Charles W. Henderson

A combination of gene therapy and drugs is showing promise in treating head and

neck cancers, researchers in Texas report.

If borne out in further trials, the findings could point the way to more effective

treatment of almost 500,000 people who suffer head and neck cancers annually.

Scientists at the M.D. Anderson Cancer Clinic in Houston, Texas, report that the

combination therapy caused tumors to shrink in 25 of 30 patients tested. Their

findings are published in the August 2000 issue of the journal Nature Medicine.

Cancerous tumors, some as large as 2-1/2 inches, disappeared in eight patients,

the scientists reported. In others, the tumors shrank by up to half.

The positive findings are reported as gene therapy has come under close scrutiny

after the death of a patient undergoing experimental treatment last September. The

field has been criticized for too much hype and two few successes, said W. French

Anderson of the University of Southern California, who was not involved in the

newly reported research.

But, Anderson added in an article accompanying the new paper, "Gene therapy

seems to be turning the corner after a very bad year."

In the Nature paper, a team led by the Houston clinic's Fadlo Khuri used a

specially engineered virus called ONYX-015, which destroys cells with a mutated

tumor suppressor gene called p53. That mutated gene occurs in up to 70% of head

and neck tumors, the scientists noted. ONYX-015 does not damage normal cells.

Along with the gene therapy, the team added the traditional chemotherapy drugs,

cisplatin and 5-fluorouracil, in patients with recurrent squamous cell cancer of the

head and neck. These cancers are often associated with the use of tobacco and

alcohol.

They reported that ONYX-015 in combination with chemotherapy is more

effective than either treatment alone. Current chemotherapy produces results in

only 30% to 40% of patients, and tumors often recur. By comparison, none of the

responding tumors treated with the combination therapy had reappeared after six

months.

In the trial, the ONYX-015 was injected directly into the tumors. The scientists

suggested that future trials look into other methods of administration and the

possibility of using the gene therapy in combination with other methods of

treatment, including radiation and surgery.

This article was prepared by Gene Therapy Weekly editors from staff and other

reports.

Copyright 2000, Gene Therapy Weekly via NewsRx.com.

Article A63988351

The use of gene-transfer technology to repair salivary-gland tissue, allowing a

pathway for saliva to flow, in patients undergoing radiation therapy for head

and neck cancer, is possible in principle, according to Bruce J. Baum, DMD,

PhD.

Baum, who is chief of the Gene Therapy and Therapeutics Branch, National

Institute of Dental and Craniofacial Research, U.S. National Institutes of

Health, Bethesda, Maryland, spoke about the at the American Dental

Association's National Media Conference, held June 15.

"We hypothesized that the major impediment to saliva flow from these

irradiated, non-secreting cells was the absence of a pathway for water in their

membranes," he explained. "Our strategy was to transfer a gene for a water

channel protein into the radiation-surviving cells that would function as the

pathway."

Each year in the United States, the salivary glands of some 40,000 individuals

are exposed to ionizing radiation (IR) during therapy for head and neck cancer.

They experience irreversible salivary gland damage. In addition, patients with

dry mouth or Sjogren's syndrome (SS), (an autoimmune disorder characterized

by progressive destruction of the lacrimal and salivary glands) also suffer the

loss of salivary secretory tissue. Many patients receiving IR or those with SS

experience complete gland destruction.

The primary function of salivary glands is to make saliva, the oral fluid that

provides the major lubrication and protection for the mouth and upper

gastrointestinal tract. In the absence of saliva, patients have difficulty

swallowing food, develop mucosal infections like candidiasis, experience

rampant dental decay, and suffer considerable pain and discomfort.

Salivary glands also may be useful target sites for gene-based protein

replacement therapies (using transferred genes as drugs) with certain systemic

deficiency disorders and for local oral diseases, Baum said.

"One obvious application for this concept is to augment saliva with gene

products for upper-gastrointestinal (GI) tract disorders," he explained.

"Salivary secretions saturate the upper-GI tract lining continuously, and we

envision both preventive and healing applications. An alternative strategy is to

direct needed therapeutic proteins into the bloodstream for systemic use."

Using rodent models in studies, we showed that salivary gland repairing and

therapeutic applications are possible in principle, he said.

"In addition," Baum said, "there is a realistic opportunity to develop a

first-generation artificial salivary gland suitable for initial clinical testing

relatively soon, within about 10 years."

A pilot program to develop an artificial salivary gland for patients with little to

no remaining secretory tissue was initiated several years ago.

This article was prepared by Gene Therapy Weekly editors from staff and other

reports.

Copyright 2000, Gene Therapy Weekly via NewsRx.com.

Article A63044114

Knowing more about genes should revolutionise the treatment of disease

THE genome project's original goal--and its primary goal even today--is to

improve human health. These days the talk is of perfect diagnosis, drugs that

work first time and have no side-effects, even of predictive medicine so accurate

that it could tell you, should you want to know, when you are going to die and

of what. But the medical hype that surrounds it now is very different from the

modest goals of the public project's originators in the late 1980s. They thought

it might help people suffering from simple "Mendelian" diseases such as

sickle-cell anaemia and cystic fibrosis (so called because they are caused by the

breakdown of a single gene, and thus follow the rules of inheritance laid down

for pea plants by Gregor Mendel).

Most of these diseases, however, are rare. And in any case, identifying the

genes that cause Mendelian diseases has not brought much relief to their

sufferers. The promise of "gene therapy" to help victims by replacing the

broken copies of their non-functional genes with versions that work has, so far,

proved a pipe-dream.

The spotlight then turned to "polygenic" disorders (illnesses in which several

genes are implicated, often in combination with particular environmental

triggers). But this approach has also had its disappointments. Despite the

discovery of a few well-known and important associations between genes and

illness (genes on chromosome 19 and the X-chromosome are involved in

migraines, and late-onset diabetes is related to genes on chromosomes 2 and

12), the hoped-for flood of information about polygenic illnesses has not yet

materialised. This probably has less to do with the lack of such diseases, than

with the difficulties of organising studies large enough to disentangle the

relevant genes and tease out the environmental factors in question. Only

recently, with studies on entire national populations such as that currently being

carried out by a company called DeCode on the inhabitants of Iceland, are

researchers beginning to overcome the problem of insufficient data.

In the meantime, the net has widened still further. This wider net was woven

originally by Craig Venter, the man who is now Celera's boss. He promoted a

trick, known as expressed-sequence tagging, that identifies the active genes in a

cell by intercepting the messenger RNA that the cell is producing. As more and

more genes are discovered, it has become possible to use this trick to create

"expression profiles" of many tissues, showing which genes are active, and to

what extent. Comparing the expression profiles of diseased and healthy tissue

means that diseases can be examined at the genetic level whether or not faulty

genes are thought to be directly responsible.

Even expression-profiling, however, may soon be old-hat. The latest wheeze is

to ignore the DNA and the RNA altogether, and look directly at the ultimate

products of the gene--the proteins themselves. This field, known by analogy

with genomics as proteomics, is chemically much harder than studying DNA or

RNA, and has not yet been as thoroughly mechanised, but big bucks are being

bet on it. Earlier this year Celera raised almost $1 billion with the avowed intent

of doing the same thing to the human proteome as it has done to the human

genome, and its sister company, PE Biosystems, is busy developing the

equipment that will be needed to perform the task.

Looking at this history of rising expectations but little actual delivery, a cynic

might argue that medical genomics is like a man playing roulette who doubles

his bet every time he loses, believing he must eventually win--and thus get his

money back with a profit. And looking at the profit-and-loss accounts of many

of the companies that have sprung up over the past decade with the avowed

intent of turning genomic knowledge into money, his cynicism might seem

justified. But it is probably misplaced, for in this case raising the stakes looks

like the correct thing to do; and although many genomics firms will no doubt go

belly-up, some may yet make their masters into billionaires.

The wisdom of the sands

One route to certain knowledge and possible wealth is being taken by those who

have adopted the idea that biology is quite literally becoming an information

science. Traditionally, biology has been done either in vivo (in a living creature)

or in vitro (in a glass container). Modern geneticists, however, do a lot of their

work in silico--in other words in a computer. And no geneticists are more

modern than those at Celera. Although the company's laboratories are filled

with in-vitro sequencing machines designed to read the order of the bases on

DNA, the heart of the operation is its computer room which contains, according

to Dr Venter, the most powerful cluster of machines outside a government

nuclear-weapons laboratory. These machines take the output of the sequencers

(random strings of information a few hundred bases long) and patch them back

together again in the correct order. Since the correct order is over 3 billion bases

long, this is no mean feat.

But you cannot sell something that is 98% junk, so Celera's computers are also

looking for the genes hidden in the sequence. There are two main ways of

doing this. One is to search for so-called open reading- frames (lengths of DNA

bracketed by sequences that mark the places where transcription starts and

stops). The other is to look for sequences similar to those found in known

genes. Though that sounds relatively simple, it is actually tedious and difficult.

Indeed, the estimate of the number of human genes ranges from as few as

35,000 to as many as 150,000. So if you are a drug company, it is worth

paying somebody else to do it for you. At least, Celera hopes it is.

Once a gene has been found and its sequence worked out, the next stage is to

find out what it does. Doing that is helped by the fact that, as knowledge has

accumulated, it has become clear that genes (and therefore proteins) come in

families. This should not be a surprise, as natural selection can work only by

modifying what is already there. But it assists enormously with the task of

deciding what a gene or protein is for.

One important class is the 7-transmembrane receptor. These proteins float in the

surface membranes of cells, with their amino-acid chains snaking to and fro

across the membrane, so that part of the protein is inside the cell and part

outside. Many 7-transmembrane receptors act as the pick-up points for

hormones and other molecules that pass signals from cell to cell. The outer part

of the protein is shaped to fit the relevant signal molecule, and the inner part

sends a chemical message to the rest of the cell that the signal molecule has

arrived.

These proteins are of particular interest to drug companies. They turn out to be

the targets of many existing "small-molecule" drugs (which either stimulate or

jam the receptor by being about the same size and shape as the appropriate

signal molecule), so the hope is that previously unknown receptors will provide

a key to treating previously intractable diseases. And all 7-transmembrane

receptors have enough in common that a computer program designed to look for

them in recently sequenced DNA can pick them out with ease.

In fact, the programs can do much better than that. The public databases are

now so full of probable proteins that a newly discovered gene can be checked

almost instantly to see what it resembles. That often gives an accurate idea of

what it does. The results, nevertheless, must be handled with care. Few would

have predicted that crystallin, a clear protein in the lens of the eye, is a slightly

modified version of the enzyme that degrades alcohol.

