published 07/23/1999

High Noon For Gene Therapy

Hemophilia, which plagued European royalty in the nineteenth century and changed the course of nations, again has a chance to make history. Biotech history. Three human trials of gene therapy for hemophilia are underway, and at least four others are in the planning stage. The gene therapy field, overhyped in the early 90s and only now regaining momentum, views these trials as its best chance to prove that sticking genes into patients can actually cure disease. If it does, it'll go a long way towards stanching biotech's hemorrhage of investment capital. To stop the bleeding all these companies have to do is -- stop the bleeding.

Gene therapy needs a win. Since 1990, more than 313 U.S. trials involving more than 3,000 patients have come up short. "There have been no convincing successes of gene therapy," says Katherine High, a gene therapist and an associate professor of pediatrics at the University of Pennsylvania. "[There has been] no unequivocal demonstration that somebody was cured."

Some quibble with that judgment: They point to Jeffrey Isner's experiments, in which he inserted genes in human heart muscle to grow blood vessels, or to the various cancer trials where tumors clearly shrank after gene therapy. (Isner, the chief of cardiovascular research at St. Elizabeth's Medical Center of Boston and a professor of pathology and medicine at Tufts University School of Medicine, is a co-founder of Vascular Genetics Inc. of Durham, NC.)

But these are hardly cures. Isner’s genes, while effective, make it into a limited number of cells and shut down after a few weeks, and the cancer results are more anecdote than proof. "We must concede that true success, as measured by proof of concept in humans and eventually the commercial development of effective gene therapy products, has not been accomplished," said James Wilson, director of the Institute for Human Gene Therapy at the University of Pennsylvania and president of the American Society of Gene Therapy (ASGT).


The field is littered with failures, most notably cystic fibrosis (CF). The 1989 cloning of the CF gene led to predictions of a quick cure through delivery of the normal gene to the lungs of CF patients -- and half a dozen biotech companies tested the theory in clinical trials. However, not only did gene therapy fail to make CF patients better, but also scientists still don’t know which cells in the lung to deliver the normal gene to, whether they can be reached, or how to even measure if gene therapy is working at all. "We will not go back into the clinic until we make substantial progress on a number of these barriers," said Genzyme Corp.'s chief scientific officer Alan Smith at the ASGT's annual meeting held in June in Washington, D.C.

Despite such defeats, the field is on the move. The number of clinical trials, down since the National Institutes of Health’s devastatingly critical Orkin-Motulsky report of 1995, is rising again. And there is a sense that technology is starting to catch up to hype. "The field has matured and we have tremendous momentum," said Wilson to his colleagues at the June meeting. "It is time now to deliver."


Which genetic disease will be first to be cured by gene therapy? Not heart disease, which has too many genes involved. Not cancer--too variable and unstable. Probably not familial hypercholesterolemia or muscular dystrophy, where the bar is high in terms of getting enough gene expression to make a difference. Many now think that the first success will be hemophilia.

Hemophilia should be easy. It’s a single gene disease, and the disease gene was long ago cloned. You don’t need to generate much protein to make a big difference; just five percent of normal levels can prevent or shorten most bleeding episodes. "The beauty of hemophilia is that we’re absolutely sure that if we can deliver the gene we can cure the disease," says Roland Scollay, vice president of research for GTI/SyStemix. But if, by accident, you make too much, there’s no damage done. (Hemophilia is a great example of what pharmacologists call a "wide therapeutic window.") Finally, it’ll be easy to tell if the therapy is working. Just take a blood sample, measure the level of Factor VIII protein (or Factor IX, in the case of hemophilia B), and do a blood clotting test. "If it didn’t work, there’s no question it didn’t work," says Penn's Wilson. "And if it did work, there’s no question that it did."

A ready and eager market for a hemophilia gene therapy product exists. While the U.S. patient population is only 17,000, stemming their bleeding episodes is astoundingly expensive. Most treat their bleeds with recombinant Factor VIII (or IX) protein, at $1,000 or more per treatment. That translates to annual costs often exceeding $100,000.

