Psst! Gene Therapy Research Lives

Volume 16 | Issue 13 | 12 | Jun. 24, 2002 Previous | Next Psst!

By | June 24, 2002

Volume 16 | Issue 13 | 12 | Jun. 24, 2002

Psst! Gene Therapy Research Lives

COVER STORY | Progress registers experiment by experiment, trial by trial | By Josh P. Roberts

Image: Getty Images

In 1990, three men--W. French Anderson, R. Michael Blaese, and Kenneth Culver--led a trial in which the genetically corrected adenosine deaminase (ADA) T cells, belonging to a 4-year-old girl, were returned to her.1 Today, the 16-year-old teenager is alive and well.

It took another decade or so for any accomplishments as dramatic as that first trial to be reported, due in part to a relatively empty toolkit. In April, following a trial of gene therapy that occurred two years prior, French researchers announced that the immune systems of several children severely affected with X-linked severe combined immunodeficiency (SCID) were nearly normal, and that no supplementary therapies were involved.2 Other, less headline-grabbing reports also occurred, including work on curing fatal congenital diseases,3 reversing infertility in mice,4 treating patients with hemophilia,5 and combining different therapies with gene therapy.6

The short history of gene therapy is like a roller coaster, with quick, adrenaline-creating ascents and tortured, heart-in-the-mouth descents. Since gene therapy patient Jesse Gelsinger died in 1999 at the University of Pennsylvania, this field has been subjected to intense scrutiny, with new regulations established to police experiments and new protocols to follow. Moreover, media coverage of the slow, steady research progress--what Anderson calls routine--had taken a back seat to headlines that sensational setbacks such as Gelsinger's death have received.

But (with apologies to Mark Twain) the death of gene therapy has been greatly exaggerated. At least 2,000 labs are engaged in gene-therapy research worldwide; an Internet search of "gene therapy" in PubMed indicates that nearly twice as many papers were published in 2001 than five years earlier. At the National Institutes of Health, monies devoted to lab and clinical research increased 22% from $349.4 million in fiscal year 2001, to an estimated $427.4 million in 2003. Moreover, membership in the American Society of Gene Therapy (ASGT) has grown from 1,000 when it was founded in 1996, to 3,000 members in 2002. More than 600 gene therapy trials are ongoing worldwide. Of the 509 trials listed in the NIH's pilot Human Gene Transfer database (, about two-thirds are for the treatment of various forms and stages of cancer. A wide variety of other trials are ongoing as well, most of which fall under the headings of cardiovascular disease (46 trials), infectious disease--nearly all HIV-related (40 trials), and inherited autosomal recessive (44 trials).

Recently, the press has begun paying more attention to some of the successes, printing headlines including "For Gene Therapy, a Humble Return,"7 and "Gene Therapy Gives Heart Patients Hope."8 Reports of successfully treating the rare X-SCID--from INSERM in Paris2 as well as similar news from London's Great Ormond Street Hospital--were greeted worldwide with headlines such as "Gene Therapy Rids 'Bubble Boy' Disease."9

"I think we should hold up the fact that you can actually do gene therapy and it really works. That's pretty exciting," says Fred Hutchinson Cancer Research Center virologist Dusty Miller, whose vectors have been used in numerous gene therapy trials. All is not glorious, however: No clinical trials have yet to complete the large Phase III trials necessary to win the Food and Drug Administration's (FDA) approval of therapeutics.

ANCIENT HISTORY Clinical gene transfer had its official beginning when, in 1989, five patients with terminal melanoma were given autologous lymphocytes that had been "marked" ex vivo with a gene encoding resistance to the antibiotic G418. This study was designed primarily to trace the cells in the patients' bodies and to show the safety of gene transfer, and in that sense it was successful. No helper viruses were found, no reverse transcriptase activity was detected, no toxicity was experienced, and the transduced cells remained otherwise "normal."10

Photo: Courtesy of W. French Anderson
 CORRECTED GENES; SAVED LIVES: W. French Anderson with patients Ashanti De Silva (left) and Cindy Cutshall, in 1992.