That used to be as far as you could go in silico. But glassware is becoming

more redundant every day. It is now possible to predict not only the order of the

amino acids, but also the shape of a protein, from the sequence of bases in its

gene. America's National Centre for Biotechnology Information has a program

known, rather archly, as CN3D. This can recognise the exons in an open

reading-frame, work out the resulting sequence of amino acids and then

calculate how the resulting chain should fold up. The shape of a protein is

usually critical to its job. And, since one picture is frequently worth a thousand

words, the result, displayed on a screen, can tell the trained eye instantly what

an unknown protein's properties are likely to be.

Predict and provide

All this information is very impressive; but at some point you have to get your

hands dirty, and start developing something practical and useful. Those

practical and useful things tend to come in two varieties: diagnostics and drugs.

A disease must be diagnosed before it can be treated, and it is in the field of

diagnostics that genomics is having its biggest immediate impact. For genomics

can allow you to spot a problem--or, at least, the risk of a problem--before it

arises. A firm called Myriad Genetics, which is based in Salt Lake City, Utah,

markets tests for mutations in genes known as BRCA1, BRCA2 and AGT. The

BRCA1 and BRCA2 mutations are known to predispose women to breast

cancer. That is useful knowledge, since such cancers, if caught early, can be

treated successfully. The AGT mutation predisposes to heart disease, which is

probably also useful to know.

Another test that is now available is for particular varieties of a gene called

apoE. The protein that this gene codes for is involved in transporting cholesterol

in the bloodstream. For some as yet unknown reason, however, it also plays a

role in Alzheimer's disease. And one of its three versions is a strong indicator

that the individual involved will develop the disease in later life. At the moment,

Alzheimer's is more or less untreatable. But a lot of drug companies are

working very hard to change that, so it is likely, soon, that the forewarning of

the disease that the test for apoE provides will also be useful knowledge.

It is the optimistic multiplication of this sort of thing by several hundred as yet

unknown, but confidently expected, examples that lies at the heart of the idea

that someone's medical life-history (accidents aside) might be predicted at birth.

Even resistance to infectious disease can have a genetic component. The gene

for sickle-cell anaemia protects against malaria. And one version of a receptor

protein called CCR5 protects against HIV, the virus that causes AIDS.

The development that the optimists point to in order to justify their optimism is

the biochip. These chips, which were pioneered by Affymetrix, of Santa Clara,

California, are laid out by a technique similar to the photolithography used to

make computer chips (although the chip itself is usually made of glass rather

than silicon). Each spot on a chip is a forest of short, single-stranded DNA or

RNA molecules known as probes. These will stick to complementary strands of

DNA or RNA if they are available. Wash a solution containing the relevant

complementary strands over the chip and the chip will act as a detector,

especially if, as is fairly easily achieved, the complementary strands have small

fluorescent molecules attached to them, so that they can be seen by laser

scanning.

Ultimately, it will be possible to put probes for all the main varieties of all

human genes on one of these chips. (It is already possible to put more than

10,000 different probes on one.) An individual's DNA, chopped into suitable

fragments and tagged with fluorescent labels, could then be washed over such a

chip, his or her genetic complement read off, and the risk factors for relevant

diseases calculated. And biochips should be able to assist with infectious

diseases, too. If someone became ill through infection, a chip holding probes

from appropriate pathogens could easily tell which organism was causing the

illness.

That is still a little way in the future. But chips that do expression- profiling, by

having probes for messenger RNA, are already starting to have diagnostic uses.

Last year, two of Eric Lander's colleagues at the Whitehead Institute, Todd

Golub and Donna Slonim, used a chip that could recognise messenger RNA

from almost 7,000 genes to devise a simple test to distinguish between two

types of leukaemia, known as AML and ALL. Making such a distinction with

traditional techniques is hard work, but it matters, because the treatments of the

two types of leukaemia are different, and applying the wrong one significantly

reduces the chance of recovery. Dr Golub and Dr Slonim found that the

expression profiles of 50 genes were so distinct between the two leukaemias

that it was possible to tell the difference between them unambiguously.

Millennium Pharmaceuticals, a firm that is also based in Cambridge, is trying to

extend this idea to other diseases. One example the company is studying--with

success according to Bob Tepper, its chief scientific officer--is prostate cancer.

This can be lethal, but is more frequently something that men die with (ie, it is

discovered post-mortem) than die of. Since prostate-cancer treatment is

unpleasant and damaging, a reliable way of distinguishing between the lethal

and less lethal forms would be welcome.

Popping pills

Diagnosis is, however, of little value without treatment. And it is for the

development of new drugs that people are looking most eagerly to genomics.

Genomic knowledge is assisting drug discovery in many ways. First, it is

identifying new targets for the traditional sort of small- molecule drug. Second,

it is helping to work out why those drugs work in some people but not in

others. Third, it is helping to explain side- effects. And fourth, it is allowing the

introduction of a whole new class of drug: therapeutic proteins.

The search for new targets is aided by the enhanced understanding of disease

mechanisms that genomics is bringing. Millennium Pharmaceuticals, for

example, is concentrating much of its effort on treatments for obesity.

Although being too fat is not usually regarded as a real "disease" by most

people--even those who are--those same people would probably rather pop a

pill to reduce weight than go on a strict regime of diet and exercise. Millennium

hopes to oblige them. It has identified and patented several of the

appetite-regulating genes which prompt people to eat too much or store too

much fat in their tissues against a risk of famine that is never going to happen in

a rich country. And, in co- operation with Hoffmann-La Roche, an

old-established drug company, the firm is probing the proteins that those genes

encode to see if any of them can be subverted.

The second and third ways in which the new knowledge is helping the process

of drug development are bracketed together under the label

"pharmacogenomics". Biochips will help a lot here. The reason drugs work in

some people but not in others is often because the same set of symptoms can

have different causes.

Sometimes, as in the case of AML and ALL, genuinely different diseases are

involved. In this case, expression-profiling can tease out the differences and

help with the identification of drug targets. In other cases a single disease may

have several possible causes, because a broken gene for any of the proteins in

the relevant biochemical pathway will result in a similar outcome. In the case of

Alzheimer's, for example, a faulty gene for a protein called presenillin results in

apparently identical symptoms (although at an earlier age) to those experienced

by people with the risky form of apoE. Again, that gives the drug companies

new and more precise targets to aim at. Yet another reason is that target proteins

can come in slightly different varieties according to the exact sequences of their

parent genes. These varieties may all be equally functional, but nevertheless

respond differently from one another to a drug.

Using the new knowledge, rapid screening for likely side-effects should also be

possible. Side-effects are the result of a drug interacting with a molecule other

than its target. Proteomics, if it works, will yield at least an approximation to

the full set of human proteins. (This, despite the "central dogma" of genetics

that one gene yields one protein, is likely to number around 1m. Many genes

can be read more than one way, and proteins are frequently modified after

release from the ribosome by having bits chopped off and molecules such as

sugars plastered on them.) Stick that full set on a chip, and it will be possible to

see if a drug candidate interacts with any protein other than the one it is aimed

at.

Not everyone, however, thinks that small-molecule drugs are still the way

forward. Bill Haseltine, the boss of Human Genome Sciences (HGS), which

is, like Celera, based in Rockville, is betting his company on a whole new class

of drugs that genomics is promising to throw up-- therapeutic proteins.

Strictly speaking, therapeutic proteins are not that new. Insulin, the treatment

for early-onset diabetes, is one. Another is erythropoetin, a treatment for some

forms of anaemia. But there are only about half a dozen examples at the

moment, and all are proteins that were known about before anyone sought to

turn them into drugs. HGS is actively seeking therapeutic proteins by

expression-profiling. One of these, KGF2, promotes the growth of skin cells; it

is being tested for the treatment of chronic ulcers, such as those suffered by

diabetics. A second, MPIF, helps to regulate blood-cell numbers and should,

the company hopes, mitigate the cell-depleting effects of cancer treatments. The

third, VEGF, stimulates the growth of blood vessels and may thus allow the

body to bypass clogged arteries such as those responsible for heart disease.

Dr Haseltine reckons that if these drugs are successful, there will be room for

many more. His reasoning, at least in part, is based on the growing problem of

side-effects when using traditional drugs.

Small-molecule drugs are alien invaders as far as the body is concerned. They

have to be neutralised and degraded, and there are only so many biochemical

pathways capable of doing this, most of which are found in the liver. One

reason that drugs have side-effects is cross- interaction between them if more

than one is prescribed, and this, again, is frequently due to the degradation

pathways getting "cluttered up". Each time a new drug is tested, the regulations

require that it be checked for cross-interactions with existing drugs. If such

cross- interactions are found, approval is frequently withheld. So each new

small-molecule drug that is approved helps to scupper the chances of its

successors.

That, according to Dr Haseltine, is the reason why established drug companies

have introduced fewer new drugs to the market over the past decade than in

previous ones. But proteins are less alien to the body than small-molecule

drugs, and there are protein-degradation mechanisms everywhere. HGS is

betting that there will be fewer cross-interaction problems with therapeutic

proteins than with traditional drugs, and that the market for therapeutic

proteins--currently about $20 billion-- will boom.

Move over Hippocrates

If the true believers in genomics are correct, the coming century should see a

plethora of diagnostics and precisely tailored drugs. It should therefore also see

two aphorisms favoured by medical practitioners, but honoured at the moment

as much in the breach as in the execution, come true. These are that prevention

is better than cure, and that you treat the patient, not the disease--or, at least,

that you can and should personalise the treatment to the patient.

That would be good news for both sides. But the new diagnostics and

treatments may also cause a shift in the relationship between patient and doctor.

With diagnosis turned from a black art into an exact science, and drugs tailored

reliably to an individual's genome and biochemical symptoms, much of the skill

that doctors now deploy will have become automated. Not all of it, of course.

And computers are not renowned for their cosy bedside manner. But many

branches of the profession may find that their jobs, like those of so many

others, have been superseded by chips of one sort or another.

Article A63068617

Genomics is revealing the unity and the diversity of life. Both are essential to

the exploitation of the new knowledge

THE phrase "Human Genome Project" has a pleasingly anthropocentric ring to

it: the sort of good, relevant science that funding agencies admire. But from the

beginning, the project has never been exclusively "human". It has always been

interested in the genomes of other species.

Much of this interest is medical. The genomes of pathogenic bacteria, for

example, yield both drug targets and a better understanding of the diseases

those bacteria cause. But it is in the area of comparative genomics that the

biggest strides are being made. This is because a lot of research is done on what

are referred to, somewhat slightingly, as "model" organisms (as though these

creatures, such as laboratory mice, had evolved for the sole purpose of serving

medical science). Yet, oddly, the more that is discovered about these

organisms, the more it looks as though they could indeed have evolved for just

that purpose.

