THE FUTURE OF GENE THERAPY FOR NEUROMUSCULAR DISORDERS

P. Joel Ross and Robin J. Parks

Molecular Medicine Program, Ottawa Health Research Institute and Department of Biochemistry, Microbiology, and Immunology and Centre for Neuromuscular Disease, University of Ottawa, 501 Smyth Road, Ottawa, ON Canada

Correspondence: rparks@ohri.ca

INTRODUCTION

Our health depends upon the proper functioning of every cell in our bodies; in order to work properly, our cells need correct instructions. All of the instructions required for the development of a human being, from one cell to a full-grown adult, are written in our genes. When genes that encode incorrect information are inherited (or arise spontaneously), genetic disorders can occur. Currently, genetic disorders cannot be cured; their symptoms can often be controlled, but cures are out of reach.

Many neuromuscular disorders arise as a result of the functional loss of genes encoding information important for muscles, the brain, or communication between the two. Conceptually, the best way to cure these disorders is to replace the missing gene. This is the rational basis of an emerging technology, called gene therapy, which offers hope to sufferers of heritable neuromuscular disorders.

There are four primary goals of this article. We intend to explain to patients, their friends, and their families: (1) how neuromuscular disorders can result from differences in genetic information; (2) how gene therapy may be used to treat these disorders; (3) exactly how this may be accomplished using current technology; and (4) what progress has been made towards the goal of treating neuromuscular disorders using gene therapy.

Much recent effort within the scientific community has focused upon the treatment of Duchenne muscular dystrophy (Duchenne muscular dystrophy), a relatively common and severe disorder. Therefore, much of the available literature (which is summarized here) focuses upon this disorder. For the sake of simplicity, Duchenne muscular dystrophy will be used as a model of the how genetic disorders arise, and how gene therapy can treat them. However, much of what is discussed can be applied to the treatment of numerous other genetic disorders, including several other forms of neuromuscular disease, such as Becker muscular dystrophy, limb girdle muscular dystrophy, spinal muscular atrophy, amyotrophic lateral sclerosis, etc.

GENES AND DISEASE

In each of the millions of cells that make up the tissues and organs of our bodies, there is a chemical called deoxyribonucleic acid (DNA), which stores all of the information for our growth, development, and maintenance. Within our cells, our DNA is packaged into structures called chromosomes. Portions of DNA that encode sufficient information to direct the production of a single building block (usually a protein) of our cells are called genes. Each chromosome contains thousands of genes.

As we grow, our cells need to replicate. When a cell makes a new copy of itself, the DNA must also be copied so that each "daughter" cell contains a full catalogue of chromosomes and genes. However, errors sometimes occur during duplication of the DNA. These errors are called mutations. If a mutation occurs in a gene encoding a protein, the protein could be changed or, even worse, not produced at all. Our cells depend upon proteins to keep things running smoothly, so mutations can have very serious side effects.

DUCHENNE MUSCULAR DYSTROPHY

Duchenne muscular dystrophy (Duchenne muscular dystrophy) is a relatively common and severe neuromuscular disorder that afflicts one in every 3500 boys (Duchenne muscular dystrophy is an “X-linked” 1 disorder, like colour-blindness, and is therefore very rare in girls). Duchenne muscular dystrophy is characterized by progressive muscle weakening, which forces patients into wheelchairs between the ages of eight and twelve; patients typically succumb to the disorder in their mid to late twenties due to failure of crucial muscles in heart or the diaphragm.

1 X-linked refers to the fact that the gene that is missing in patients with Duchenne muscular dystrophy, dystrophin, is found on the X chromosome. Women have two X chromosomes, while men have only one (men have what is called a Y chromosome; the presence of a Y chromosome is what makes men men). Since men have only one X chromosome, they have only one dystrophin gene . Therefore, if men encode an incorrect copy of the gene, they have no second gene to compensate for it, while women do.

