGenes andMedicine

2 Genes and Medicine

3Genes and Medicine

4HEMOPHILIAand the Last Czar’s Son

6A Healing Gene

8The Challenges to Gene Therapy

10Naked DNATherapy for Blood Vessels

12Cancer and theGuardian of the Genome

14Could We? Should We?

16References

TABLE OF CONTENTS:

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BIOTECHNOLOGY & YOU

Volume 8, Issue No. 1

Your World/Our World describes the application ofbiotechnology to problems facing our world. Wehope that you find it an interesting way to learnabout science and engineering.Development by:The Pennsylvania Biotechnology Association,The PBA Education Committee, andSnavely Associates, Ltd.Writing & Editing by:The Writing Company, Cathryn M. Delude andKenneth W. Mirvis, Ed.D.Design by:Snavely Associates, Ltd.Illustrations by:Patrick W. BrittenScience Advisors:Jim Wilson, M.D., Ph.D., Director, Jane Glick,Ph.D., Nelson Wivel, M.D., Institute for HumanGene Therapy, University of PennsylvaniaSpecial Thanks:The PBA is grateful to the members of theEducation Committee for their contributions:John C. Campbell, SmithKline BeechamKathy Cattell, SmithKline BeechamCeil M. Ciociola, PRIME, Inc.Jeff Davidson, Pennsylvania BiotechnologyAssociationAlan Gardner, SmithKline BeechamAnthony Green, Puresyn, Inc.Mary Ann Mihaly Hegedus, BioprocessingResource CenterLinda C. Hendricks, SmithKline BeechamDaniel M. Keller, Keller BroadcastingRichard KralColleen McAndrew, SmithKline BeechamBarbara McHale, Gwynedd Mercy CollegeJune Rae Merwin, The West CompanyM. Kay OluwoleLois H. Peck, Philadelphia College ofPharmacy & ScienceRhône-Poulenc Rorer GencellJean L. Scholz, University of PennsylvaniaJohn Tedesco, Brandywine Consultants, Inc.Adam Yorke, SmithKline BeechamLaurence A. Weinberger, Esquire,Committee Chair

If you would like to make suggestions orcomments about Your World/Our World, pleasecontact us at:Internet: [email protected] write to:Pennsylvania Biotechnology Association1524 W. College Avenue, Suite 206State College, PA 16801

Copyright 1998, PBA. All rights reserved.

SUBSCRIPTION INFO

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Your World/Our World is designed to bring biotechnology tolife by featuring scientific discoveries and applications in aclear and informative way. Issues on different topics in bio-technology are published each Fall and Spring. If you wouldlike information on subscribing (individual, teacher, and librarysets are available) or on sponsoring distribution to teachers inyour area, please call the Alliance for Science Education/Pennsylvania Biotechnology Association at 800-796-5806.Thirteen previous issues have been published and some backissues are available.

On the cover: Scientists are climbing theDNA “ladder of information” and using thetools of molecular biology to “work” onour genes which control inheritance andplay an important role in health and disease.


T hat’s a phrase we hear often these days.The remark may refer to an inherited dis-ease, musical talent, a poor attention span, ormathematical ability. “Genetic” is becoming a commonexplanation because scientists are identifying more and morehuman genes and figuring out the roles they play in ourlives. A fifteen-year-long international effort called theHuman Genome Project (HGP) has quickened the pace ofthese discoveries. The HGP is identifying and “mapping”

and Medicine

“It’s genetic!”

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all of our 100,000 genes to their exact positions on our chromosomes. Scientists call thisentire set of genes the genome and this field of study genomics.

A newly identified gene creates widespread excitement because genes have such an importantfunction in human health and disease. Understanding genes can lead to treatments forpreviously untreatable diseases. Indeed, genetic science has led to a whole new conceptabout treating disease. Called gene therapy, this approach is aimed at treating theactual cause of a disease rather than easing its symptoms.

At first, scientists thought about using gene therapy to treat inherited diseases.Cystic fibrosis, sickle cell anemia, and hemophilia are a few of over 4,000inherited diseases caused by a defect in a single gene. Then scientists realizedthat gene therapy might also treat non-inherited diseases that people acquireover the course of their lifetimes, such as cancer, heart disease, and even infec-tious diseases such as AIDS. (New studies also show that faulty genes makesome people more likely to develop cancer and heart disease.) These diseases canbe understood, and maybe treated, at the level of genes.

Gene therapy could greatly reduce human pain and suffering. But it is still a youngfield with many technological challenges to solve. In the next decades, gene therapywill have a huge impact on human health and our understanding of disease. It willalso raise many questions. Which diseases should we treat with gene therapy? Will itbe expensive and who will pay for it? Should we try to prevent certain diseases frombeing inherited from one generation to the next? Such questions will force society toexamine closely the promises and pitfalls of gene therapy.

Gene therapy is such a new field that research is just beginning to move out of thelaboratory and into trials on human participants. This issue of Your World/Our Worldprovides a snapshot of some of this new research, as well as glimpses of how leadingexperts view the future of gene therapy. e

An exploded view of a cell,its nucleus, and a chromo-some (which forms the “X”shape during cell division).Scientists are mapping genesto specific places on thechromosomes and learningto use them to treat disease.