One of the most surprising results of the genome project has been the discovery

of just how similar many living things are to one another at the genetic level

and, paradoxically, how diverse life really is. Before genomics, the living

world was divided into two. One group, the eukaryotes, consisted of organisms

whose cells have "proper" nuclei. These are the animals, the plants, the fungi

and single-celled creatures such as ciliates. The other group, the prokaryotes,

were the bacteria. These creatures, thought of --often literally--as the scum of

the earth, have no proper nucleus. Their DNA is a single molecule.

Genomics has reversed the picture. Fishing for genes by a process similar to

expression-profiling, and doing so in such unlikely sites as hot springs and the

waste-water outlets of chemical factories, has shown that traditional methods of

growing bacteria have revealed only about 1% of the diversity of bacterial life.

In other words, the living world is really ruled by prokaryotes. It is the

eukaryotes, and particularly the multicellular eukaryotes, that are the "scum".

They form an outlying twig on a tree of life whose trunk and branches are

otherwise largely bacterial. Indeed, the prokaryotes divide into two entirely

different groups, now known as the eubacteria and the archaea. And these

groups, in turn, have proliferating branches unsuspected by biologists a mere

ten years ago.

Many of the creatures on these new branches make their livings in ways which,

viewed from the lofty heights of the eukaryotes, look bizarre. Methanotrophs,

for example, rely on methane (the main component of natural gas) as their food.

Methanogens, on the other hand, excrete methane; they feed on hydrogen gas

and carbon dioxide. And many prokaryotes can endure conditions thought, until

recently, to be completely hostile to life. Deinococcus radiodurans, for instance,

is almost immune to radioactivity. Even if its DNA is blasted to pieces by

radiation, those pieces can reform themselves into a working chromosome like

the scuttling limbs of an undead creature from a horror movie.

Such bacteria call into question the idea that a narrow range of temperatures and

the presence of sunlight are essential for life. Their discovery is therefore

exciting for those who hope to find living creatures on other planets, for they

show that the variety of conditions in which organisms can thrive is far greater

than previously suspected.

Supermodels

Besides putting mankind in its place in a way similar to the work of Copernicus

and Darwin, this new knowledge has two practical consequences. First, by

showing how closely related all the eukaryotes really are, it justifies the

comparisons between species as superficially distinct as yeast and people:

comparisons which biologists are now eager to make. Second, by showing

how diverse the prokaryotes are, it reveals a group of organisms that could be

of enormous industrial importance.

Trying it out on the dog (or, more usually, the mouse) has long been a mainstay

of medical science. Trying it out on the worm, however, is a new idea. But that

is what Exelixis, a firm based in the city of South San Francisco, California, is

doing.

C. elegans, a soil-dwelling threadworm a millimetre long, is one of the most

important animals in biology. It was first used to work out how a multicellular

animal develops from a single fertilised egg. That was done in the 1970s and

1980s by John Sulston, who is currently head of the Sanger Centre, the main

British laboratory involved in the Human Genome Project. Using a microscope

and a lot of patience, Dr Sulston followed the fates of individual cells in the

worm's body as they divided and specialised for their particular tasks. An adult

worm has 959 cells, so the job of doing this, while taxing, was not impossible.

Once it was done, it made C. elegans the obvious candidate for a genome

project all to itself. And in December 1998 the worm became the first animal to

have its DNA completely read. The result was surprising. For even then, when

the genetic databases were relatively empty, 42% of the genes discovered had

some sort of match to genes in organisms only distantly related to threadworms.

Now the databases are brimming, and the genetic unity of the eukaryotes has

become even clearer. When the genome of the fruit fly Drosophila was

published earlier this year, 83% of its genes matched those of other species.

These matches are not perfect, of course. In the millions of years since flies and

mice had a common ancestor, the DNA sequences of genes that do the same job

in each have drifted apart. But they still do the same job for all that, as was

shown a few years ago when a gene involved in eye development in mice was

substituted for its homologue in flies, and the flies were born with normal,

functional eyes.

This means that other eukaryotes can stand as models of what goes on in

people. One way this is exploited is by "knocking out" individual mouse genes

that have known human homologues in order to find out what happens.

Another technique is to use rapidly breeding species such as worms, flies and

yeast to do quick-and-dirty assessments of the actions of chemicals that might

be turned into drugs--which is what Exelixis is up to.

By finding worm, fly or yeast genes that are homologues of human genes that

cause disease (for example, the presenillin gene implicated in Alzheimer's), and

then engineering them so that the resemblance to the defective, disease-causing

version of the human gene is perfect, the company's geneticists can

mass-produce model organisms. The exact effect of the engineered gene is then

observed by knocking out every other gene in the species, one at a time

(something that can be done only with full knowledge of its genome). That

shows which other genes are important in the disease, and thus those that might

form drug targets.

But genomics is not only about medicine--at least, its practitioners hope it isn't.

It should have an enormous impact on agriculture, although it has had a rocky

start there. And the chemical industry also has high hopes of it.

The real genetic engineers

It is a nice irony, given that scientific genetics started with the manipulation of a

crop plant, the pea, that the most vehement public opposition to it in recent

years has come from those who object to the genetic manipulation of crops.

At the moment, so-called genetically modified (GM) crops are in disgrace.

Consumers, particularly in Europe, are wary of buying food that may contain

them. Environmental activists are ripping up fields where they are being tested

experimentally. And companies that design them are selling off their GM

subsidiaries, or even themselves, to anyone willing to take on the risk.

Yet the chances are that this is just a passing fad. No trial has shown a health

risk from a commercially approved GM crop (or, more correctly, a transgenic

crop, as all crop plants have been genetically modified by selective breeding

since time immemorial). And while the environmental risks, such as

cross-pollination with wild species and the promotion of insecticide-resistant

strains of pest, look more plausible, they also look no worse than the sorts of

environmental havoc wreaked by more traditional sorts of agriculture.

In any case, research is ploughing ahead. Existing GM crops are designed for

the advantage of the farmer. They are equipped with genes that produce

insecticides (cotton and maize) or resistance to herbicides (soyabeans). The next

generation will have genes that bring benefits to the consumer. Barbara Mazur

and her team at DuPont, a large American chemical firm, for example, have

engineered a variety of soya that produces more than three times as much oleic

acid as normal varieties. They did this by the paradoxical means of adding a

gene that would normally result in the degradation of oleic acid. But since such

a gene is already present in soya, the two cancel each other out by a

little-understood but much valued process called co-suppression. Oleic acid is at

a premium because it is more stable than most soya oils, so food made with it

lasts longer. And it may also have industrial uses as a lubricant.

Others also see a future in using GM crops as a source of industrial raw

materials (which would not run into worries that they were somehow poisoning

people). Anthony Sinskey, of the Massachusetts Institute of Technology, and

his colleagues at the Palm Oil Research Institute of Malaysia are planning to pull

a similar trick to Dr Mazur's on their oil palms. They intend to improve the

palms' yields of oleic acid, and also of stearic acid, which is one of the main

ingredients of soap. Since palms already produce ten times more oil per hectare

than soya, that would be a very productive trick.

Plants are also being investigated for use in cleaning up sites polluted by heavy

metals. Cadmium, copper, mercury and so on are poisonous to most creatures,

but some plants have proteins called phytochelatins which bind them up and

squirrel them away in places where they can do no harm. The genes for the

enzymes that make phytochelatins have now been identified, and several groups

of researchers are working on transferring them into species that can be grown

on the polluted ground and perhaps even harvested to recover the metals.

Plants, however, are relatively slow-growing. And even if they are boosted

with bacterial genes (as is the case with insect-resistant maize), they suffer from

the biochemical narrow-mindedness of all eukaryotes. So, rather than work

with prokaryotes at one remove, a number of researchers are turning to them

directly.

The leader in this area is DuPont. Its bacterial genomics group, led by Ethel

Jackson, is making bugs that have completely new biochemical pathways.

These can, in principle, turn out any chemical produced by any bacterium

anywhere, using any chemical input which at least some bacterium can digest.

And there are so many different bacteria in the world that the range of possible

products is vast.

A biochemical pathway is actually a series of enzymes. Each enzyme acts as a

catalyst for a particular chemical reaction, the product of which is the raw

material for the next enzyme in the pathway. So constructing an artificial

pathway means finding a set of enzymes that provides all the necessary reactive

steps from cheap molecule "A", the input, to valuable molecule "B", the output.

Having found these enzymes, each of which can come from a different

organism, you snip out the genes that code for them and stitch them into

whatever sort of bacterium you think most suitable for the fermentation vats in

your factory.

This really is genetic engineering, rather than merely prissing about transferring

one gene at a time. And Dr Jackson and her colleagues are starting to make it

work. They have experimental bacteria that can turn out adipic acid (one of the

ingredients of nylon), teraphthalic acid (a component of a specialist polyester)

and even spider silk. They are also close to commercialising production of 1,3

propandiol, teraphthalic acid's partner in polyester. And they are working on

the input as well as the output by studying the genome (now completely

sequenced) of a methanotroph. Few raw materials come cheaper than natural

gas. And methanotrophs would find it so hard to live in the sort of environment

favoured by people that the chance of them doing any harm if they escaped is

negligible.

Even this, however, is not good enough for some. A firm called Maxygen,

based in Redwood City, California, is not content with Nature's bounty of

enzymes. It is working on ways of transcending those natural limits by

applying Darwinian principles to the creation of new ones.

Maxygen uses analogues of the main drivers of evolution: sexual reproduction

and natural selection. First, it establishes its commercial objective (a better

enzyme for washing powders is one example). Then it takes the gene for a

protein that shows some inkling of the desired activity, and searches the

databases for homologues of that gene. With one or more homologues, the

process can begin.

Each of the available genes is broken up into pieces. The pieces are then mixed

up in the presence of an enzyme that encourages them to recombine. (This is the

part of the process analogous to sexual reproduction.) The recombined genes

are then inserted into bacteria to produce their novel proteins, which are tested

to see if they are better at the desired task than the original protein. The best of

the bunch are picked out and allowed to "breed" by being recombined again.

(This is the part of the process analogous to natural selection.)

Repeat two or three times and you often end up with something far better than

the originals. Maxygen's washing enzymes, for example, were able to

outperform existing ones, even though those existing ones were the product of

decades of conventional research.

Mary Shelley's ghost

Maxygen's synthetic genes lead naturally to the question of synthetic life. Until

the first synthesis of an organic compound (urea) from inorganic ingredients in

1828, most people believed that living matter was infused with some sort of

vital spark. Few would profess to believe that now, but a lot of people still have

a gut feeling that life is not merely quantitatively, but qualitatively different from

non-life. A synthetic organism, made from inorganic ingredients in the way that

Friedrich Wohler made urea, would render that idea untenable. And the first

such organism is likely to come out of the laboratories of the Institute for

Genomic Research, yet another of the cluster of gene-labs in Rockville,

Maryland.