Figure 1. This diagram depicts muscle structure and where dystrophin resides within muscle. There are numerous muscle groups within the human body (in bright red on the person to the left of the picture). All muscles are made of muscle cells; each muscle is basically the same at the cellular level. To the right of the picture, we see that muscles are tightly packed collections of muscle fibre bundles. Each muscle fibre is an individual cell, and is surrounded by a membrane. This membrane separates the inside of the cell from the outside. Dystrophin sits just inside of the membrane, and anchors structural proteins on the inside of the cell to proteins on the surface of the cell (which sit within the membrane). Although not depicted in this diagram, the proteins in the fibre membrane bind to important structural proteins that lie outside of the cell. All of these proteins must work together to ensure the structural integrity of muscle cells. Without dystrophin, muscle cells are highly susceptible to muscle damage. Used with permission of the Muscular Dystrophy Association.

These symptoms of Duchenne muscular dystrophy are the clinical manifestation of mutations in the gene encoding the protein dystrophin. Dystrophin is a very large protein that acts as an anchor for numerous structural proteins that help to stabilize muscle (Fig. 1). Mutant copies of the dystrophin gene can be inherited from the mother or can arise spontaneously (which occurs in approximately one third of cases). Mutant genes that give rise to Duchenne muscular dystrophy often encode a shortened version of dystrophin that is unable to stabilize muscle. Without dystrophin, muscle cells are highly susceptible to damage due to daily wear and tear. Damaged muscle cells undergo a specialized form of programmed cell death; this results in muscle degeneration. After degeneration, the muscle tries to rebuild, or regenerate, itself. The pathology associated with Duchenne muscular dystrophy is the outcome of a constant cycle of degeneration and regeneration in the muscles of patients. During this process, more degeneration than regeneration occurs, and much of the muscle is slowly replaced by fat and fibrous tissue (neither of which is able to compensate for the loss of muscle). Also, muscle cells are not immortal. Eventually, cells are unable to produce daughter cells, and subsequently undergo a form of “genetic death” called senescence. When the muscle is replaced with too much fibrous tissue or fails to regenerate at all, it wastes away.

Gene Therapy

Gene therapy is any disease treatment-regimen that uses genetic information as a therapeutic. The rational basis of gene therapy, as mentioned above, is that the only way to cure a genetic disorder that results from the loss of a crucial gene, is to supply a good copy of that gene.

However, gene therapy is easier said than done: DNA is not easy to deliver to human cells. DNA cannot be swallowed like a pill, as it is degraded in the harsh environment of the stomach. And even if the DNA is not degraded, it would probably not find its way to muscle, where dystrophin expression is required for correction of the disorder.

So, how can a functional copy of dystrophin be delivered to muscle cells? Scientists have great interest in using viruses to accomplish this goal. Viruses consist of nothing more than genetic material (DNA or a highly similar molecule called RNA) packaged in some sort of molecular “shell” (called a virion; often made of protein and/or lipids). The virion serves to both protect the genetic information within and to shuttle it into a cell. Viruses can therefore be used as “vectors” to deliver therapeutic genes to cells. (The term vector is simply used to describe anything that acts as a carrier).

Viruses are experts at delivering genetic material to human cells, as the life cycle of Adenovirus (Ad), a DNA virus that is used in gene therapy. The steps of a natural virus infection are as follows: (1) the virus binds to the outside of a cell, and tricks this cell into letting it inside; (2) once inside, the virus convinces the cell that it should be trafficked to the nucleus (the information centre of the cell, where all of the DNA is located); (3) once it reaches the nucleus, the virus is able to have its genes read, and the proteins that these genes encode are made; this is all accomplished using cellular machinery (for future reference, this process, of turning-on genes, is referred to as “gene expression”); (4) these viral proteins are involved in hijacking the cell, making many copies of the viral DNA (up to

10,000 new copies of the viral DNA in a single infected cell), and packaging that DNA into new virions; (5) once enough viruses fill the cell, it dies and the viruses are then able to escape and infect new cells. For a visual representation of this life cycle, please refer to Figure 2.

USING VIRUSES TO DELIVER THERAPEUTIC GENES

How do you turn a disease-causing virus into a therapeutic gene-delivery vehicle? As described above, viral DNA contains genes that interfere with our cells and help the virus to replicate. Viral genes are therefore potentially dangerous. The easiest way to both attenuate the virus so that it does not make the patient ill, and to get the virus to deliver a therapeutic gene, is to simply exchange the bad DNA for the good DNA. For example, in the case of Duchenne muscular dystrophy, the viral genes are replaced with DNA encoding dystrophin (Fig. 4).