I n 1917, therevotionary Bol-sheviks roundedup the last Czarof Russia, Nicho-las II, and hisfamily. The revo-lutionaries shotthe whole familyand then hid thebodies to erasetraces of theiridentities. Somepeople believedthat the Czar’s daughter Anastasia survived, but scientists werecertain that her brother, Alexis, did not. The reason? He was ahemophiliac. He did not have a certain blood clotting proteinin his blood. This protein, now called Factor VIII, is one ofmany blood clotting factors required to make blood thicken.Without any one of these factors in their blood, people can’tstop bleeding (clot) even after minor cuts, and they suffer frompainful bleeding into joints, muscles, and the brain. Even ifAlexis survived the shooting, he would have bled to death.

INHERITING A DISEASE GENEAlexis inherited hemophilia from his mother, Alexandra.Alexandra and her grandmother, Queen Victoria of England,carried a recessive disease gene for hemophilia on one of their Xchromosomes (in the 23rd pair). But they did not have the dis-ease, because they had a healthy gene on the other X chromosomethat produced the proper clotting factor.

Chromosomes are like filing cabinets for genes, and genes are likepapers containing the genetic instructions that guide our growthand development. Except for the genes on the 23rd chromosomepair, we have a double copy of every gene, one from each parent.The double copy creates a type of safety net: If one copy of thegene contains a defect that makes it malfunction, the other copycan often make up for it.

The gene for Factor VIII lies on the X chromosome which breaksthe safety net rule. Females have two X chromosomes but maleshave only one, which they inherit from the mother. The othermale chromosome, a Y, comes from the father and has a differentset of genes. When a woman carries a hemophilia gene on one ofher X chromosomes, each of her sons has a 50/50 chance of get-ting the defective gene, with no safety net gene from the father.Alexis got the unhealthy gene when he was conceived.

HOW GENES AFFECT HEALTHAs Alexis developedfrom a fertilized egg,each cell in his bodyreceived a copy of hisoriginal set of chromo-somes, including thedefective gene for FactorVIII on his X chromo-some. When that geneis healthy, the liver cellsturn it on to producethe clotting factor pro-tein. To turn on a gene,the cells use other mol-ecules to read andtranslate the gene’sinstruction codes. Thiscode is written in thelanguage of life, DNA(DeoxyriboNucleicAcid).

The DNA language isboth simple andcomplex. It uses analphabet of only four“letters,” A, T, C, andG, which refer tochemical bases (adenine,thymine, cytosine, andguanine). But it usesthese same letters tobuild a string 3 billion

THIERRY SOURSAC, M.D, PH.D.,M.B.A,GENERAL MANAGER,RHôNE-POULENC RORER

GENCELL

“Our DNA is like a compactdisk containing all the infor-mation for our cells to makemusic. Just as the stereoreads different tracks on theCD, the body reads differentgenes in the DNA. Instead ofsending a flow of electricityto the speaker, the genesends a flow of proteins tocells. When we have agenetic disease, drugs treatus by adjusting the dials orwires rather than by repair-ing the scratch on the CD.They may help some, butthey also have side effectsbecause they are foreign tothe system/body. Genetherapy will allow us toinsert a new track into theCD or record a blank over atrack to stop it from playing,thus treating disease at themost basic level.”

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H E M O P H I L I A THE LAST CZAR’S SON&

The last Russian Czar and his family:Alexandra and Nicholas II (center), Maria,Tatiana, Olga (top row), Alexis, andAnastasia (bottom row).


Queen Victoria

Alexandra

Victoria Edward

AliceLeopold Beatrice

IreneElizabethVictoria

Olga Tatiana

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Anastasia

Mary

Marie

Earnest Frederick

Alexis

= female

= male

= female carrier

= male hemophiliac

= Possible Carrier

KEY

Family Tree: This family tree showshow Alexis, the heir to the Russianthrone, inherited the hemophiliagene through the female line.

bases long that contains all the genetic information for an indi-vidual. In English, we can combine our alphabet’s 26 letters inmany ways, but only some combinations create meaningfulwords. Likewise, only certain DNA sequences (orders of basecodes) create functioning genes.

A gene is like a paragraph containing the recipe for making onespecific protein. This paragraph contains sentences that help acell know when to read the gene or “turn it on.” When a cellturns on a gene, the gene expresses a protein just as we expressthoughts when we speak or write. This process of producing aprotein is called gene expression.

Genetic diseases result from mutations or “misspellings” in theDNA sequence that change the gene’s recipe for the protein. Inhemophilia, these mutations produce blood clotting proteins thatdon’t work properly, or they fail to produce any at all.