Over the past few years, some of the researchers at that institute have been

involved in a study known as the Minimal Genome Project. This has taken

Mycoplasm genitalium, one of the bacteria whose genomes have been

sequenced there, and knocked out each of its 517 genes, one at a time, to see

how many of them are essential to its existence. The answer, if the bug is

cosseted in a laboratory, is around half of them.

The next stage of the project is to synthesise a minimal genome composed of the

essential genes from "off-the-shelf" chemicals. Stuck inside a synthetic cell

membrane made of fat-like molecules, and kick- started with the relevant

enzymes and some ribosomes, such a genome should start work churning out

proteins. With luck, the whole arrangement would settle down, find its

equilibrium, and start dividing. And the last refuge of the vitalists would have

vanished.

Article A63068618

2000 JUN 11 - (NewsRx.com) --

DNA vaccination using T-cell epitopes may be a viable gene therapy for

allergies, researchers in the United States and Korea suggested.

"Genetic vaccination with naked plasmid DNA provided longstanding cellular

and humoral immune responses and promoted a shift in the pattern of cytokines

produced by the T cells," said SoonSeog Kwon and colleagues from the

Catholic University of Korea and the Univeristy of Tennessee, USA. "Recently

peptides derived from T-cell epitopes [were shown to] downregulate cytokine

production and prevent specific antibody formation and administration of a

single dominant epitope may tolerize the response to all the T-cell determinants

within that protein.

"Therefore," they continued, "to determine whether the vaccination of naked

plasmid DNA coding only a T-cell epitope peptide is able to suppress the

allergic reaction in vivo, we have used mixed naked DNA plasmids encoding

the five classes of human T-cell epitopes on Der p1 and Der p2 as a genetic

vaccine in BALB/c mice."

Kwon et al. presented data from their study at the 56th Annual Meeting of the

American Academy of Allergy, Asthma and Immunology, held in San Diego,

California. The title of their presentation was "Vaccination with DNA encoding

T-cell epitopes suppresses Der p induced IgE production."

A blank pcDNA 3.1 vector was used to inject control mice. The researchers

observed a reduction in the total and Der p-specific immunoglobulin E (IgE)

synthesis in the treated mice as compared with the control mice. Evaluation of

Der p-specific-IgG2a antibody responses in the vaccinated mice showed that

they had more prominent responses than did the control mice. Also, in the

treated mice there were elevated levels of interferon-{{gamma}}.

Interferon-{{gamma}} is a Th1 cytokine associated with the suppression of IgE

production. This was determined by analysis of serum levels of cytokines after

immunization with Der p extract.

"The histologic studies showed reduced infiltration of inflammatory cells in the

lung tissue of vaccinated mice as compared to control mice," concluded Kwon

et al. "These results suggested that the vaccination with the DNA encoding

T-cell epitopes were effective in the inhibition of the allergen induced IgE

synthesis as well as less cells infiltration in the lung tissue than the control mice,

thus gene therapy using T-cell epitope encoding DNA could be an ideal way of

combating allergic disease in the future."

The corresponding author for this study is SoonSeog Kwon, Department of

Internal Medicine, Catholic University of Korea, Medical College, Korea.

A search of the www.NewsRx.com online database using the term "DNA

vaccine" generated 276 articles.

Key points reported in this study are:

* DNA vaccination using T-cell epitopes may be a viable gene therapy for

allergies

* T-cell epitope peptides down regulated cytokine production and prevented

specific antibody formation

* Allergen induced IgE synthesis was reduced by genetic vaccination with

T-cell epitopes

This article was prepared by Immunotherapy Weekly editors from staff and

other reports.

Copyright 2000, Immunotherapy Weekly via NewsRx.com.

Article A62502841

A CUNNING way of sneaking genes into the brain should make it easier to

give people gene therapies for diseases such as Parkinson's and Alzheimer's.

The brain is protected from potentially dangerous substances in the blood by the

tight junctions between capillary cells. Only recognised molecules are allowed

in, so to get genes into the brain, researchers either have to inject them through

holes drilled into the skull or give them intravenously along with drugs that

disrupt the blood-brain barrier.

Now William Pardridge and Ningya Shi at the University of California School

of Medicine in Los Angeles have found a way to trick the barrier into letting

therapeutic genes through while still protecting against harmful substances. The

researchers first packaged the genes inside a fatty sphere called a liposome.

Pardridge and Shi then tethered this package to an antibody that latches onto

receptors on the brain's capillary cells and tells the cells to let the package into

the brain. "It piggybacks through without interfering with the endogenous

transport system," says Pardridge.

When the researchers injected a package containing a gene for luciferase into

rats' bloodstream, they found the protein appeared throughout the animals'

brains. Their results will appear in a forthcoming issue of Proceedings of the

National Academy of Sciences.

"This approach is intriguing, and a potentially useful means of distributing

genes widely throughout the brain," says Mark Tuszynski, a neuroscientist at

the University of California, San Diego. Pardridge says they have already

developed antibodies for primate receptors that could work in humans.

Article A63671032

It's been more than 30 years since scientists discovered bone morphogenetic

proteins (BMPs), molecules that spur bone production. After much

experimentation, tests in people show that BMPs can regrow missing or

damaged bone. Some severely injured bone does not respond to this therapy,

however, because BMPs need a foundation of living cells to stimulate bone

formation.

Using rats and mice as models, researchers at the University of Michigan

School of Dentistry in Ann Arbor have now devised a gene therapy that delivers

cells making both BMPs and bone itself. The study suggests a new line of

treatment for hard-to-repair fractures or degenerated bone, both of which would

otherwise require that surgeons transfer, or graft, bone or bone marrow from

one part of the body to another, says study coauthor R. Bruce Rutherford, a

dental scientist at Michigan.

Rutherford and his colleagues knew that BMPs injected into odd places, such as

skin or muscle, could induce these tissues to make bone. They took skin cells

from rats and combined them in a laboratory dish with a genetically engineered

adenovirus to which the researchers had added the gene for a BMP family

member called BMP-7. Although the virus can't replicate, it infects the cells and

induces some of them to mass-produce BMP-7 and others to take on the role of

bone-building cells.

Rutherford and his colleagues added the genetically engineered cells to a

mixture of protein-rich foam, then applied it to the heads of six rats that had had

the tops of their skulls removed. The therapy spurred bone formation so well

that 90 percent of the missing skull bone grew back within a month.

"The rapidity with which it filled in was quite surprising to me," Rutherford

says. Six untreated rats with similar injuries regrew very little bone, the

scientists report in the May 20 HUMAN GENE THERAPY.

In another BMP-7 experiment, human gum cells treated with the virus in a lab

dish launched bone growth in 30 mice.

In both experiments, the virus-infected cells stopped making BMP-7 after 2 to 3

weeks. It's not clear what ends the process, Rutherford says.

The study "represents a new approach for BMPs," says Pamela G. Robey of

the National Institute of Dental and Craniofacial Research in Bethesda, Md.

"There are many [patients] that BMPs by themselves don't work on."

These include people whose broken bones become infected or are heavily

scarred, says George H. Rudkin, a plastic surgeon at the University of

California, Los Angeles School of Medicine.

The gene therapy might also help cancer patients in whom bone has been

removed surgically or has been weakened by radiation treatment, which

diminishes the number of bone-making cells and limits blood supply to the few

remaining, Rudkin says.

The scientists are trying to reduce the need for bone grafts because the

procedure causes considerable discomfort and can lead to infection. The

researchers next plan to apply the experimental gene therapy to broken

thighbones in rodents.

Article A63184603

USA Today (Magazine), June 2000 v128 i2661 p6

High-Tech Treatment for Sports Injuries. (Brief Article)

Full Text: COPYRIGHT 2000 Society for the Advancement of Education

Whether weekend athletes or sports professionals, most people eventually are faced

with an injury. Traditional treatments of surgery or a cast followed by lengthy

rehabilitation are being replaced with gene therapy.

"We're using basic scientific research to develop new orthopedic therapies that use

genes, stem cells, and tissue engineering to create improved healing in sports

injuries of all kinds," notes orthopedist Johnny Huard. "With a typical muscle

injury, muscle regeneration begins within two weeks. Often, the healed muscle has

a lot of scar tissue that causes complications for professional and recreational

athletes. Our research shows that, by adding gene therapy to the treatment

program, muscles heal with less scar tissue, creating a nearcomplete recovery of

the muscle."

The development of improved methods of transferring genes into cells has created

new options to aid in the healing capacity of muscu-Ioskeletal tissues like muscles,

ligaments, and cartilage. Gene therapy also has the potential to revolutionize the

treatment of bone loss problems related to fractures that don't heal, hip and knee

replacements, and spinal fusion. Orthopedic surgeons use cells harvested in a

patient's body, and the modified cells are placed back in a particular area of the

body to create a biological response in that site. Once the gene is in place, it

produces a response that stimulates the healing process.

"The more we

understand about the

biology of bone

formation, cartilage

repair, and tendon

healing, the more

we will be able to

develop the

appropriate tissue

engineering and

gene therapy

strategies to treat

specific injuries,"

suggests orthopedist

Jay Lieberman.

"Gene therapy has

the potential to allow

orthopedic surgeons

to harness or

stimulate the body's

inherent healing

potential." Gene and cell therapies remain in the experimental stages, although

researchers expect them to be available for use as a standard treatment within a

decade.

Article A62685283

Charles W. Henderson

Researchers in Italy have used a gene therapy approach to produce

interferon-producing cells in vivo, which created an anti-angiogenic and

anti-tumor effect.

"We developed an in vivo gene therapy approach to characterize and optimize

the anti-angiogenic activity of class I interferons (IFNs), using packaging cell

lines producing an amphotropic LXSN-based retrovirus expressing either

IFN-alpha 1 (alpha 1Am21), IFN-beta (beta Am12) murine cDNAs, or the

vector alone (neoAm12)," stated A. Albini and colleagues, Institute Nazional

Ricerche Cancer, Center Biotecnology Avanzate, Italy.

Albini et al. published the results of their study in American Journal of

Pathology ("Inhibition of angiogenesis and vascular tumor growth by

interferon-producing cells - A gene therapy approach," Amer J Pathol, April

2000;156(4):1381-1393).

The researchers pretreated endothelial-like Eahy926 cells in vitro with

conditioned media (CM). The medium was derived from alpha 1Am12 or beta

Am12 cells. The cells were pretreated for 48 hours. They observed that this

significantly inhibited the tumor cells migration and invasion as compared to

control neoAm12-CM-treated cells.

They found that "beta Am12-CM also inhibited the formation of capillary-like

structures on Matrigel by EAhy926 cells."

According to the researchers, when they included beta Am12 cells in vivo, it

strongly inhibited the angiogenic response in the Matrigel sponge model. When

alpha 1Am12 was included, it partially inhibited the angiogenic response in the

same model. They performed the same experiments in both immune-competent

and athymic nude mice.