The necessary modifications described above are made by cutting and splicing the viral DNA (Fig. 5). Molecular biologists have many tools and techniques available for cutting DNA at specific, predictable sites, and then later sticking two pieces of DNA together. Using these techniques, the viral genes are easily removed and replaced with a therapeutic gene.

Once this modified DNA is constructed, scientists are able to trick viral proteins into packaging that DNA. The result is a virus that contains no bad viral genes, but which contains a therapeutic gene, such as dystrophin. The modified virus will still be able to reach the nucleus of a human cell, just like a regular virus. However, the DNA that is delivered to the nucleus may help people with Duchenne muscular dystrophy.

Figure 2. The adenovirus life cycle. The adenovirus life cycle begins with Ad binding to the outside of the cell (1). This triggers internalization of the virus; the virus is subsequently trafficked to the nucleus, where its DNA is released(2). The viral DNA encodes genes that are read by cellular proteins to produce viral proteins that take over the host cell. These proteins change the host cell environment so that it is more hospitable to the virus. These proteins also replicate and package the viral DNA; the result is that many copies of the virus begin to fill the cell (3). Once many virus particles accumulate within the cell, the cell is killed and the virus is released (4). These new viruses are now free to infect another cell and start the life cycle again.

PRE-CLINICAL STUDIES

Once these viruses are made, they are not yet ready to be tested in patients. First, they must be tested in animal models of human disease. In the case of Duchenne muscular dystrophy, three models are currently available. These models are lines of mice, dogs, and cats that do not express dystrophin. The most commonly used model for most preliminary experiments is the mouse, which is called the mdx mouse. When further studies are required to verify results obtained in the mouse model, the dog is typically used. Few studies to date have used the cat model of Duchenne muscular dystrophy.

Although they do not look much like people, mice, dogs, and cats are biologically very similar to us. They have the same organs, bones, muscles, circulation, and immune systems. Most importantly, mice, dogs, and cats have nearly all of the same genes that we do, and they have nearly identical machinery for reading and copying DNA, and for making proteins. Therefore, these animal models can be used to test viral vectors to be sure that they are safe and effective before risking the well-being of a patient.

So far, Ad vectors encoding dystrophin have been tested in both the mdx mouse and in the dog model of muscular dystrophy. In both models, these vectors delivered dystrophin to muscle; the good copy of the gene was read to produce functional dystrophin proteins; and some correction of the health of the muscle was noted.

These initial promising results have not been improved upon over the past few years. Preclinical studies have shown that Ad vectors are unable to efficiently enter muscle cells. These studies have also shown that once inside the cell, Ad vectors are unable to sustain production of dystrophin for more than a few weeks. The root causes and potential solutions to these problems will be discussed in the section to follow.

LIMITATIONS OF AD VECTORS AND HOW THEY CAN BE OVERCOME

Pre-clinical studies have pointed to several key problems that must be fixed before Ad vectors can be tested in the clinic. The three major hurdles to successful gene therapy using Ad are poor entry into muscle, inability to infect every muscle cell, and immune responses. Although these barriers have significantly hindered gene therapy in pre-clinical studies, current research is focusing upon ways to improve effectiveness by modifying the virus or the ways in which the virus is delivered. These limitations and how they can be circumvented are discussed in detail below.

POOR ENTRY INTO MUSCLE

The first major limitation to successful gene therapy with Ad vectors to be identified in preclinical studies with mice is inefficient entry into muscle. The key to this problem is that muscle cells do not produce much of the receptor that Ad uses to gain entry into cells.

Figure 4. This picture diagrams the conceptual basis of using modified viruses to deliver dystrophin for gene therapy of Duchenne muscular dystrophy. Successful gene therapy depends upon a method for efficiently delivering DNA to diseased cells; viruses provide such a method. However, viruses contain genes that are not safe for delivery to human cells. Viruses can be made safer and more efficient for use in gene therapy by removing viral genes and replacing them with therapeutic genes, such as dystrophin. The result is a modified virus that packages the therapeutic DNA as if it was viral DNA. This modified virus can still efficiently deliver DNA to a cell, just like during a normal infection.