TREATING HEMOPHILIA THEN AND NOWSince biblical times, people knew that hemophilia caused exces-sive bleeding, but there was no treatment for it. As scientistslearned about chromosomes, they recognized that hemophilia ison the X chromosome because of the way it is inherited. In the1950s, scientists mapped the gene for Factor VIII to a generallocation on the X chromosome. By then, doctors treated hemo-philia by giving patients blood or blood products containingclotting factors. However, hemophiliacs required repeated dosesbecause the factors did not last in their blood. Unfortunately,some of the blood and blood products supplied during the1980s contained the HIV virus, so thousands of hemophiliacshave died from AIDS.

In the early 1980s, genomic researchers deciphered the DNAsequence of the gene for Factor VIII, and then they pinpointed itslocation on the X chromosome. At that time, this gene wasamong the few that had been both sequenced and mapped. Thisgenomic information led to a safer treatment for hemophilia.

Scientists copied the healthy gene for the clotting factor andinserted it into cultured cells. As these cells grew in the laboratory,the gene expressed the blood clotting factor. This technique isknown as recombinant DNA because scientists “combine” DNAfrom different sources. This process works because all organismsshare the universal genetic language. They can also express thegenes from different sources and produce recombinant proteins.

In this way, scientists produce disease-free clotting factors whichfunction just as the body’s own proteins would. Unfortunately,these recombinant proteins cost too much to use on an ongoingbasis, so they are reserved for times of crisis and injury. Patientsstill suffer joint damage, and they risk more severe problems.

Gene therapy may soon provide a better way. It involves transferringcopies of a healthy gene into cells in the body to produce a healingprotein. The next article discusses one of the methods being developedfor hemophilia. e



VIRAL DNA

THE THERAPEUTIC GENE FOR FACTOR VIII IS PLACED INTO THE VIRUS.

SCIENTISTS REMOVE THE DISEASE-CAUSING GENES FROM A VIRUS.

GENE DELIVERY VECTOR

PACKAGING CELL LINE1

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I f a male descendent of Queen Victoria were born today, hemight become a candidate for gene therapy. Here’s a scenariothat may become commonplace in a few years...

Doctors will probably know that his mother is a carrier for the he-mophilia gene, so they will test all her male children either before orjust after birth. When they learn that the baby boy inherited thisgene, they will add a working version of the gene to his cells.

To deliver the gene to the cells, scientists will package it in adelivery vehicle called a vector. They will use a vehicle thatalready delivers genetic information to cells: a virus. Viruses entera cell, deliver genes, and hijack the cell’s machinery so the cellproduces the viral proteins. That’s how we get a cold or flu. Tomake a viral vector, scientists remove the viral genes that causedisease, as well as those that allow the virus to reproduce andspread infection. In that way, scientists “tame” a virus so it nolonger can cause harm but can still deliver genes.

Next, scientists will insert a helpful or therapeutic gene into thevirus, in this case, the gene for Factor VIII. With this new geneon board, the virus still infects a cell, but instead of spreadingillness, it contributes to health!

For this little boy, doctors will inject the virus into his muscles. Itmay seem odd that they won’t put it in his liver, since liver cellsnormally produce the clotting proteins. Still, the liver cells them-selves don’t use the blood clotting factors. Rather, the proteinsleave the liver and circulate in the blood to do their job. It does notmatter whether they are made by the liver or another organ. Afterthe injection, the boy’s muscles will become permanent factoriesfor the blood clotting protein ‘round the clock.

The boy may think this gene therapy shot is just one more child-

hood vaccine! He may need a“booster” shot as he becomesan adult. Otherwise, he will befree to play without fearingevery scraped knee.

These techniques may be adaptedto treat other diseases that needspecific proteins in the blood.Still other diseases require quitedifferent approaches, and youcan read about these challenges onthe next page. e

A Healing GeneIll

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This map of the X chro-mosome shows the loca-tion of the Factor VIII genethat causes Hemophilia A,as well as some otherdisease genes.

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Albinism of the Eye

Duchenne MuscularDystrophy

Cleft Palate

Hemophilia B

Fragile X MentalRetardation

Hemophilia A


MANY COPIES OF THE VECTORS ARE PRODUCED INA LABORATORY CELL CULTURE. A LARGE AMOUNT OFTHE VECTOR IS NOW AVAILABLE FOR GENE THERAPY.

3

4THE VECTOR IS INJECTED INTO A MUSCLE,

AND THE MUSCLE CELLS PRODUCE (EXPRESS) THE BLOOD CLOTTING PROTEIN.

JIM WILSON, M.D., PH.D., DIRECTOR,INSTITUTE FOR HUMAN GENE THERAPY AND

JOHN HERR MUSSER PROFESSOR, CHAIRMAN,DEPARTMENT OF MOLECULAR AND CELLULAR

ENGINEERING, UNIVERSITY OF PENNSYLVANIA

“Gene therapy will eventually be able to treatdiseases that need careful regulation of aprotein. For example, treating diabetesrequires carefully adjusting the amount ofinsulin in the blood. We will construct thetherapeutic gene with regulatory elementsthat we can control externally. We couldinsert a gene into your cells, but the gene willstay off until you take a pill that triggers thecell to turn it on. The higher the dose of thepill you take, the more protein the gene willexpress. You could monitor the protein levelin the bloodstream and adjust the dose of pillto achieve just the right balance. This couldprovide an easier, safer, and more effectivetreatment for diabetes and hemophilia, or itcould activate a cancer therapy.”