A reduction of host cell infiltration in alpha 1Am12- and beta Am12-containing

sponges was demonstrated by electron microscopy. The same test also showed

a reduction of invading tubular clefts in the host cells as compared to controls.

"Finally, inoculation of either alpha 1Am12 or beta Am12 cells (10%) along

with a highly angiogenic Kaposi's sarcoma cell line (90%) resulted in a

powerful reduction of tumor growth in nude mice in vivo, as did infection with

the interferon-alpha-producing retroviruses," Albini et al. concluded. "These

data suggest that a gene therapy approach using class I interferons can

effectively inhibit tumor angiogenesis and growth of vascular tumors."

The corresponding author for this report is D.M. Noonan, Institute Nazional

Ricerche Cancer, Center Biotecnology Avanzate, Molecular Biology

Laboratory, I-16132 Genoa, Italy.

A search of the www.NewsRx.com online database using the term

"anti-angiogenesis" generated 225 articles.

Key points reported in this study are:

* A gene therapy approach which produced interferon producing cells in vivo

stimulated an anti-angiogenesis and anti-tumor effect

* The researchers used packaging cell lines to produce an amphotropic

LXSN-based retrovirus expressing either IFN-alpha 1 (alpha 1Am21) or

IFN-beta (beta Am12) murine cDNAs

* Beta Am12 cells strongly inhibited the angiogenic response in the Matrigel

sponge model and alpha 1Am12 partially inhibited the angiogenic response in

the same model

This article was prepared by Cancer Weekly editors from staff and other

reports.

Copyright 2000, Cancer Weekly via NewsRx.com.

Article A62329164

Charles W. Henderson

Researchers in South Korea have evaluated a suicide gene therapy protocol in

an animal model of human prostate cancer.

"We sought to determine the feasibility and efficacy of suicide gene therapy

using adenovirus (Ad)-mediated herpes simplex virus thymidine kinase

(HSV-TK) and the prodrug acyclovir, and to evaluated changes in the biological

phenotype for tumor cell proliferative activity after suicide gene therapy in

animal models of human prostate cancer," wrote J. Cheon and colleagues,

Korea University Hospital, Korea ("Adenovirus-mediated suicide gene therapy

using the herpes simplex virus thymidine kinase gene in cell and animal models

of human prostate cancer: Changes in tumor cell proliferative activity," British

Journal of Urology International, April 2000;85(6):759-766).

The researchers used a replication-defective adenoviral vector (cytomegalovirus,

CMV). The vector contained the beta-galactosidase gene (Ad-CMV-beta-gal) as

a control and Ad-CMV-TK as the therapeutic vector. The vector was under the

transcriptional control of the CMV promoter.

First, they evaluated transduction efficiency in vitro. They infected LNCaP and

PC-3 androgen-dependent and independent human prostate cancer cells with

Ad-CMV-beta-gal. X-gal staining was conducted to evaluate transduction

efficiency.

For prostate cancer cells infected with Ad-CMV-TK, the researchers determined

the TK activity by measuring TK-mediated [H-3]-gancyclovir phosphorylation.

They evaluated the sensitivity of the LNCaP and PC-3 cells to Ad-CMV-TK

after infection in vitro with the therapeutic vector with or without acyclovir.

"The inhibition of PC-3 tumor growth in vivo induced by the

Ad-CMV-TK/acyclovir suicide-gene system was assessed in separate and

controlled experiments using human prostate cancer mouse models," said

Cheon et al. "Ki-67 proliferative antigen and proliferating cell nuclear antigen

(PCNA), both useful proliferative indices, were evaluated using

immunohistochemical staining (MIB-1 monoclonal antibody and monoclonal

anti-PCNA antibody) in formalin-fixed, paraffin-embedded tissues from gene

therapy-treated and control animals."

The researchers found a significantly increased mean TK activity in the LNCaP

and PC-3 cells that were infected with Ad-CMV-TK as compared to the cells

infected with control Ad-CMV-beta-gal (P<0.05). They also observed that the

addition of acyclovir significantly inhibited the growth of the cells infected with

Ad-CMV-TK in vitro (P<0.05).

"In the in vivo experiments using the PC-3 human prostate cancer mouse

model, tumor volume and growth was lower in mice treated with

Ad-CMV-TK/acyclovir than in those treated with Ad-CMV-TK only, acyclovir

only or untreated (controls) (P<0.05)," wrote Cheon et al.

Histochemical staining of the infected tumor tissues showed that Ad-CMV-TK

combined with acyclovir killed PC-3 tumors, they reported. The process

included apoptosis with local lymphatic infiltration.

In vivo, the researchers observed that the mean PCNA labeling index in prostate

cancer cells of the Ad-CMV-TK/acyclovir treated mice was significantly lower

than that in untreated controls (P<0.05, Mann-Whitney U-test). The Ki-67

labeling index was also lower (P<0.05, Student's t-test).

"Adenovirus-mediated suicide gene therapy using the HSV-TK gene decreased

the proliferative activity of PC-3 human prostatic cancer cells in vivo,"

concluded Cheon et al. "Adenovirus-mediated suicide-gene therapy using an

HSV-TK/acyclovir system provided effective therapy in an experimental human

prostate cancer mouse model, by significantly inhibiting tumor growth and

decreasing the proliferative activity of human prostate cancer cells. Such therapy

could be developed as a novel method for treating patients with

androgen-independent prostate cancer."

For additional information, contact J. Cheon, Korea University Hospital,

Department of Urology, 126-1, 5KA, Anam Dong, Sungbuk Ku, Seoul

136705, South Korea.

A search of the www.NewsRx.com online database using the terms "gene

therapy" and "prostate" generated 117 articles.

Key points reported in this study are:

* A suicide gene therapy protocol was evaluated in an animal model of human

prostate cancer

* The researchers used an adenovirus (Ad)-mediated herpes simplex virus

thymidine kinase (HSV-TK) vector and the prodrug acyclovir

* The suicide gene therapy protocol was effective both in vitro and in vivo

This article was prepared by Cancer Weekly editors from staff and other

reports.

Copyright 2000, Cancer Weekly via NewsRx.com.

Article A62329166

Researchers in Germany have evaluated a suicide gene therapy approach using

adenoviral expression of bacterial cytosine deaminase in a metastatic colon

carcinoma model.

"Colon carcinoma accounts for 20% of deaths due to malignancies in the

Western world," stated A. Block and colleagues, University of Hamburg,

Germany. "Once metastases occur, therapeutic options are limited, with an

approximate five-year survival of only 5%. To investigate the potential of new

gene therapeutic approaches, a hepatic micrometastasis model of colon

carcinoma in BALB/c mice was established."

Block et al. published the results of their study in Cancer Gene Therapy ("Gene

therapy of metastatic colon carcinoma: Regression of multiple hepatic

metastases by adenoviral expression of bacterial cytosine deaminase," Can

Gene Ther, 2000;7(3):438-445).

Tto form a multiple hepatic metastases model, the researchers inoculated

syngeneic MCA26 colon carcinoma cells into the spleens of 18- to 20-week-old

mice. A B-galactosidase-expressing recombinant adenovirus was used to

demonstrate selective transduction of developing hepatic metastases in the

animals.

The basis for this particular suicide gene therapy approach is founded on the

ability of cytosine deaminase (CD) to metabolize the prodrug 5-fluorocytosine

into the chemotherapeutic reagent 5-fluorouracil (5FU), the researchers

explained. To explore the antitumoral potential of this suicide gene therapy

approach, Block et al. systemically administered a recombinant

replication-deficient adenovirus vector that encoded the bacterial CD gene under

the control of the cytomegalovirus promoter (Ad.CMV-CD).

Application of this particular suicide gene therapy resulted in delayed tumor

growth in the mice. There was also a significant reduction in hepatic metastases

in the same animals.

"The potential of this experimental approach for possible future clinical

applications was evaluated by investigating adenoviral transduction efficiency,

5FU sensitivity, and 5-fluorocytosine-dependent Ad.CMV-CD toxicity in a

variety of human colon cancer cell lines," added Block et al.

Results from the in vitro experiments showed that the murine cell lines MCA26

and CC36 were highly sensitive to 5FU. However, the human colon cancer cell

lines the researchers tested were one to 100 times more resistance to 5FU.

They reported that 5FU toxicity correlated specifically with Ad.CMV-CD

toxicity. The transduction efficiency was 10-1,700 times higher in the human

colon carcinoma cell lines as compared with the murine colon carcinoma cell

lines. This permitted the human colon carcinoma cell lines to compensate for

5FU resistance, the researchers said.

"In conclusion, suicide gene therapy using CD may be promising as an adjuvant

treatment regimen for hepatic micrometastases of human colon carcinoma,"

Block et al. concluded.

The corresponding author for this report is A. Block, University of Hamburg,

Hospital Eppendorf, Department of Medicine, Martinistr 52, D-20246

Hamburg, Germany.

A search of the www.NewsRx.com online database using the terms "gene

therapy" and "colon" generated 124 articles.

Key points reported in this study are:

* A suicide gene therapy approach using adenoviral expression of bacterial

cytosine deaminase was evaluated in a metastatic colon carcinoma model

* Delayed tumor growth and reduced hepatic metastases resulted from systemic

administration of the therapy

* Murine cells were more sensitive to 5FU than were the human cells; however,

the human cells were more sensitive to viral transduction

This article was prepared by Cancer Weekly editors from staff and other

reports.

Copyright 2000, Cancer Weekly via NewsRx.com.

Article A62329169

Charles W. Henderson

New research from Children's Hospital of Columbus, Ohio, reports that safe

transfer of healthy genes in the womb may help treat genetic diseases such as

Tay-Sachs disease, a deadly birth defect which leads to the destruction of the

central nervous system, prior to the onset of long-term damage.

The study was presented at the 2000 Pediatric Academic Societies and American

Academy of Pediatrics Joint Meeting on May 14 by Children's researcher Bruce

Bunnell, PhD.

More specifically, the pre-clinical study concluded that healthy genes can be

safely administered to animal fetuses in the womb by transferring the healthy

genetic material through a retroviral vector system, or non-disease causing

virus, directly to the damaged cells. The results were seen pre-gestation, during

gestation, and after the fetus was born.

"Treating genetic diseases in the womb improves the likelihood that the new

genes will be absorbed and do their repair work since fetal cells divide much

more rapidly than adult cells," explained Bunnell. "In the past, a lot of attention

has centered around the safety of transmitting these cells, as the risk of

mutations increases and partial cures are often expressed. Our research shows

the transmission of foreign genetic material through a retroviral vector can

induce many fast-dividing fetal cells to take up the gene and help the fetus to

overcome symptoms of its inherited deficiency or disease."