A great deal of current Ad research focuses upon changing the protein “shell” of the virus so that it is able to enter the cell by another means - one that is independent of the normal adenovirus receptor (Fig. 6 and 7). This can be accomplished in many ways. To date, one modification of the virus itself has been quite effective: this modification eliminates binding to the normal Ad receptor and simultaneously targets the virus to another receptor, which is more abundant in muscle (Fig.7).

INABILITY TO INFECT ALL MUSCLE CELLS OF THE BODY

The only way that Duchenne muscular dystrophy can be cured using gene therapy is if every muscle cell is infected by an Ad encoding dystrophin. However, this is seen as nearly impossible. At the very least, treatment of Duchenne muscular dystrophy would require good overall infection of muscles throughout the body, so that nearly every muscle group contains at least some healthy muscle fibres that are resistant to stress. This could best be achieved by intravenous, or "systemic”, injection of the virus; however, for Ad, this has proven to be problematic. There are two main reasons for poor results after systemic administration of Ad: Firstly, Ad has very poor stability in blood, although reasons for this instability are poorly understood; secondly, Ad tends to accumulate in the liver soon after systemic administration. Blood flows throughout the veins and arteries of the body, and eventually ends up in the blood's filter, the liver is unable to circulate with the blood to the rest of the body.

Hopefully, scientific breakthroughs in the years to come will improve Ad stability and improve overall circulation throughout the body, so that Ad can be delivered systemically in gene therapy studies in the clinic

Figure 5. This diagram depicts exactly how viral DNA can be modified in order to turn an Ad into a gene delivery vector. As long as appropriate DNA sequences exist on the ends flanking viral genes, DNA-cutting enzymes called restriction enzymes can be used to remove viral genes (1). Incubation of DNA with the appropriate restriction enzymes results in cleavage of DNA at specific, known sites (2). The remnants of the viral DNA, which do not contain any viral genes, can be incubated with DNA encoding the dystrophin gene (3), in the presence of DNA “glue”, called ligase. Ligase glues the dystrophin gene into the region that formerly contained viral genes (4). This modified AdDNA can now be packaged into Ad capsids, for delivery to muscle cells (5).

THE IMMUNE SYSTEM

Introduction to the immune system

The primary limitation to the use of Ad in gene therapy appears to be the immune system. Our bodies are equipped with systems for detecting micro organisms (such as viruses and bacteria), stopping the infection, and remembering the invader, so that it can be immediately recognized and destroyed if it appears again. This system is called acquired immunity, and it confers long-term defense against foreign invaders such as bacteria and viruses.

Acquired immunity depends upon two types of cells that circulate in the blood, called B-cells and T-cells. B-cells produce and secrete proteins called antibodies, which bind to foreign molecules. T-cells are able to recognize and kill cells that are infected by viruses or bacteria.

Pre-existing immunity to Ad

The first way in which the immune system limits Ad efficiency is by keeping the virus from ever infecting cells. Ad is a relatively common cold virus. Therefore, many people are naturally infected at some point in their lives; these people have pre-existing immunity to Ad. Circulating antibodies can bind to Ad and destroy the virus before it ever gets to a cell. In fact, mice with pre-existing immunity to Ad are not infected as efficiently as “naïve” mice that have never been exposed to Ad.

Pre-existing immunity is being addressed in the lab by modifying both the vectors and the ways in which they are delivered. So-called “stealth” Ads have been developed in which the virus is coated in a non-toxic chemical that hides viral proteins that may be recognized by the immune system. Also, physicians routinely perform procedures called apheresis, and this can be used to remove all circulating antibodies before administration of a gene therapy vector.

Figure 6. Adenovirus retargeting. Ad can be targeted to specific cell types by modification of the virus. Each cell has a membrane at its surface that separates the inside of the cell from the outside of the cell. This membrane contains proteins called receptors that recognize molecules that should be allowed into the cell, and internalizes them (A, B). Ad binds to a receptor that is abundant on liver cells (among many other cell types) (B), but is absent on the surface of muscle cells (C). Ad can be modified, so that it no longer binds to the normal Ad receptor, but rather, binds to are ceptor that is prevalent on muscle (D). These modified Ads are able to enter muscle, which they could not efficiently enter before being modified.