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■ Every cell in our body (except red blood cells) has anucleus.

■ Each nucleus contains 23 pairs of chromosomes: onechromosome in each pair from our mother and one fromour father.

■ Chromosomes are made of DNA (DeoxyriboNucleic Acid).

■ DNA forms the shape of a twisted ladder or “double helix.”

■ Four bases [adenine (A), thymine (T), cytosine (C), andguanine (G)] are attached at each rung position.

■ The bases attached to the two strands are complementary,with A opposite T and C opposite G.

■ These complementary combinations, called base pairs,form the “rungs” of the ladder.

■ The sequence (order) of bases along the DNA strandscontains the coded information of life.

■ Sections of DNA form genes, which are arranged in aspecific order on a chromosome.

■ Each gene contains thousands of base pairs and theirsequence creates a pattern for building a specific protein.

■ Small mutations or “misspellings” in the DNAsequence can change the final protein and may lead to agenetic disease.

■ If a defective gene in the germline (egg or sperm) causes adisease, it is inherited.

■ Environmental factors that damage genes in other cells ofthe body (called somatic cells) can lead to disease, and thatdisease is not inherited.

Genes, Inheritance, and Disease

This illustration shows one gene therapy experiment to treathemophilia, a disease caused by a defective gene for the bloodclotting protein, Factor VIII.


REGULATING PROTEIN PRODUCTION LEVELSIn hemophilia, even a small amount of the blood clotting protein allows a person tolive a normal, active life. Thus, the added gene does not need to produce the same

amount of blood clotting protein as a healthy gene naturally would. In otherdiseases, scientists must fine-tune the amount of protein a gene produces.

Take for instance sickle cell anemia, the most common inherited disease amongpeople of African descent. A defective gene produces a misshapen protein thatmakes red blood cells form “sickles” instead of round shapes. (See illustrationon page 9.) These cells cannot pass through small blood vessels easily, whichcauses pain and injures organs. It is not enough for gene therapy to simply add a

healthy gene. If the added gene produces too much protein, it can do moredamage than the disease, such as causing a stroke. If it produces too little,it will not treat the disease.

To solve this kind of problem, scientists are studying the DNA codesthat control or regulate the gene’s activity level. Regulatory codes

work like the symbols that tell amusician to speed up, slowdown, and play soft or loud.

CHOOSING A DISEASE TO TREAT WITH GENE THERAPY HAS NOT BEEN

EASY. IN MANY WAYS, HEMOPHILIA IS A GOOD CANDIDATE. OTHER

DISEASES POSE TOUGHER CHALLENGES.

Viruses can enter a cell because theirsurface proteins unlock the receptor’sdoor on the cell membrane.

T H E C H A L L E N G E S T O G E N E T H E R A P Y



Finding thesecodes and thenusing them to adjustgene expression will open up newpossibilities for gene therapy.

TARGETING CELLSFor hemophilia, scientists could add thegene to any cell that secretes proteins. Buttreating many diseases requires placing the genein the specific cells that need the protein to func-tion. In that case, scientists must use a deliveryvehicle that “targets” those cells.

For instance, cystic fibrosis (CF) seriously affects the cellsthat line the lung, so scientists tried to use the adenovirus,which is a virus that naturally infects the lungs. Viruses canenter a cell through a door or receptor in the cell’s membranebecause they have specially shaped proteins on their surface that act

like a key fitting intothe receptor’s lock. (Seeillustration on page 8.)Viruses have adaptedtheir surface proteins tofit receptors so they cansneak into cells. Someviruses have proteinsthat can unlock specificcells that other virusescan’t enter. Scientists areidentifying the genes thatbuild various surfaceproteins. Then, they canselect genes to customizethe surface proteins on avector. They hope todevelop a catalog ofvectors that target spe-cific cells, like an arsenalof “smart” missiles.

EVADING THEIMMUNE SYSTEMScientists face anotherproblem in gene delivery:the immune system.Immune cells can tellwhen a virus has invadeda cell because it expressesforeign-looking proteins.The immune cells recog-nize those viral proteinsand kill the invaded cell.This immune responsekeeps us from gettingdiseases like chicken poxmore than once. But

MICHAEL BLAESE, M.D., CHIEF OF

CLINICAL GENE THERAPY

BRANCH, NATIONAL HUMAN

GENOME RESEARCH INSTITUTE,NATIONAL INSTITUTES OF HEALTH

“So far, gene therapyresearch focuses on addingan entire gene. But genes arelarge molecules that aredifficult to get where they’reneeded. In the future, wemay have a molecular“white-out” and just fix thetypo that leads to a disease.We will use DNA repairproteins that cells naturallyproduce to fix damagedgenes. These proteins are sosmall they don’t need a vec-tor. This technique will in-crease the number ofdiseases we can work with.For instance, we can’t yetdevelop a gene therapy forsickle cell anemia becausewe can’t manipulate theregulatory elements. But thedefective gene only has onemisspelled letter. If we couldcorrect that letter, wewouldn’t have to worryabout the regulatory ele-ments. Using DNA repairmolecules will allow us toapproach many diseases withsingle-letter mutations.”