The viral vector allows the healthy genes to enter the cells, while the protein

from the vector allows it to be expressed, and the body can begin to replace the

damaged genes with new, therapeutic ones. According to the research, blood,

tissue and marrow samples collected throughout the study indicate that the

positive results are not only expressed while the fetus is in gestational stages but

also after birth. The study is in its first stage, as researchers have yet to

determine the long-term effects of the transfer, as well as determining if the

gene transfer can occur in the reproductive cells.

According to the National Human Genome Research Institute, there are

approximately 4,000 genetic diseases known worldwide, including hemophilia,

cystic fibrosis, celiac disease, and muscular dystrophy. The absence or

inappropriate presence of a natural protein causes many human genetic diseases.

Today, gene therapy is the ultimate method of protein delivery where the

delivered gene enters the body's cells and turns them into small "factories" that

produce the therapeutic protein needed for specific diseases.

Bunnell said: "Our study is the first of its kind to illustrate safe and effective

genetic transfer techniques on a subject closest to resembling a human. It is our

hope that we can help give those affected by genetic disorders a second chance

by catching the disease in the womb and eliminating cellular and tissue

damage."

This article was prepared by Gene Therapy Weekly editors from staff and other

reports.

Copyright 2000, Gene Therapy Weekly via NewsRx.com.

Article A62204648

Age-related deterioration in critical brain networks may be restored by gene

therapy, according to a study in monkeys presented at the American Academy

of Neurology's 52nd Annual Meeting in San Diego, California, April 29 - May

6, 2000.

This finding lends support to a study just underway to treat Alzheimer's disease

using a similar gene therapy approach, say the study's authors.

Researchers from the University of California in San Diego found that normal

aging in monkeys causes a 28% decline in the density of certain brain networks

originating from nerve cells called neurons deep in the brain.

The scientists found that they were able to restore these connections by

transplanting brain cells genetically programmed to release a protein called

"nerve growth factor."

"It would be inappropriate to suggest that this approach could be used to treat

the course of normal aging, but it is not a far stretch to suggest that this may be

useful in the treatment of Alzheimer's disease," said Mark Tuszynski, MD,

PhD, a researcher at the Center for Neural Repair at UCSD and principal author

of the report. "Indeed, we are now beginning clinical trials to determine whether

nerve growth factor gene therapy will be useful in combating Alzheimer's

disease in humans."

Nerve growth factor nourishes neurons and allows brain cells to grow and

maintain fibers called axons that link neurons in one area to neurons in other

areas of the brain.

In previous studies, Tuszynski's group found that the normal aging process

involves atrophy and a loss of function within a particular set of brain cells deep

in the brain known as cholinergic neurons. These cells, which are connected by

axon connections to the outer layers, or cortex, of the brain, are believed to be

critical to many of the memory and other mental functions that deteriorate

gradually and to a mild degree with age, but much more rapidly and severely in

Alzheimer's disease. Indeed, other researchers have shown that the cholinergic

neurons are particularly hard hit by Alzheimer's disease.

Tuscynski's group measured the density of cholinergic axons in the cortex,

comparing the brains of normal aged monkeys with aged monkeys that had

received transplants of brain cells engineered to produce large amounts of nerve

growth factor.

"We show that we can reverse these age-related losses of connections in the

cortex by delivering nerve growth factor to cell bodies deep within the brain,"

said Tuszynski.

The next steps, he said, are to determine whether nerve growth factor gene

therapy actually improves mental function in aged monkeys and to proceed with

clinical trials to determine if this therapy is safe and effective in humans with

Alzheimer's disease.

This article was prepared by Gene Therapy Weekly editors from staff and other

reports.

Copyright 2000, Gene Therapy Weekly via NewsRx.com.

Article A62949979

Researchers in France have developed a method of gene therapy to treat human

severe combined immunodeficiency (SCID) X1, a life-threatening disease

inherited on the X chromosome.

Usually, patients with SCID are forced to live within tightly controlled and

sterile "bubbles" to avoid any threats to their nonexistent immune systems until

a bone marrow transplant is attempted. The new therapy was described in the

April 28, 2000, issue of Science.

Two infants, aged eight and 11 months, were the beneficiaries of this treatment,

which provides a normal copy of the defective gene that causes SCID X1 that

quickly proliferates within the patient's body. The new gene "unblocks" the

development of other immune cells, restoring the immune system to normal

functioning. The infants' return to a normal immune system has lasted over 11

months without side effects, says study co-author Alain Fischer of the Hospital

Necker in Paris, France. The Science report notes that a third patient is

experiencing similar progress four months after the gene transfer.

The defective gene encodes part of a cell receptor that sends out signals to the

parents of T and NK cells, crucial components of the immune system that

destroy invaders and rally other immune defenses. Without this gene's

direction, these cells do not develop, grow, or spread, and SCID X1 patients

are left fatally vulnerable to even slight infectious insults to the body such as a

cold sore or common childhood diseases like chicken pox.

The researchers began the therapy by harvesting bone marrow from the patients

and sorting out a set of blood stem cells from the marrow. After bathing in a

growth factor in containers coated with a fibronectin fragment, a threadlike

protein that encourages efficient gene transfer, the cells were infected with a

retrovirus carrying the replacement gene. After three days of repeated infection,

the scientists transplanted the cells back into the patients without any prior drug

treatment. "It was important to show success in the absence of any

chemotherapy," explains Fischer.

As early as 15 days later, he and his colleagues detected new cells bearing the

correct version of the gene, along with rising numbers of fully functional and

diverse immune cells. Currently, the two patients have T, B, and NK cell

counts comparable to normal children of their age. The scientists also tested the

patients' rebounding immune systems with tetanus, diphtheria, and polio

vaccinations, and found that the infants produced the correct antibodies for

each.

Fischer believes that the key to the therapy's success lies "not in the technique,

but in the disease itself." In the SCID X1 cases, cells with the normal gene

seem to enjoy a significant selective advantage, multiplying rapidly until their

numbers overwhelm their mutated cousins. Researchers had an inkling of this

advantage when one faulty version of the gene spontaneously reversed itself in

a previous SCID X1 patient, leading to a lasting rally of the patient's immune

system. "This means that even a poorly efficient gene therapy technique - one

that only introduces a few cells with the right gene-may work as a treatment,"

says Fischer, who notes that this might bode well for the success of this therapy

for other genetically similar immune diseases.

According to the study, the two young SCID X1 patients have experienced

"striking" clinical improvements. No longer in protective isolation, they both

live at home without any treatment, enjoying normal growth and development

for their ages. Ideally, Fischer says, the children will be monitored for the rest

of their lives, both to ensure their continued health and to monitor the long-term

success of the therapy.

This article was prepared by Gene Therapy Weekly editors from staff and other

reports.

Copyright 2000, Gene Therapy Weekly via NewsRx.com.

Article A62951113

The promise of gene therapy--the replacement of dysfunctional genes with useful

ones--has gone largely unfulfilled because the microbial delivery agents used to

insert the desirable genes into needy cells haven't been up to the job.

Early in the research, scientists seized on viruses as ideal vectors to deliver genes

to patients since these microbes insert their genome into a host cell. However, the

agents have proved less than perfect. Viruses can be expensive to prepare and

store. Moreover, one of the most promising viruses isn't big enough to tote the

large genes required to overcome some troubling diseases.

More serious obstacles have also arisen. Even viruses that scientists have partially

disabled sometimes replicate, and the microbes can attract unwanted attention from

a patient's immune system.

A research team at Stanford University School of Medicine now reports success at

circumventing the viral approach altogether, while other groups are testing ways to

expand a virus's cargo capacity.

The Stanford work employs a transposon, or naturally mobile piece of DNA, as

the gene-delivery truck. Geneticist Mark A. Kay and his colleagues reasoned that a

selected gene delivery truck might be packaged into such DNA, which then could

easily insert itself into a patient's chromosome.

They performed experiments on more than 50 mice, some with hemophilia, a

disease in which the blood doesn't clot properly. The researchers sought to replace

a defective version of the gene for a coagulation protein called factor IX. Using

transposon DNA as a carrier for the functional gene, they implanted the whole

package into liver cells in the mice.

The transposon, which in this study consists of DNA engineered from a fish gene,

encodes an enzyme called transposase. Once produced, this enzyme attached the

coagulation-factor gene to the host chromosome. The transfer was successful in 5

to 6 percent of liver cells sampled, Kay and his colleagues report in the May

NATURE GENETICS.

Mice treated with the transposon gene therapy showed vastly improved blood

coagulation. It didn't seem to matter precisely where on the chromosome the gene

attached, Kay says.

The implanted genes have so far functioned correctly, directing the production of

factor IX for at least 5 months--a long time in the typical 2-year life span of a

mouse. Kay suggests that the gene might work indefinitely, which would make

such treatment essentially a cure.

"Our experience is that anything that integrates into the liver of a mouse lasts as

long as the mouse lives," he says. Now 8 months after the gene therapy, Kay has

still detected no immune backlash in the mice.

Hemophilia provides a good test for gene therapy. The absence of a single factor

can sabotage the body's ability to stanch bleeding. Correcting this genetic

abnormality yields clear results, Kay says.

Because of the problems of using viruses, any advance in nonviral gene therapy is

welcome, says virologist David T. Curiel of the University of Alabama at

Birmingham. Using a transposon to carry a gene is a "very significant

accomplishment," he says.

Molecular biologist Xiao Xiao of the University of Pittsburgh agrees that the

experiments are "a nice piece of work" but adds that the high volume of fluid that

the researchers pumped into the mouse veins may require that the method be

modified for use in people.

Meanwhile, three other studies address a problem nagging current gene therapy:

the inability of an otherwise ideal virus to carry large genes into a cell. All three

studies use recombinant adeno-associated virus (rAAV), a genetically engineered

virus incapable of replicating but able to deliver a selected gene. This virus is being

used in some ongoing trials in people.

Two of the studies split a gene from its promoter region, the nearby DNA that

switches on the gene. Two rAAV vectors then deliver the separate cargoes into

mouse cells, where the gene and its promoter reunite. Kay and his Stanford

colleagues in experiments described in the May NATURE BIOTECHNOLOGY

were able to deliver the gene for the enzyme betagalactosidase. In the May

NATURE MEDICINE, John F. Engelhardt and his team at the University of Iowa

in Iowa City reported successful transfer of the erythropoietin gene.

Taking another tack, Xiao and his colleagues split a large gene in two and used

rAAV to deliver the parts, one of which included the promoter. In mouse muscle,

the two pieces produce a complete protein. The transplanted gene encodes factor

VIII, another coagulation protein.

"These studies really expand the utility of rAAV," says Brian K. Kaspar, a

neurobiologist at the Salk Institute for Biological Studies in La Jolla, Calif. Cystic

fibrosis and a common form of muscular dystrophy--both of which stem from

defects in large genes--may also make good targets for these new technologies, he

says.