Immune responses to treated cells

Our immune system responds to any molecule that is deemed “foreign”. The immune system only knows what is foreign based on what it has and has not previously encountered. Since Duchenne muscular dystrophy patients do not produce full-length dystrophin, the protein encoded by the Ad vector is seen as foreign, and the cells producing this protein are targeted for destruction by T-cells. Several studies have shown that mdx mice treated with Ad vectors encoding dystrophin develop immune responses against the introduced protein. This severely limits the therapeutic potential of Ad vectors.

Long-term immunosuppression (which is a drug-induced, life-long weakening of the immune system) has been used to inhibit the development of immunity against dystrophin in mdx mice treated with Ad vectors encoding dystrophin. In one study, immunosuppression greatly enhanced long-term expression of dystrophin, and inhibited T-cell-mediated elimination of cells that were infected by the dystrophin- expressing Ad.

Figure 7. Modified viruses targeted to muscle are able to enter muscle cells much more efficiently. The viruses used to infect the cells shown above do not encode dystrophin; rather they encode a “reporter” gene called lacZ that causes infected cells to turn blue. Any white spot represents cells that have not been infected (although the individual white cells are difficult to see in this photograph). Clearly, normal, untargeted Ad does not efficiently enter muscle cells (left). However, the modified virus (right) is able to enter muscle cells with ease.

Even if T-cell responses to dystrophin can never be circumvented, an alternative measure exists. There is a gene encoded by our cells called utrophin. The utrophin protein is very similar to dystrophin, but is expressed in different parts of the body, and at different times during development. When expressed at high levels in muscle, utrophin is actually capable of compensating for dystrophin in mdx mice. However, since utrophin is not seen as "foreign" by the immune system, it can be delivered by Ad vectors without the risk of developing an immune response.

Although many scientists are pursuing utrophin-based therapies, it is still not certain that utrophin will provide the same long-term benefits to Duchenne muscular dystrophy patients as dystrophin. This is due to the fact that utrophin tends to localize to different parts of the muscle than does dystrophin. Also, utrophin is most important for communication between muscle cells and the cells of the nervous system, not for stabilizing muscle. Utrophin preferentially resides in a region called the neuromuscular junction, where signals are sent between muscle cells and neurons. Over-expression of utrophin to levels necessary for stabilization of muscle may therefore have as of yet unknown, detrimental effects on this communication.

Ringing the alarm bell

Ad readily infects cells called macrophages. The function of these cells is to patrol about the body, pick up protein wherever it can find it, and show these proteins to the cells of the immune system. Ad-infected macrophages announce loud and clear to the immune system that there is a foreign invader present in the body. It is hoped that some of strategies used to retarget Ad (such as that described above for improving muscle entry; Fig. 6) may also significantly reduce entry into macrophages, thereby preventing the macrophage from sounding the alarm.

Even if they are able to avoid circulating antibodies and macrophages and get to the muscle, Ad vectors are limited by the fact that they are highly inflammatory. This means that the mere act of getting into the cell indicates to that cell that there may be a foreign invader trying to start an infection. Due to the inflammatory nature of Ad, the infected cell produces “SOS” signals that attract and activate immune cells.

Recruitment of immune cells due to inflammatory signaling induced by Ad infections greatly enhances the probability that the patient will develop acquired immunity to dystrophin after delivery of the gene with an Ad vector. Interestingly, gene therapy studies using transient immuno-suppression (a short-term inhibition of the cells involved in the development of acquired immunity) have shown that immunosuppression may only be required immediately after vector administration, and that this effectively inhibits the long-term development of acquired immunity. This underscores the importance of elimination of the early inflammatory response induced by Ad for future clinical successes.

Many basic scientists are examining in detail exactly how Ad induces this potent inflammatory response. This research will provide the foundation upon which clinically-applicable developments will be built. Over the next few years, we expect to see great strides made in this area of research. Hopefully, this will result in the production of safer, more efficient Ad vectors.