"Factor" is a $1.2 billion market, dominated by Baxter Healthcare Corp. and a few smaller players. While gene therapy isn’t likely to be cheaper than protein therapy, by preventing bleeds it could offer a powerful competitive advantage. Due to cost, most hemophilic Americans take factor only after a bleed starts, and many pay a heavy price in both pain and long-term joint damage.

At least seven biotech companies have entered the race for hemophilia gene therapy (see table below).

Company	Location	Vector/delivery system	Status	Clinical site
Chiron Corp.	Emeryville, CA	Retrovirus	Phase I	Univ. of Pittsburgh Medical Center
Avigen Inc.	Alameda, CA	Adeno-associated virus	Phase I	Children's Hospital of Philadelphia
Transkaryotic  Therapies Inc.	Cambridge, MA	Ex vivo transfection	Phase I	Beth Israel Deaconess Medical Center
GTI/SyStemix (Novartis AG)	Gaithersburg, MD/Palo Alto, CA	Adenovirus	Preclinical	N/A
Cell Genesys Inc.	Foster City, CA	Adeno-associated virus	Preclinical	N/A
Kimeragen Inc.	Newtown, PA	Chimeric oligonucleotides	Preclinical	N/A
GenStar Therapeutics	San Diego, CA	Adenovirus	Preclinical	N/A

Besides a commercial payoff, the prestige factor looms large: publicity, good personnel and investment capital would flow to the winner. And curing hemophilia could help the field of gene therapy, and biotechnology in general, shake off the stigma of unfulfilled promise. "The stakes are incredibly high," said Wilson. "For once I may say what I really think: I hope to God this works."

Wilson was only half-joking. The field of gene therapy, and the biotech firms invested in it, could suffer a backlash if the hemophilia trials come up blank. "We need to get gene therapy to work now," says one biotech researcher. "The public is tired of [just] hearing about it."

Careers and fortunes are also at stake, partly because the hemophilia race is also shaping up as a battle of gene delivery systems. The seven companies will be trying five very different vector systems: retrovirus, adenovirus, adeno-associated virus, ex vivo transfection and chimeric oligonucleotides. And while failure might not send any of them to the scientific dustbin, success could put one over the top and make it the vector of choice for the next generation of gene therapies.


Chiron, the oldest company in competition, is betting on the oldest vector: the retrovirus. In early June, doctors at the University of Pittsburgh Medical Center injected a hemophilic patient with Chiron’s retroviral vector encoding Factor VIII. Two more patients have since received treatment, with six more to come. Chiron beat Avigen by a few days to become the first company to do in vivo human gene therapy for hemophilia.

Retroviral vectors have plenty to recommend them. They’re easy to make; their large genomes, stripped of replication genes, accommodate plenty of therapeutic DNA; and they deliver their product to the nucleus of the cell, where it integrates into the host genome, leading to long-term gene expression. In theory, anyway. Unfortunately, by 1991, gene therapists had discovered that cells infected by retroviral vectors, although prodigiously active in culture, mysteriously stopped producing protein after a few weeks in mice -- even in mice with no functioning immune system. This infuriating behavior, dubbed "retroviral shutdown," has yet to be fully explained, but its undeniable reality contributed to mass desertions of scientists from the retroviral camp.

All was not lost. By tweaking their vectors -- for example, playing with different promoter regions -- the retrovirus faithful have greatly improved on results. But a bigger problem looms: Retroviral vectors only work in dividing cells, because their RNA is unable to pass the host cell's nuclear membrane unless it's in the process of splitting. (HIV is an exception.) Liver, the ideal site for production of Factor VIII, has very few dividing cells.

"That’s a key issue," admits Margaret Liu, Chiron’s vice president of vaccines and gene therapy. "Are we going to hit enough cells?" For animals, the answer appears to be yes: In rabbits, normal dogs and hemophilic dogs, Chiron has seen long-lasting gene expression (up to two years), and clotting time has dropped. The company has vectors that are pure, stable in human serum, and can be made in high numbers. But Chiron’s dose may not be enough to help human patients. Although humans are three to ten times larger than dogs, they’ll get only slightly more vector. "This is a Phase I [safety] study, so we’re not really pushing it," says Liu. "But we think that [an effect] is possible."