In gene therapy until now, "everybody was forced to work within certain gene size

limitations," says Richard Jude Samulski, a molecular virologist at the University

of North Carolina in Chapel Hill. "I think now they can approach [techniques

using rAAV] without that reservation."

However, these virus-loading methods may introduce new problems. For

example, splitting a promoter region from its gene and then trying to reunite the

two pieces might leave the promoter free to switch on another gene, with

unforeseen consequences, Samulski says.

All these methods will require animal testing "until they come up squeaky clean,"

he concludes.

Article A62791546

Researchers at Brown University have demonstrated that adenoviral vectors can

facilitate the delivery of an Escherichia coli purine nucleoside phosphorylase

(PNP)/fludarabine system in mice that shows potential for treating

hepatocellular carcinoma (HCC).

According to L. Mohr and colleagues at Brown, "The E. coli purine nucleoside

phosphorylase (PNP) converts purine analogs into freely diffusible metabolites,

which are highly toxic to dividing and nondividing cells."

Investigators analyzed the effect of PNP in three cell HCC cell lines: HepG2,

Hep3B, and HuH-7. PNP effects were compared with the effect of using

herpes simplex thymidine kinase (TK).

"The genes for PNP, TK, and enhanced green fluorescent protein (EGFP) were

delivered to HCC cells by identical adenoviral vectors," they said.

In addition, Mohr's group used fludarabine and ganciclovir (GCV) as

prodrugs, according to the protocol.

Study results indicated that fludarabine concentrations between 0.5 and 1

(micro)g/mL killed 100% of the cells which expressed PNP. There was no

toxicity indicated in the control cells that expressed EGFP, the researchers

reported.

"Expression of TK followed by GCV treatment produced a potent growth

inhibition but failed to kill all TK-expressing HCC cells," Mohr and colleagues

noted.

Finally, they determined that there was a highly effective bystander effect

created by PNP expression that was not seen in TK expression.

In tests on nude mice, the PNP/fludarabine adenoviral delivery system

prevented new tumor formation and was therapeutic for established tumors,

Mohr et al. stated.

"These results demonstrate the potential of the PNP/fludarabine system for the

treatment of HCC," they concluded ("Gene therapy of hepatocellular carcinoma

in vitro and in vivo in nude mice by adenoviral transfer of the Escherichia coli

purine nucleoside phosphorylase gene," Hepatology, 2000;31(3):606-614).

The corresponding author for this study is J.R. Wands, Brown University,

Liver Research Center, 55 Claverick Street, Providence, Rhode Island 02903,

USA.

Key points reported in this study are:

* Escherichia coli purine nucleoside phosphorylase, when delivered by an

adenoviral vector into hepatocellular carcinoma cells treated with fludarabine,

shows promise as a system for treating cancer

* Purine nucleoside phosphorylase expression causes a bystander effect that

effectively kills adjacent cancer cells treated with fludarabine

* Purine nucleoside phosphorylase expression is not toxic to cells that have not

been treated with fludarabine

This article was prepared by Gene Therapy Weekly editors from staff and other

reports.

Copyright 2000, Gene Therapy Weekly via NewsRx.com.

Article A61796868

BECAUSE OF THE ADULT CENTRAL NERVOUS SYSTEM'S (CNS's)

limited ability to repair itself following traumatic injury, spinal cord injuries can

be devastating, and the prospects for recovery are generally grim. However, the

observation that a few regions in the CNS continue to produce neurons

throughout life offers exciting prospects for repairing an injured spinal cord.

Considerable progress has been made in developing efficient methods for

culturing the neural stem cells of rodents, genetically modifying them to

produce therapeutic genes, and transplanting them into animal models of brain

diseases. These same gene therapy and grafting methods are now being pursued

for restoring function following traumatic spinal cord injury.

Neural Stem Cells

Stem cells are multipotential cells that have the capacity to proliferate in an

undifferentiated state, to self-renew, and to give rise to all the cell types of a

particular tissue. [1] In the developing embryo, neuroepithelial cells of the

neural tube generate a variety of lineage-restricted precursor cells that migrate

and differentiate into neurons, astrocytes, and oligodendrocytes (FIGURE 1).

[2] CNS stem cells have now been discovered in the human CNS and appear to

behave similarly to their rodent counterparts. [3] These stem cells could

potentially be used to promote neurogenesis following injury and disease.

Transplantation studies have demonstrated that neural stem cells and precursors

have the capacity to alter their fate in response to the environment into which

they are reintroduced and to integrate appropriately with the host tissue. [4]

Neural stem cells can be isolated from different areas and propagated for long

periods in culture without losing their multi-potentiality. Thus, when

transplanted back into the CNS, these stem cells have the capacity to migrate, to

integrate with the host tissue, and to respond to local cues for differentiation.

Transplantation of Stem Cells

Neural stem cell grafts have been studied in a variety of animal models. One

application involves grafting neural stem cells into a specific area of

degeneration to replace a missing or deficient product. For example, in an

animal model of Parkinson disease, precursor cells grafted into the striatum can

replace degenerated dopamine-producing neurons in the nigrostriatal pathway

and promote limited functional recovery. [5] Grafts of neural stem cells may

also be effective in cases of widespread neural degeneration. For example, in a

genetic model of demyelination, both the pathology and symptoms can be

reversed by transplantation of neural stem cells into the cerebral ventricles at

birth. [6] The grafted stem cells migrate extensively throughout the brain,

integrate into the host cytoarchitecture, and correct the myelination process

during subsequent developmental stages.

Grafted neural stem cells could potentially replace cells lost to injury,

reconstitute the neuronal circuitry, and provide a relay station between the

injured pathways above and below the lesion. Furthermore, intraspinal stem cell

transplants can be genetically modified to provide therapeutic factors that

prevent cell death and promote regeneration.

Cells to be transplanted into the injured spinal cord need to be readily obtained,

easily expanded and stored, and amenable to genetic modification. They should

also be able to survive for extended periods within the injury site, to integrate

with host tissue, to rescue injured neurons from cell death and atrophy, to

promote axonal regeneration, and, ultimately, to restore function. Neural stem

cells and neural precursors theoretically fit many of the above requirements; the

challenge is to demonstrate their efficacy and safety for clinical applications.

Spinal Cord Repair

Among the most promising sources of cells for spinal cord repair are

neuronal-restricted precursors (NRPs) derived from the developing spinal cord.

These cells can be expanded in vitro and have the potential to differentiate into

numerous neuronal types (FIGURE 1), including motoneurons . [7] In the ex

vivo modality of gene therapy, therapeutic genes are introduced into cultured

cells that are subsequently transplanted into the CNS. Researchers in our

laboratory, in collaboration with Mahendra Rao, MBBS, PhD, at the University

of Utah School of Medicine, are studying the developmental potential of NRP

cells and plan to use the ex vivo approach to examine the therapeutic potential of

these cells grafted into a rat model of spinal cord injury. Preliminary

observations demonstrate survival of grafted NRP cells in the lesion site for at

least 1 month (FIGURE 2).

Genetically modified stem cells have not yet been grafted into the injured spinal

cord; however, transplantation of brain-derived neurotropic factor-producing

fibroblasts has been carried out in our laboratory using a rat spinal cord injury

model of partial cervical hemisection. These grafts resulted in long distance

regeneration of axons from brainstem neurons and partial recovery of motor

function. [8] Ongoing experiments with genetically modified fibroblasts are

examining the effects of other growth factors, as well as adhesion molecules

and growth-associated genes.

Conclusion

Transplantation of neural stem cells and precursor cells together with gene

therapy offers great promise for spinal cord repair. Specific research goals

include improving neuronal survival, promoting functional recovery through

axonal regeneration, compensating for demyelination, and replacing lost cells.

[9] Many issues will need to be resolved before stem cells can be considered for

use in human subjects, but continued basic research on the properties of these

cells and development of appropriate animal models of repair will pave the way

for successful clinical application.

Acknowledgment. Preliminary results from research reported here were done in

collaboration with Mahendra S. Rao, MBBS, PhD, and Stella Y. Chow, PhD.

We thank Marion Murray, PhD, Alan Tessler, MD, and Alfred Kim for their

comments on the manuscript.

Corresponding author: stevehan@drexel.edu.

REFERENCES

(1.) Morrison SJ, Shah NM, Anderson DJ. Regulatory mechanisms in stem cell

biology. Cell. 1997;88:287-298.

(2.) Rao MS. Multipotent and restricted precursors in the central nervous

system. Anat Rec. 1999;257:137-148.

(3.) Svendsen CN, Caldwell MA, Ostenfeld T. Human neural stem cells:

isolation, expansion, and transplantation. Brain Pathol. 1999;9:499-513.

(4.) Suhonen JO, Peterson DA, Ray J, Gage FH. Differentiation of adult

hippocampus-derived progenitors into olfactory neurons in vivo. Nature.

1996;383:624-627.

(5.) Studer L, Tabar V, McKay RD. Transplantation of expanded

mesencephalic precursors leads to recovery in Parkinsonian rats. Nat Neurosci.

1998;1:290-295.

(6.) Yandava BD, Billinghurst LL, Snyder EY. "Global" cell replacement is

feasible via neural stem cell transplantation: evidence from the dysmyelinated

shiverer mouse brain. Proc Natl Acad Sci U S A. 1999;96:7029-7034.

(7.) Kalyani AJ, Piper D, Mujtaba T, Lucero MT, Rao MS. Spinal cord

neuronal precursors generate multiple neuronal phenotypes in culture. J

Neurosci. 1998;18:7856-7868.

(8.) Liu Y, Kim D, Himes BT, et al. Transplants of fibroblasts genetically

modified to express BDNF promote regeneration of adult rat rubrospinal axons

and recovery of forelimb function. J Neurosci. 1999;19:4370-4387.

(9.) Murray M. Therapies to promote CNS repair. In: Ingoglia N and Murray

M, eds. Nerve Regeneration. New York, NY: Marcel Dekker; 2000. In press.

Mag.Coll.: 102K6107

Article A61947528

Charles W. Henderson

Whether you are a weekend athlete or a sports professional, most people

eventually are faced with an injury. Traditional treatments of surgery or a cast

followed by lengthy rehabilitation are being replaced with gene therapy that

reengineers damaged muscles, cartilage, and ligaments.

During a media briefing at the 67th annual meeting of the American Academy of

Orthopedic Surgeons, a panel of orthopedic surgeons explained the new research

on gene therapy and tissue engineering. Moderated by Freddie Fu, MD, the panel

included Jay Lieberman, MD; Johnny Huard, PhD; and Jacques Menterey, MD.

The Academy held its annual meeting, March 15-19, 2000 in Orlando, Florida.