That trial paved the way for the world's first sanctioned gene-therapy trial. In 1990, 4-year-old Ashanti de Silva's ADA T cells were genetically corrected and then returned to her.1 She is still on a low-dose regimen of intravenous PEG-ADA therapy, her immune system is now fully functional, with 20% to 25% of all her T cells containing the gene that was introduced by retroviral transfer nearly 12 years ago, says Anderson, who is now at the University of Southern California.

At the time, ADA deficiency was "really the only disease that we could think of that we thought we had a shot of helping," recalls Blaese, former chief of NIH's Clinical Gene Therapy Branch. For one thing, transduction efficiencies were "terrible," he explains. Culver, now at Norvartis, comments that although relatively few T cells would become transduced, these had a tremendous selective advantage over their endogenous counterparts. As a bonus, the introduced ADA gene product helped endogenous T cells to thrive as well.

T cells can also be easily removed and isolated, then grown and expanded in culture. This is important because retroviral vectors, the only ones available at the time that were capable of stable transduction, could transfer genes only to dividing cells. And T cells can be reintroduced into their proper places in the body.

Photo: Courtesy of Kenneth Culver
 Kenneth Culver

STEADY PROGRESS The recent advances witnessed in gene therapy reflect a large number of incremental steps in many areas, rather than one or two "great strides," Blaese and others note; these include more and better viruses. Anderson cites as an example the incremental improvement of culturing conditions, including growth factors, which allows for significantly greater transduction. What were once mystery factors in tissue culture supernatant, such as the T-cell growth factor IL-2, can now often be purchased off-the-shelf in purified form.

Anderson also points to scientists' hard-won ability to determine which vector will work best in which type of application. "There isn't going to be a 'magic vector' that is useful for every situation." Not only do many vector classes exist today--the big five are retroviral, adenoviral, adeno-associated viral (AAV), lentiviral, and nonviral, with many others being investigated as well--but the vectors themselves have improved, says Anderson, founder and editor of Human Gene Therapy.

Some improvements include "gutless" adenoviruses that allow for larger genes to be inserted and have less potential to evoke an immune reaction to the vector (which is thought to have contributed to Gelsinger's death).11 Viral genes are now generally supplied by the packaging cell in trans (as has been the case for most other vectors), giving added assurance against transferring active virus to the patient. And viral vectors can now be routinely pseudotyped to achieve a desired tropism. Investigators can now choose from a "whole toolkit of viruses," notes Miller.

Although many would disagree, Miller does not think that nonviral technologies, such as introducing naked or plasmid DNA by gene gun or liposomal transfer, are, at this point, very efficient for gene therapy. These technologies have no specific integration mechanism, and the gene tends not to persist in the target cell, he observes. "Nature has developed some pretty good tools. Viruses have figured out over billions of years what to do, and it will take a while for scientists to do the same."

However, one of the four early-stage clinical success stories cited by Anderson in a Nature Medicine commentary12 involved using naked plasmid DNA to induce angiogenesis in cardiovascular disease patients. Illustrating the toolkit concept, the other trials involved retroviral transfer (the X-SCID trial), AAV (used to treat hemophilia), and an oncolytic adenovirus.

CANCER AND GENE THERAPY The latter trial, which Anderson calls the first successful Phase II trial for cancer, used the mutant adenovirus to specifically replicate in and lyse carcinomas that had lost the ability to make the tumor suppressor p53.3 The therapy was not effective on its own, but it did show a significant benefit when combined with standard chemotherapy.

Since the field's seminal days, oncologists have investigated gene therapy's potential to treat their patients. Most early lab and animal experiments--few actually made it to humans--were variations on one theme: Researchers tried to make tumors more immunogenic, partially because most of these did not require the long-term transduction of many cells. Most experiments have met with only limited success. Blaese cites the fundamental premise of inducing the immune system to fight the disease--rather than problems of gene transfer--as the major limitation of these early studies, but he does admit that "there are some studies along those lines showing some levels of efficacy."