OTHER VECTORS USED IN GENE THERAPY

This entire article has focused on Ad, because we see it as one of the vectors with the best potential for the treatment of neuromuscular disorders, particularly Duchenne muscular dystrophy. However, other viruses, and some non-viral methods, have been examined in similar pre-clinical trials.

Non-viral methods of delivery of DNA for the treatment of Duchenne muscular dystrophy are highly inefficient; therefore, very little time will be spent discussing them. Most non-viral methods of gene delivery involve coating DNA (that encodes dystrophin) with a lipid-based chemical, which is able to get the DNA into the cell. However, unlike DNA delivered by viral vectors, DNA delivered in this manner is not efficiently trafficked to the nucleus. These methods are safe, easy, and cost-effective, making them quite attractive. However, until chemicals can be developed that get DNA into the nucleus, these delivery systems will remain inefficient for gene therapy.

The herpes simplex virus type 1 (HSV-1), which causes cold sores in humans, is one of a few viruses, in addition to Ad, that have been explored in pre-clinical studies using animal models of Duchenne muscular dystrophy. HSV-1 is a very large virus, and has room within its substantial DNA to encode very large genes, like dystrophin. Therefore, this virus was of great interest in early gene therapy studies. However, HSV-1 is limited by two factors: it is highly inflammatory (even more-so than Ad) and it is very difficult to “disarm” by genetic manipulation to the point at which it is completely safe. For these reasons, many scientists have abandoned HSV-1, in favour of safer viruses.

The only virus that has received as much attention as Ad in the treatment of Duchenne muscular dystrophy is the adeno-associated virus (AAV). AAV is subject to some of the same limitations as Ad: it is unable to infect every muscle cell within the body after a single systemic administration and many people have pre-existing immunity to AAV. However, AAV has many advantages over Ad: it is not associated with any human disease; it does not induce a significant inflammatory response; it is very small, so it easily passes through the basal lamina (a protective sheath that surrounds each fibre within a muscle); it binds to a receptor that is abundant on the surface of muscle cells; and a recent study showed that after a single injection of a mouse, AAV was able to enter cells of every muscle group.

Despite these advantages, AAV is limited by one major shortcoming: it is very small, and therefore, cannot package the full-sized dystrophin gene. AAV can only be used to deliver condensed dystrophin genes, called “mini--genes” and even smaller genes, called “micro-genes”. There are large portions of dystrophin that are not completely necessary for the function of the protein. These portions can be removed without significantly altering the ability of dystrophin to stabilize muscle. The parts of the gene that encode these “unnecessary” portions of the protein can be removed; this makes the gene small enough to be delivered by AAV vectors.

Many studies have shown that mini- and micro-dystrophin genes can help to improve the symptoms of Duchenne muscular dystrophy in animal models. mdx mice that have been engineered to encode mini- and micro-dystrophin genes are much healthier than regular mdx mice. Also, mice treated with AAV encoding mini-dystrophin show improved health. It should be noted that the pathology of mdx mice is much milder than Duchenne muscular dystrophy in humans. Therefore, treatments that cure the symptoms of Duchenne muscular dystrophy in mice may not be sufficient to cure Duchenne muscular dystrophy in humans. These mini-genes have yet to be tested in the dog model of Duchenne muscular dystrophy, which is considered a more realistic model of Duchenne muscular dystrophy.

There may be limitations to the therapeutic effectiveness of mini-gene therapy of Duchenne muscular dystrophy. The original mini-gene was derived from a patient with a mild form of a disorder called Becker muscular dystrophy (BMD), which is similar to Duchenne muscular dystrophy, but is much less severe. BMD is the result of mutations in dystrophin that remove pieces from the central portions of the dystrophin protein (unlike Duchenne muscular dystrophy, which is the result of mutations that severely shorten the dystrophin protein, removing the part of the protein that anchors internal proteins to cell-membrane proteins). It has not yet been shown whether mini-gene therapy is an actual cure for Duchenne muscular dystrophy, or if those treated with mini-genes could still develop a milder form of the disorder, similar to BMD. Only time will tell.