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misspelled letter produces faultyproteins that lead to misshapenred blood cells.

immune cells can’t know that a virus used in gene therapy is beinghelpful, so they respond as if it were dangerous. This response foiledan early gene therapy trial for cystic fibrosis. An adenovirus placed ahealing gene in the lung cells, which then expressed the new protein

along with a few other viral proteins. After the first dose, theimmune cells thought the cells expressing these proteins

had been invaded by an infec-tious virus. They destroyed those

cells, and ended the gene therapy.

This outcome sent scientists backto the laboratory with a new plan:

removing the viral genes that pro-duce the telltale proteins recognized

by the immune system. Withoutthose proteins, the healing

gene might survive like anundercover agent. If so, thegene therapy could have along-term effect.

A RISING STAR: AAVMeanwhile, scientists dis-covered that a virus calledadeno-associated virus(AAV) does not trigger theimmune response the way

the adenovirus does. Because patients don’t reject the AAV, scien-tists are using it in several experiments, including the hemophiliaresearch described in the last article.

The AAV also inserts genes directly into the chromosome of ahost cell. Thus, the new gene is copied when the cell divides andcontinues expressing the protein, so the therapy could have along-term effect. Furthermore, AAV naturally places genes into aspecific site on chromosome 19, so scientists can predict how itwill behave. Other viruses behave randomly. If AAV used as avector still acts predictably, it could lead to more precise tech-niques for adding genes.

PHYSICAL BARRIERSSometimes physical barriers protect a tissue, so even a targetedvector might not work. For example, CF patients have thickmucus in their lungs that may block viral vectors from reachingtarget cells. Likewise, the brain is protected by the skull and abarrier that screens most molecules, including viruses, out of theblood. Such defenses create special challenges for gene therapy.

Sometimes disadvantages can be turned into advantages. A short-term treatment may not work for inherited diseases that last alifetime, but it may be good for diseases that people develop duringtheir lives. The next two articles discuss such cases. e


G ene therapy may bring relief to millions whosuffer from blood vessel disorders, which are a lead-ing cause of early death in the U.S. In one suchdisorder, called arteriosclerosis, plaque forms on theinside of the artery wall, causing the artery toharden, thicken, and block circulation. When thisplaque blocks the coronary arteries that supply bloodto the heart muscle, it causes heart attacks, heartdisease, and pain. When a blockage occurs in thelegs, it leads to constant leg pains, sores that can’theal, and eventually amputation.

People once lost their limbs – and lives – to arterio-sclerosis. Then scientists developed an artery bypassprocedure, using a healthy blood vessel from else-where in the body to go around the blocked section ofthe artery. More recently, surgeons use balloonangioplasty to clear a passage in a vessel by threading asmall balloon through the artery and inflating it.Both surgical techniques have negative side effects,and they usually are not permanent solutions.

NAKED DNA THERAPY FORBLOOD VESSELSVEGF: MAKING A NEW PASSAGENew gene therapy research combines the “bypass” concept with bal-loon angioplasty. In one experiment on a group of people withextremely advanced leg arteriosclerosis, researchers covered the outsideof the balloon with naked DNA. In naked DNA, the therapeutic geneis not packaged in a vector but is simply spliced into a round section ofDNA known as a plasmid. (See sidebar on page 11.) At first, no oneexpected naked DNA to deliver genes into cells in the living body. Butwhen the plasmids come into contact with a cell, they do! NakedDNA thus bypasses the problems involved in developing a guidedmissile that targets blood vessel cells.

Surgeons snaked the balloon covered with naked DNA to the block-age and inflated it. The inflated balloon pressed the plasmids ontothe vessel wall. The DNAentered the cells of thevessel wall and deposited agene for a growth factorprotein called VEGF (pro-nounced “veg F”) thatmakes new blood vessels

grow. Once inside the cells, the geneexpressed small amounts of thegrowth factor, and that was enoughto make new blood vessels growaround the blockage.

In later experiments, researchers usedan even easier technique, injecting thenaked DNA into leg muscles, whichthen produced the growth factor andled to new blood vessels. Researchershope the new vessels will restore circu-lation to the legs on a long-term basisso the patients don’t need to faceamputation and can resume morenormal lives. Researchers also hope toinject VEGF into the heart muscle tocreate a bypass for coronary arteries,saving future patients from the risk of

open-heart surgery!

Other research uses genesthat help reduce plaque buildup

in arteries or that prevent the arterywalls from thickening. The next article

deals with another area where scientistsare working within current limitationsof gene therapy technology to treat verydifficult diseases. e

THOUGHTS ON THE FUTURE:RACHEL KING, MBA, CHIEF

EXECUTIVE OFFICER, GENE

THERAPY, INC. (NOVARTIS)

“In the early days, many smallgene therapy companiesspecialized in vectors thatcould transfer genes to cells,but they didn’t have expertisein disease biology. Today,more companies workclosely with large pharmaceu-tical companies because theyhave experts in cell biologyand immunology. Thoseexperts help us determinewhich genes to transfer towhich cells to affect thedisease. This merging ofdifferent sciences is movingthe field forward. It will helpus develop gene deliveryvehicles that combine aspectsof different vectors. Forexample, we might take asurface protein from thehepatitis virus that targetsliver cells, combine it with avirus that can place a gene ina specific site on the chromo-some, and put it in a syntheticpackage that doesn’t cause animmune reaction.”