The term "sports injury" conjures up thoughts of professional athletes being

helped off the field during the big game. In reality, most sports injuries involve

less famous people participating in activities like an informal softball game with

friends after school or an organized football league with co-workers. Between

10% and 55% of all injuries are sustained in sports-related incidents.

In addition, the longer, healthier lives of average Americans also means that

orthopedic surgeons treat a large number of senior citizens. An estimated 53,000

people age 65 and older were treated in U.S. hospital emergency rooms in 1996.

That's a 54% increase from 1990.

The basic treatment for many injuries is called "R.I.C.E." which stands for Rest,

Ice, Compression and Elevation. Treatments for more significant injuries include

surgery, strengthening exercises, support braces or a cast, and medications. Many

of these treatments result in scar tissue in the injured muscle. Serious injuries may

result in cartilage defects that require a total joint replacement.

"We're using basic scientific research to develop new orthopedic therapies that use

genes, stem cells, and tissue engineering to create improved healing in sports

injuries of all kinds," said Huard. "With a typical muscle injury, muscle

regeneration begins within two weeks. Often the healed muscle has a lot of scar

tissue that causes complications for professional and recreational athletes. Our

research shows that by adding gene therapy to the treatment program, muscles heal

with less scar tissue, creating a near complete recovery of the muscle."

There are three stages of the healing process of muscles. The first is the

destruction phase in which swelling, bruising, and pain take place. In the second,

or repair phase, regeneration of the muscle and blood vessels begins and scar

tissue is developed. The final remodeling phase occurs when the regenerated

muscle matures and contracts into its final form.

The development of improved methods of transferring genes into cells has created

new options to aid in the healing capacity of musculoskeletal tissues like muscles,

ligaments, and cartilage.

Gene therapy also has the potential to revolutionize the treatment of bone loss

problems related to fractures that don't heal, hip and knee replacements, and spinal

fusion. Orthopedic surgeons use cells harvested in a patient's body, and the

modified cells are placed back in a particular area of the body to create a biological

response in that site. Once the gene is in place, it produces a response that

stimulates the healing process.

"The more we understand about the biology of bone formation, cartilage repair,

and tendon healing, the more we will be able to develop the appropriate tissue

engineering and gene therapy strategies to treat specific injuries," said Lieberman.

"Gene therapy has the potential to allow orthopedic surgeons to harness or

stimulate the body's inherent healing potential."

While gene and cell therapies are still in the research stages, researchers expect it to

be available for use as a standard treatment within a decade.

This article was prepared by Gene Therapy Weekly editors from staff and other

reports.

Copyright 2000, Gene Therapy Weekly via NewsRx.com.

Article A61374545

Scientists have created strains of mice that can eat a high-fat diet without getting

chubby using a single gene that might lead to a new obesity treatment for

people.

In its normal form, the gene, called HMGIC, apparently helps mice make more

cells to store fat when they have been eating a fatty diet, researchers said. But

the mice in the experiment had a defective version of the gene. They apparently

failed to create storage cells in response to the high-fat diet, and so avoided

putting on weight, the researchers said in the April 2000 issue of the journal

Nature Genetics.

The finding could lead to a human obesity treatment if scientists can find a drug

that interferes with the effect of the normal HMGIC gene, said Kiran Chada, a

biochemistry professor at the University of Medicine and Dentistry of New

Jersey and senior author of the paper. He is also president of a company formed

to develop products related to the gene.

Dr. Bradford Lowell, an obesity expert at Harvard Medical School, said it will

take a lot more work to find out how promising Chada's approach would be for

humans.

Dr. Rudy Leibel, an obesity expert at Columbia University in New York, said if

a person's supply of fat-storing cells were restricted, fat might build up in the

liver instead. That could seriously interfere with liver function, he said. Chada

said he has seen no sign of fatty liver buildup in the mice.

Mice born with the genetic defect develop only about 10% of the normal amount

of body fat but are otherwise normal, Chada said. Mutant mice that ate a

high-fat diet for six months didn't put on any more weight than mutants that ate

a standard diet. Normal mice, in contrast, did become obese on the high-fat

diet. All three groups of mice ate about the same amount.

This article was prepared by Gene Therapy Weekly editors from staff and other

reports.

Copyright 2000, Gene Therapy Weekly via NewsRx.com.

Article A61374552

Harvard Health Publications Group

Until recently, one of the most durable assumptions in neuroscience had been

that brain cells (neurons) are incapable of renewing themselves and that aging

brings with it irreversible neuronal loss and inevitable memory problems.

Fortunately, neuroscientists began putting sizeable dents in this dogma in the

1990s with new research showing not only that the brain is continually making

new cells but also that it is possible to renew brain cells that have begun to

atrophy. One of the most exciting potential treatments to come out of this

research is about to be tested in California.

Scientists at the University of California at San Diego are in the process of

carefully recruiting a small number of patients with evidence of early

Alzheimer's disease for a phase I trial of gene therapy to restore brain cell

activity. Doctors will inject cells genetically engineered to produce human nerve

growth factor (NGF) into the brains of the trial subjects. The goal is to prevent

the death of neurons affected in Alzheimer's disease and improve the function

of others.

The experimental treatment builds on previous extensive work in animals. Mark

Tuszynski, M.D., Ph.D., and colleagues reported in the Sept. 14, 1999,

Proceedings of the National Academy of Sciences on a study in which cells

secreting NGF were injected into the brains of normal rhesus monkeys with

evidence of age-related neuronal atrophy. The brain area involved - the

subcortex - regulates memory and attention via the neurotransmitter

acetylcholine. Decline in this cholinergic system can occur with normal aging,

and is seen extensively in the brain tissue of Alzheimer's patients. The

researchers discovered a 43% rate of decline in subcortical cholinergic neurons

in aged monkeys and were able to reverse this loss almost completely by

treatment with the genetically altered cells.

Although many questions remain about activity in the subcortical region and

how it actually affects cognitive functioning, the next step, say the researchers,

is to study this treatment in humans, since no animal models for Alzheimer's

disease exist. The Recombinant DNA Advisory Committee, an advisory board

to the National Institutes of Health, has reviewed the patient trial. The phase I

study will enroll only eight patients.

Article A61456849

2000 Benjamin Franklin Literary & Medical Society, Inc.

Dr. Keith March of the Krannert Institute of Cardiology in Indianapolis is invest/gating

whether genes injected into the pericardium (the fluid-filled sac around the heart) might

stimulate growth of new blood vessels where they are needed.

"Placing DNA in the pericardial sac allows a soaking from the outside into the vessels,"

explains Dr. March. "There is no blood flow in that space, so there is plenty of time for

access into the tissue." Data from their animal studies may help identify the best way to

use gene therapy for people with coronary artery disease.

Dr. March, director of the Indiana Center for

Vascular Biology and Medicine, is also associate

professor of medicine at the Indiana University

School of Medicine. He was interviewed

following his return from a conference on

cardiac gene and molecular therapy in Geneva,

Switzerland.

Q: What is the focus of your research?

A: For some time, one of the real issues with

gene therapy has been to identify the proper and

most efficient way to deliver the genes you want

to insert into the heart or into the blood vessels.

One approach--that used by Dr. Jeffrey Isner--is

direct injection into the myocardium.

We have been pursuing a similar approach. We

have been looking very carefully at the

possibility of introducing--or transducing--genes

into the membrane that surrounds the heart. This

membrane, called the pericardium, surrounds

everyone's heart unless they have had a bypass

surgery.

There is a small volume of fluid in this

membrane in which the heart is always "taking a

bath." Over a long period of time, then, the

whole heart would take a bath in the gene products made by the pericardium.

Q: Will that cause new blood vessels to grow around the heart?

A: We think biologically, at this point, that those areas which need the growth factors will

use them.

Q: Are you using naked DNA to transport the new genes into the body, like that used at

St. Elizabeth's?

A: We have been interested in using viruses and nonviral

approaches. Nonviral is essentially along the lines of

"naked DNA." No proteins of a virus are added to help

the DNA get into cells. On the other hand, the viral

approach, to date, has been more efficient.

"I have plenty of go power since I had the gene implants.

I can run 24 hours a day if I want to, and it doesn't bother

me. I have never been back to a doctor besides Dr. Isner

for a checkup. My last checkup was several months ago.

Since the gene implants, I haven't had a problem. I ran

combines all fall for about 30 days. We started at about

10:00 a.m. and ran until 2:00 the next morning."

--Floyd Stokes, Texas peanut farmer and Dr. Isner's seventh patient to undergo the gene

therapy treatment at St. Elizabeth's.

In our work on the pericardial sac, we have worked with viruses--adenoviruses--and

found that we can transduce large numbers of the cells within the sac extremely easily.

However, we have also found that we can transduce the pericardial sac, although less

efficiently, with the plasmid vectors.

In a way, that is important. It only takes a few of these cells or "soldiers" to affect a large

area of tissue--or, as it were, a little bit of yeast to leaven the bread.

Gene Therapy for Heart Failure?

Q: In addition to facilitating the growth of new blood vessels around heart blockages,

how else might gene therapy someday help people with heart disease?

A: Gene therapy might also be useful in heart

failure. If we could find ways to give genes to

the heart to help it contract more efficiently and

effectively, despite the fact that some of the heart

muscle has already died, we would be able to

treat those patients.

Another area of interest in gene therapy is the

prolongation of the longevity of bypass grafts by

pre-treating bypass arteries and veins with

beneficial genes before they are implanted.

Researchers are also interested in the possibility

of using coronary gene therapy to help the heart

muscle regrow even after cell injury or death

from a heart attack. If we could introduce genes

that would signal the heart muscle cells to grow

back, we could possibly fix a heart that has

already had a heart attack.

In addition, perhaps those earlier in their disease

course will be able to benefit from it.

Q: We hear that gene therapy trials for other

diseases are having problems. Are we having

problems in coronary gene therapy, too?

A: To the best of my knowledge, no substantial clinical toxicity due to the gene therapy

has been noted in any of the coronary gene therapy trials. Although we have had some

news recently of difficulty with certain gene therapy trials, I am not aware of any problem

in any situation where the coronary gene therapy approach can be linked to difficulties a

patient has had.

RELATED

ARTICLE:

HOPING

FOR

CONGRESSIONAL

HEARING

Rev.

Charles

Wilson

(Feb.

2000 MU)

has called

his

congressional

representative,

Sue

Myrick.

Denyse

Trejo is

doing

research

on

coronary gene therapy in the North Carolina representative's office. We hope that if

enough representatives and senators can hear about Dr. Isner's work, a congressional

hearing will be called.

Call your representative, or you can contact Representative Myrick and her assistant at

this address:

Rep. Sue Myrick 230 Cannon House Office Building Washington, D.C. 20515

Telephone: 202-25-1976 Fax: 202-225-3389 E-mail: myrick@mail.house.gov

Article A61931783