Gene therapy using AAV vectors that deliver mini-dystrophin genes appear to be nearly ready for clinical trials. Such a treatment offers the very real possibility of improving the length and quality of the lives of Duchenne muscular dystrophy patients. However, the only foreseeable way of curing Duchenne muscular dystrophy with full-length dystrophin is by using Ad vectors. Therefore, we will continue with our discussion of the future of gene therapy using Ad.

CONCLUDING REMARKS REGARDING PRE-CLINICAL STUDIES

Although some negative results have been obtained in animal studies using Ad in gene therapy of Duchenne muscular dystrophy, much hope remains. These studies have simply revealed the weak points of Ad as a gene therapy vector. Current and future research is focused upon rectifying these problems. Once these problems are overcome, and Ad becomes the safe and effective vector that it promises to be, it may be ready for clinical trials in humans.

TAKING ADENOVIRUS TO THE CLINIC

If an experimental treatment is both safe and effective in preliminary trials using at least two animal models, scientists are ready to take it to the clinic. Clinical trials are carefully regulated attempts to determine first, whether the therapeutic is safe and - only after safety has been proven - to determine whether the therapy is effective in humans.

CLINICAL TRIALS

Clinical trials designed to test new drugs are divided into three phases: Phases I, II, and III. Phase I trials are performed using a very small number of people (20-80) and are designed to evaluate safety, determine a safe dosage range, and determine whether there any side effects. If all goes well in Phase I trials, Phase II trials are initiated. As in Phase I trials, safety is the primary concern of the Phase II trials. However, Phase II trials involve more people (100-300) and efficacy is also examined. Phase III trials involve large numbers of people (1000-3000) and these trials are designed to confirm effectiveness and safety, and typically involve comparing the new treatment with a proven, “gold standard” treatment.

Ad vectors have been used in clinical studies for a variety of disorders, mostly for cancer. Some positive results have been obtained in the treatment cancers of the head and neck and the lung. However, some setbacks have also been noted. Overall, Ad has been disappointing in the clinic. This is due to the limitations described above, which have been noted in lab animals, and that are currently being addressed by scientists. To date, no clinical trials have been initiated for the treatment of Duchenne muscular dystrophy or any other neuromuscular disorders using Ad.

LIMB GIRDLE MUSCULAR DYSTROPHY CLINICAL TRIAL

Phase I trials have been initiated for the treatment of limb girdle muscular dystrophy (LGMD). These trials involved the use of the aforementioned AAV. LGMD is the name of a group of maladies that all result in weakening of the muscles of the hip and shoulder girdles. Much like Duchenne muscular dystrophy, LGMD is the result of a loss of proteins (due to mutation) involved in muscle stability.

Although no results have been published from this trial, the experimental procedure is outlined below. Patients (6-12 in total) in this trial were given a direct injection of virus into a small muscle in the foot. Although the disease pathology does not involve the foot, this muscle is as good as any for the goals of the study: The most important part of this study is to determine whether the patients exhibit any immune response to the virus or to the therapeutic protein; of secondary importance is whether the virus is able to deliver the therapeutic genes. Both of these issues can be addressed using the procedure as outlined. We anxiously await the publication of news from this study.

PERSPECTIVES

In the mid-1980s, when the idea of using modified viruses to deliver therapeutic genes was first suggested, it was thought that we were on the verge of a completely new type of medicine. However, the reality is that delivering therapeutic genes using viruses is not as easy as we thought it would be.

Although no clinical trials have been initiated for the treatment of Duchenne muscular dystrophy or any other neuromuscular disorders using Ad vectors, there is no need for discouragement. Science tends to be a very slow process: scientists and physicians always want to be sure that their discoveries are true and that rigorous science has been undertaken before rushing ahead with therapies that could possibly bring further harm to patients; as the Hippocratic Oath states: “first, do no harm”. The key is to hone and perfect these therapies in the animal models before taking them to the clinic.

All of the limitations discussed above are being addressed in the lab. Soon, we hope that Ad vectors will be much safer and even more effective. With these improved vectors in hand, it is foreseeable that gene therapy trials for neuromuscular disorders could begin in the near future. However, only time will tell whether Ad can be used to deliver therapeutic genes for the treatment of neuromuscular disorders.

ACKNOWLEDGEMENTS