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These photographs show the growth of blood vesselsfollowing gene therapy with the VEGF gene.

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Plasmids are circular DNA molecules that come from bacteriaand are separate from the bacteria’s chromosome. When thebacteria reproduce, they make copies of the plasmid.

To study or use a particular gene, scientists insert it in a plasmid –with the help of special “cut and paste” proteins called enzymes.“Cut” enzymes take the gene out of the chromosome and open up asection of the plasmid. A “paste” enzyme places the gene in theplasmid.

Then scientists put this plasmid into a bacterium that makes manycopies of its plasmid. These plasmids can serve as naked DNA vectors– or they can produce recombinant proteins, as discussed on page 5.

Genomic ResearchIdentify genes and proteins involved in disease.

Cellular ResearchUsing laboratory cell cultures, test vectors and see whether

the added gene makes the protein.

Animal ModelsDevelop techniques for delivering genes to animals.

Verify the production of desired proteins.

Study safety in two mammals, usually rodents and primates.

Human Clinical Trials

Phase I TrialsFocus on whether the therapy has a toxic effect on patientsor causes other harm. Participants are often critically ill and/

or have not responded to other forms of therapy.

Phase II TrialsTest whether patients express the added gene and if their

health improves. To make sure these results are valid,participants must stop other treatment.

Phase III TrialsTest a larger number of participants in “double blind” studies*

and compare the new treatment with existing treatments.

FDA Approval for General Clinical Use

* Double blind: To increase validity of results, neither the patient nor theinvestigator knows which patients receive the treatment and which

receive a “blank” placebo.

RESEARCH& TRIALSHere are some steps in the long process ofdeveloping and approving a new medical

treatment for people.

if fails

if fails

if fails

if succeeds

if succeeds

if succeeds


CANCERp53 VECTOR

TUMOR

TUMOR CELL

GROWTH ARREST

WHEN THE p53 PROTEIN RECOGNIZES DNA DAMAGE, IT STOPS CELL DIVISION UNTIL THE DAMAGE CAN BE REPAIRED.

IF THE DAMAGE CANNOT BE REPAIRED, THE p53 PROTEIN PROGRAMS THE CELL TO BE DESTROYED.

This diagram represents a method being tested in Phase I human clinical trials.

CELL DEATH

AFTER ENTERING TUMOR CELLS, THE ADDED GENE PRODUCES THE p53 PROTEIN.

AN ADENOVIRUS CONTAINING THE FUNCTIONAL p53 GENE IS INJECTED INTO THE TUMOR OF A CANCER PATIENT.

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AND THE GUARDIAN OF THE GENOMEM odern medicine continues todevelop better ways to treat cancer.In fact, about half of the humantrials for gene therapy focus oncancer. There are about a hundreddifferent kinds of cancer, and noneare the same. They start whendifferent genetic mutations or “mis-spellings” occur that make cellsgrow out of control. Some peopleinherit these mutations, but most ofus acquire them during our lives.Tobacco, sunshine, alcohol, toxicchemicals, and certain viruses canall cause mutations.

It may seem that gene therapywould have to add healthy genes toreplace the many misspelled genesthat lead to cancer – a dauntingtask! But that may not be neces-sary. Instead, scientists are focusingon how a cell normally controls itsgrowth and how it loses control tobecome cancerous.

They are studying two types ofgenes that control cell division.Oncogenes stimulate cell division,while tumor suppressor genes ensurethat cells divide in an orderly fash-ion and that any DNA damage isrepaired before the cell divides. In ahealthy cell, these two types worktogether to maintain normal pat-terns of cell division. When eithertype becomes mutated, cells candivide rapidly to form canceroustumors.

p53, WHERE ARE YOU?In studying the activity of bothtypes of genes in tumor cells, scien-tists found unusually high amounts

of a protein called p53 in 60% of the tumors. Because the p53protein was so common, they thought it might be involved incausing these cancers.

But they soon realized p53 is actually a tumor suppressor gene that“puts the breaks” on cancer. A healthy cell produces a small

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AIDSAbout 10% of gene therapy trials focus onAIDS. The virus that causes AIDS sneaks into

the very immune cells that should organize theimmune response against them. Scientists areexploring two gene therapy approaches to stop

AIDS. One attempt aims to give immunecells a gene that will help them recognize

and attack the virus before it hides in theimmune cells. The second approach will

add genes that stop the virus from reproducing.

amount of p53 protein that acts like an automatic spell checker,continuously patrolling the cell’s genome. If it finds a break ormisspelling in the DNA, it signals the cell to produce more p53protein, like calling in backup police officers on a raid. Theseproteins stop cell division until the DNA damage is repaired. Ifthe damage is too severe, they make the cell “commit suicide” tostop the mutation in its tracks.