If it works, Chiron’s system should be easy to ramp up to commercial-scale production. "The retroviral vectors are the ones that we, today, have a manufacturing process [for] that can supply the entire hemophilia field in the U.S.," says Liu. "And I think that’s not true for any single other vector that’s a serious contender." Still, Chiron is bucking scientific dogma by targeting the non-dividing cells of the liver with a retroviral vector. If this trial doesn’t work, the company will have to decide whether to go to Phase II and give higher doses. Liu leaves that possibility open. "A positive will be exciting," she says. "A negative will not be a showstopper."



But retroviruses are clearly retro for the majority of the field. Their fall from favor ushered in the golden age of the adenovirus (1991-1995). Adenoviruses -- the source of the common cold -- have the happy ability to infect both dividing and nondividing cells. And they’re wonderfully efficient at delivering foreign DNA to these cells. In so-called "nude" mice lacking an immune system, adenoviral vectors produced astonishing results, in some experiments generating high enough levels of Factor IX from a single injection to stop a human bleed.

Excitement turned to dismay, however, when adenoviruses went into mice (and eventually humans) with functioning immune systems. Gene expression sputtered and then quickly crashed. By the mid-1990s scientists knew why: Both arms of the immune system went after the adenovirus with a vengeance. Killer T cells sought out genetically modified cells and ripped them open. And antibodies created when the vector was first infused then roamed the body, wiping out any later dose before it could deliver its genetic package. (Repeat doses are needed because the adenovirus doesn’t integrate its DNA into the host genome.) Some cystic fibrosis patients developed a violent inflammatory reaction from gene therapy; at least one ended up in an intensive care unit. Although improved vectors are far less dangerous, many experts now think the adenovirus will mainly be useful for delivering anti-cancer genes, because the supercharged immune response should help knock down tumors as well as vectors.

But the adenovirus is still in the picture for hemophilia gene therapy. Scientists at GTI/SyStemix (Novartis AG companies that consolidated last year) and elsewhere have found clever solutions to some of the vector’s problems. By removing most of the virus’s "backbone" genes, the adenovirus can be made less provocative to the immune system. (The extreme is the "gutless" adenovirus, which has virtually the entire genome removed.) And the antibody response can be avoided by giving the patient immunosuppressant drugs before infusing the vector, or by using a different type of adenovirus each time.

GTI’s adenoviral vector is not ready for human trials. It worked very well in hemophilic mice, basically providing a complete cure that lasted, in some cases, over a year. And it provoked very little immune response. Unfortunately, in hemophilic dogs, the vector caused liver toxicity. GTI is now testing the vector in other animals. "The question is, how will humans turn out?" says GTI’s Scollay. "Will they be like dogs, or will they be like mice? We don’t know the answer to this point. If they turn out like dogs, then obviously you don’t have much in hand. If they turn out like mice, then potentially you have a therapy there."


We are now in the era of the adeno-associated virus (AAV). This tiny virus, which can only replicate in the presence of adenovirus (hence the name), seems ideal. It infects both dividing and non-dividing cells; it causes far less of an immune response than the adenovirus; and it apparently integrates its DNA package into the genome of host cells, leading to long-lasting expression. And AAV does not cause any disease, as far as anyone knows, so it appears safer than other viral vectors.

In early June, Kathy High’s team at the Children’s Hospital of Philadelphia injected Avigen’s AAV vector into the thigh muscle of a patient with hemophilia B. In addition to twelve patients who will receive the Avigen vector in muscle, High and Stanford University’s Mark Kay (where he is the director of the program in human gene therapy) plan to deliver the vector directly to the liver in several other patients. (The liver, as the natural site of Factor IX production, has obvious advantages. But most people with hemophilia have hepatitis, contracted from contaminated factor during the 70s and 80s, and for them liver delivery might be risky.)