This system works well – until the p53 gene itself becomesmutated. Then the cell produces high levels of p53 protein toprotect the cell’s genetic information. Because the protein ismutated, however, it cannot kill the cancerous cells.

CANCER’S SMOKING GUNNo one has ever been able to tell what specific thing or eventcaused a particular case of cancer. But recently researchers founda “smoking gun” linking cigarettes to cancer. A chemical incigarettes called benzopyrene introduces a specific misspelling inthe p53 gene, changing a G to T and a C to A – a mutationfound in 50% of lung cancers. Researchers have actually seenthe benzopyrene molecule clinging to the spot where thatmutation occurs on the p53 gene! Other researchers traced amutation caused by ultraviolet light (changing CC to TT) tosome forms of skin cancer. Future studies may show other caseswhere environmental hazards and human behaviors lead to aspecific genetic mutation and cancer.

THE NEIGHBORHOOD WATCHResearchers hope to turn healthy p53 genes loose against tumors,using the body’s own defenses to kill cancer. One way to cleanup a corrupt police force is to send in honest officers. Likewise,adding a functioning p53 gene to a tumor may stop the runawaycell division and kill the cells too damaged to repair.

Some scientists believe that the p53 has a “good neighbor” effect.It may clean up the corruption in its own cell and send proteins

GAIL MADERIS, MBA,PRESIDENT,GENZYME MOLECULAR ONCOLOGY

“We are using new genomicinformation to develop waysto fight cancer. For example,we might teach immune cellsto fight cancer the way theyfight infections. In our bod-ies, the immune systemdoesn’t “see” a cancer cell asan enemy so it doesn’t attackit. We are identifying genesthat could help the immunesystem recognize cancercells as enemies. Immunecells can access the entirebody, so they could reachcancer anywhere. We alsohope to use genes to repro-gram cancer cells internally.In the future, gene therapywill attack cancer on twofronts: from the outsideusing the immune systemand from the inside using thecell’s own self-correctionmechanisms.”

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a t t a c k i n gThe p53 protein binds to DNA to check itsgenetic “spelling.”

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to patrol and protect neighboring cells. If so, scientists may notneed to make the gene produce a lot of protein, since the geneincreases its own effect in the body.

In one test, researchers injected an easy-to-reach tumor with anadenovirus carrying the p53 gene. (See illustration on page 12.)They did not yet have a vector that could seek out cancer like aguided missile, but they could “drop the bomb” directly over thesite of the cancer.

For harder-to-reachcancers, researchershope to develop othertechniques. They mayput tumor suppressorgenes inside a lipid,which is a little dot offat. When injected intothe bloodstream, somelipid vectors target can-cer cells and ignoreothers. Scientists don’tyet know why this hap-pens. But since it does,they hope to use it toseek out cancer wher-ever it lurks in the body.

Researchers are pursuingmany gene therapy strate-gies to cure differentkinds and stages of can-cer. Gene therapy mayeven lead to a vaccinethat prevents cancer inthe first place. e


I MAGINE IF PARENTS COULD ELIMINATE THE GENE

FOR A DEADLY INHERITED DISEASE SO THEIR CHILDREN

WON’T INHERIT IT. SUPPOSE THE TAY-SACHS DISEASE

RUNS IN YOUR FAMILY, WHICH CONDEMNS BABIES TO ASHORT, TORTURED LIFE WITH SEVERE BRAIN DAMAGE,ENLARGED HEADS, CONVULSIONS, AND MORE. TODAY

YOU’D HAVE LIMITED OPTIONS. BUT WHAT IF A DOCTOR

COULD ERASE THE FAULTY GENE IN THE FERTILIZED EGG

AND INSERT A HEALTHY GENE IN ITS PLACE. WOULD

THAT BE RIGHT OR WRONG?

Initially, many people rejected the concept of gene therapy asimmoral because it seemed to tamper with nature. But manyscientists believe there is an important distinction betweengermline therapy that affects sperm and egg (and thus futuregenerations) and somatic cell therapy that affects cells that aren’tpassed on. At present, research focuses on somatic cells and ontreating serious diseases with no other successful treatments.Because somatic gene therapy could help so many people suffer-ing from deadly diseases, many former critics now accept theconcept. Also, people see that the recombinant proteins used totreat diabetes, heart disease, and hemophilia work better thanother drugs because they contain the same product that the cellsthemselves make. Gene therapy seems like a logical next stepbecause it uses the body’s cellular machinery to make a morenatural product than external machines can produce.

Now, many people wonder about germline therapy. If we caneliminate diseases like Tay-Sachs from the human race, theyask, wouldn’t it be wrong not to? Others respond that it willbe impossible to draw the line once we allow such therapy.Suppose you could also program genes in your fertilized egg tomake your child grow tall, be athletically gifted, never get sun-burned, and have the perfect color eyes and hair? It may be a

EDDY RUBIN, M.D., PH.D.,HEAD OF GENOME SCIENCES DEPARTMENT,LAWRENCE BERKELEY NATIONAL

LABORATORY,UNIVERSITY OF CALIFORNIA

“By 2015 we will have convertedthe raw data from our 3 billionDNA codes into useful informationfor understanding health and dis-ease. Then, people can give a dropof blood to learn about thousandsof genes relating to their health.Today we already know that weare susceptible to heart disease ifour parents have it. In the future,genetic testing will tell us moreabout such risks and how to takecare of our medical needs. Also,scientists will know more about thegenetic basis of diseases like asthmaand schizophrenia that have beengreat mysteries and burdens tosociety, and we will have bettertreatments. Still, genetic testing willnot tell us for certain that someonewill develop a disease, because somany environmental and behavioralfactors influence most genes. Oneworry is that some employers,insurance companies, and othersmight use the uncertain geneticinformation about an individual tomake unjust decisions. Luckily,people are already taking measuresto prevent this potential misuse.”

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he future“slippery slope” from

curing diseases toenhancing featuresthat we happen tovalue in this culture.Once we begin toslide, can we stop?

For the foreseeablefuture, we will not havethese choices. To beginwith, scientists have notstarted to identify thegenes that control en-hancement traits, suchas athleticism, since theyare focusing on diseasegenes. Further, suchtraits usually result froman interaction of manygenes and the environ-ment, so they are not atall predictable. Moreimportantly, germlinetherapy would requireerasing the defectivegene and inserting a newgene exactly in the rightplace. Otherwise, theembryo’s developmentcould be drasticallydisrupted. Researcherscannot yet erase a gene;they only add genes.Without this precision,germline therapy en-


The participants in human clinical trials oftenworry about how it will affect others around

them. Here are some of their questions:

Why do I have to avoid my grandmotherwhile I’m undergoing gene therapy

treatment?

Scientists operate under the worst-case scenario when dealing with gene

therapy. Although the virus that transfers thegene should not reproduce, it could theoretically

interact with another virus that does. There is noevidence that this happens, but scientists would rather

be safe than sorry.

Can I pass on a gene to the people around me?

No, the gene can only travel in a delivery vehicle. Scientists cripplethe virus they use so it cannot reproduce and spread to other people.

Will my children inherit this gene?

No, gene therapy is currently used only on somatic cells that arenot passed on to future generations.

counters another huge obstacle: regulations. The government willnot approve a technique without a proven track record for humanclinical trials. In addition, it would be impossible to design a humanclinical trial that could predict the outcome for future generations.

SAME OLD ISSUES?The distinction between treating a disease and enhancing desir-able traits is not a new issue for somatic cell gene therapy.We’ve encountered it in traditional medicine for decades.For example, growth hormones and plastic surgery can fulfilla serious medical need, but they can also be requested fornon-medical reasons. When it fills a medical need, medicalinsurance usually covers the treatment. When the treatment is“cosmetic,” only the rich can afford it. The standards we use forcurrent medical technologies might apply to somatic cell genetherapy as well. The unanswered question is: would these stan-dards apply to germline therapy, or should we create new regula-tory and ethical standards? e

IS IT SAFE?


ReferencesWebsites

Access Excellence: www.gene.com/ae- How Can You Remember These Uses of Biotechnology: www.gene.com/ae/AB/

IWT/aapost/remember/html- Biotech Chronicles: www.gene.com/ae/AB/BC/index.html

Ethical, Legal, and Social Isssues (ELSI): www.ncgr.org/gpi/web.links/elsi.html

DOE Human Genome Program: www.er.doe.gov/production/oher/hug_top.html

National Center for Human Genome Research (NCHGR): www.nchgr.nih.gov

MendelWeb: www.netspace.org/MendelWeb/

Watching Science: www.press1.com/

■ Blueprint for Life, Time-Life Books, 1993.

■ The Ethics of Human GeneTherapy, LeRoy Waltersand Julie Gage Palmer,1997. (Includes overview ofgenetics.)

■ The Romanovs: The FinalChapter, Robert K. Massie,1995. (Includes discussionof use of DNA in identify-ing the bodies.)

■ “Special Report on GeneTherapy,” Scientific Ameri-can, June 1997.

Books and MagazinesDear Readers:

We are delighted to provide a special edition of Your World/Our World,

entirely focused on Genomics and Gene Therapy.

This issue covers the basic science behind a new world of medicine, a

world which young people will most likely have as a healthcare option in

their future.

Knowing how our genes influence our individuality is just the begin-

ning. Today’s scientists are investigating what each gene does and how

we might use them to treat our toughest diseases. From the Human

Genome Project to the beginning investigation of gene therapy, this

field of discovery will likely have a profound impact on our future health

and well being.

We thank our sponsor, RPR Gencell, the gene therapy division of Rhone-

Poulenc Rorer, for making this issue possible. Along with distribution to

students across the country, RPR Gencell will distribute this issue to

people involved and interested in the development of genetic medicine.

Sincerely,

Jeff Davidson

Executive Director

Pennsylvania Biotechnology Association

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