It’s a safety trial, although High and Kay will be measuring factor IX production, testing blood samples for clotting ability, and recording clotting time to see if the gene therapy works. After that, the biggest question -- for this and all other hemophilia trials -- will be immune response. Some hemophilic patients who receive recombinant factor develop antibodies, known as inhibitors, that attack the protein and ruin the therapy. One danger of gene therapy is that it will create inhibitors where none were before, not only canceling the normal gene but also making it harder for people to treat their bleeds with recombinant factor.

High isn’t making predictions. "Will gene therapy be a higher risk? We don’t know," she says. In theory, an "endogenous" protein created by a cell’s own molecular machinery is more natural, and hence less objectionable to the immune system, than one made by foreign cells and injected. On the other hand, High points out, cells display endogenous proteins on both MHC class I and class II molecules, while introduced proteins are only displayed on class II. (MHC, or major histocompatibility class molecules, are the immune system’s mechanism for recognizing foreign antigens, with class I priming killer T cells and class II priming CD4 "helper" T cells.) So, in terms of immune response, genes might be worse for patients than proteins. High’s lab is trying to find out.

One positive sign is that High’s experiments in hemophilic dogs didn’t cause a lasting inhibitor problem, and one dog has been producing Factor IX for over two years. The vector itself doesn’t create a violent T cell response like the adenovirus does, partly because AAV doesn’t infect antigen-presenting cells (macrophages and dendritic cells) nearly as much.


This is only the second human gene therapy trial using AAV (Targeted Genetics Corp. had the first, for cystic fibrosis), so Avigen -- a small biotech firm with fewer than sixty employees -- is risking a lot. Cell Genesys, by contrast, has pulled in its horns. Its vector is very similar to Avigen’s, and its results for mice and dogs are just as impressive. The company, in a 1997 press release, touted its animal results as a "major scientific breatkthrough." Yet while Avigen has gone ahead and funded a human trial, Cell Genesys has stalled. "We feel we have the data to go to the clinic," says Cell Genesys research scientist Brian Donahue. "[But] we’re looking for a corporate partner."

The company’s dilatoriness led to the defection of Stanford's Kay, a key collaborator and one of the country’s top gene therapists. "All the important scientific work that needed to be done to go forward with a clinical trial was done," he explains. "[But] they weren’t committed to going forward." Kay is now working with Avigen.

Vector production will be Avigen’s biggest challenge. Making AAV vector is excruciatingly hard. All viral vectors have replication sequences stripped out, both to render them safe and to make room for the therapeutic gene. To make vector in quantity, the missing genes can be supplied by transfecting them into a cell line at the same time the vector sequence goes in, waiting until the viruses replicate within the cell, then laboriously separating out the new vectors. This process, called "transient transfection," is enormously labor intensive and time-consuming, involving rooms filled with cell culture plates and dozens of technicians making plasmids, performing transfection, and scraping gobs of dead cells into test tubes for purification.


Images Courtesy Avigen Inc.

What’s needed is a "packaging" cell line, genetically modified to express the missing viral genes. Add the vector which contains the therapeutic gene, and the cell line supplies the machinery to reproduce it. (In the case of AAV, adenoviral genes must also be added.) Ideally, all this can be done in a single large fermentation vat. Unfortunately, AAV’s replication genes, rep and cap, are poisonous to mammalian cells, so making a cell line that will survive long enough to actually produce vector is a huge obstacle. And AAV purification is also hard, because of the adenovirus proteins in the mixture. (An impure preparation could ruin a clinical trial.) Although some progress has been made, the Avigen trial is still using AAV made by transient transfection -- a process that won’t work for large-scale trials, not to mention commercial use. "I don’t think the optimal cell line is out there," admits Kay, who is also skeptical that current purification methods are good enough to make vector in quantity. "[But] I think it’s a do-able thing in the future."

AAV’s other big problem is its tiny genome. It can accommodate the Factor IX gene, which is why Avigen is attacking hemophilia B first. Four times as many people suffer from hemophilia A, but they’re missing functional Factor VIII -- and the factor VIII gene is huge. By removing a large, superfluous area of the gene -- the "B" region -- it might be possible to shoehorn Factor VIII into AAV, but that might mean skimping on promoters or enhancers. Work continues, although High won’t go into detail. "I think it’s safe to assume that everyone working on Factor IX is also interested in Factor VIII," she says. "I'll just leave it at that."


Besides Chiron and Avigen, a third biotech company -- Transkaryotic Therapies (TKT) -- has a human hemophilia clinical trial underway. TKT beat the others into the clinic by half a year, treating its first patient (with hemophilia A) late in 1998. Many in the field are skeptical of this trial, because TKT is perceived as using an old and discredited technology: ex vivo transfected fibroblasts.

But TKT’s forty-year-old founder and CEO, Richard Selden, isn’t worried about what others think. His company, at the moment, is on a roll: It won a major legal victory last year when a federal judge threw out Amgen’s patent infringement claim over TKT’s "gene-activated" erythropoietin, which is now in Phase III clinical trials. (TKT recently moved to reopen the lawsuit in order to pre-empt future claims.) And TKT is finishing a gene therapy trial, with human growth hormone, which Selden says was successful.

TKT’s hemophilia approach is much more involved than a simple injection of viral vector. Doctors at Beth Deaconess Medical Center in Boston do a skin biopsy, isolate fibroblasts, introduce the factor VIII gene with an electrical pulse, then select for a transfected cell. Clones are grown, then implanted into the patient’s peritoneum using laparoscopic surgery.

Aside from the sheer unwieldiness of the procedure, skeptics feel it won’t work. Many others have failed using fibroblasts ex vivo. But Selden brushes off the critics. "People say...‘That’s not going to work, it’s too hard, it’s too expensive, there’ll be safety problems because the cells will transform,’" he says. "That’s just not right." Selden, who has wanted to do gene therapy since he was thirteen years old, has spent two decades perfecting cell culture conditions and the gene constructs used for transfection. "It’s not as if other people really tried all that hard to get an ex vivo nonviral approach to work," he says. "A problem in the field is that people jump from system to system....At the first sign of significant problems [they say], ‘Well, let’s try the next virus on the list.’"

Not that Selden is guaranteeing success in this Phase I trial. "Will it work?" he asks. "I don’t know... if we get some benefit therapeutically, it would be awesome. But it was designed for safety." The critical safety issue will, again, be inhibitors. "It’s not likely to be a significant problem," Selden says. "We have to find out. If it turns out to be a bigger problem than we think, we have to go back and redesign."

There’s one safety issue where TKT's approach claims an advantage over viral vectors. All the genetically-modified fibroblasts arise from a single cell, so they’re genetically identical. Unlike cells receiving DNA from viral vectors, these all have the same gene integration site. Before reimplantation, "we make sure that the cells have appropriate growth properties and metabolic requirements," says Selden. This minimizes (but doesn’t eliminate) the risk of "insertional mutagenesis" -- a new gene lodging near an oncogene or tumor-suppressor gene and triggering cancer. And since TKT doesn’t infuse viral vectors, there’s no chance of the new gene entering germ cells (sperm or eggs) and getting passed on to children, with potentially catastrophic results.

Most gene therapists consider these risks minimal. But, Selden stresses, they do exist. "If you’re doing an in vivo injection of adeno- or adeno-associated virus, or a retrovirus... do you get into germ cells? And I think the answer to that is yes," he says. "Do you get into cell types other than the target cell? I think the answer to that is yes. And do you have any real control over where the virus goes? I think the answer to that is no."


Safety aside, will any of these trials actually cure hemophilia? Knowing will be easy -- just take a blood sample. Such simplicity "creates some anxiety," says Jim Wilson. "Because if you flat-out don’t see a change, there’s going to be no rationalizing around it. It’s going to say that the experiment doesn’t work."

And these trials are anything but slamdunks. The doses are very low. Wilson stops short of predicting a backlash against gene therapy if it fails to cure hemophilia, but biotech could take a hit. "Where we definitely have to be careful about is our sponsors... outside the field," Wilson says. "We have to make sure they don’t overreact -- to a positive or a negative."

Another biotech researcher puts it more bluntly."If we don’t get a direct hit, we’re all going to take it on the chin," he says. "And we’d sure better not hurt anyone, or it’s going to be a dark age for gene therapy."