Immunotherapy still dominates researchers' efforts to treat malignancies with gene therapy; investigators are using strategies ranging from returning gene-enhanced irradiated tumors to patients, to injecting tumor-specific antigen-engineered pox virus into muscle.

Two other heavily investigated oncological strategies are gene replacement--the same concept used in treating patients with SCID--and direct or indirect killing of the malignant cells. In the former, a working copy of a gene that has gone awry (for example, p53) is introduced into the tumor to recheck its growth. In the latter, researchers introduce a "suicide gene" such as the thymidine kinase gene of the herpes simplex virus, whose product will poison the cell on exposure to the antiviral drug gancyclovir.

ON TRIAL The majority of new gene-therapy trials, says Anderson, involve either cancer or vaccines, and sometimes both. Initially, most of them involved ex vivo genetic manipulations followed by reintroduction of the gene-engineered tissue. Now, the tide is changing, Anderson notes, with vectors being delivered in situ and introduced directly into the target tissues.

But whether these new trials hold promise has yet to be seen. The vast majority of clinical trials have not made it to Phase II, and those dealing with rare genetic disorders such as ADA "never will," Anderson points out. The issue is money: the NIH, private foundations, or perhaps an academic institution, generally bankroll these trials. Says Anderson, "There's no money in it. No drug company is going to spend ... $100 million to go through all the pivotal Phase III trials that are necessary for approval." Such trials themselves may eventually become standard-of-care, but without "that golden piece of paper from the FDA that says your NDA [new drug application] has been approved," Anderson explains, "it is still a trial."

Photo: Courtesy of R. Michael Blaese
 Michael Blaese

Of course, pretrial work continues--the recent ASGT conference produced a record number of abstracts, Blaese says. One of these, from Miller's lab, describes investigations into treating patients with cystic fibrosis by introducing a functional cystic fibrosis transmembrane conductance regulator gene. That gene, with all of its regulatory elements, will not fit into a vector, necessitating use of the cDNA with shortened transcriptional elements. Even this does not easily fit into an AAV vector (which, he says, works best in the lung), so they developed a way to split the shortened gene into two AAV vectors and then allowed them to recombine in vivo. The lab is also trying to adapt an oncogenic sheep retrovirus, which replicates in the lung, for use in human gene therapy. "There is a new batch of vectors evaluated every year, Blaese notes.

Observers point to several new developments that hold great promise for the field: lentiviruses (such as HIV); transposons, which can stably introduce genetic material into quiescent cells; and continued improvements in nonviral delivery systems. Blaese also expects to see a lot of "transgenomic viruses," combining aspects of different viruses, as well as combinations of viral with nonviral vectors and strategies. The field is now vibrant and healthy, he says, "working its way through its growing problems."

Josh P. Roberts ( is a freelance writer in Minneapolis.

1. R.M. Blaese et al., "T lymphocyte-directed gene therapy for ADA-SCID: Initial trial results after 4 years," Science, 270:475-80, Oct. 20, 1995.

2. S. Hacein-Bey-Abini et al., "Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy," New England Journal of Medicine, 346:1185-93, April 18, 2002.

3. T.J. Fraites Jr. et al., "Correction of the enzymatic and functional deficits in a model of pompe disease using adeno-associated virus vectors," Molecular Therapy, 5:571-8, May 2002.

4. M. Ikawa et al., "Restoration of spermatogenesis by lentiviral gene transfer: Offspring from infertile mice," Proceedings of the National Academy of Sciences, 99:7524-9, May 28, 2002.

5. J.D. Mount et al., "Sustained phenotypic correction of hemophilia B dogs with a factor IX null mutation by liver-directed gene therapy," Blood, 99:2670-6, April 15, 2002.

6. W.M. Rideout III et al., "Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy," Cell, 109, 17-27, April 2002.

7. M. Waldholz, "Gene therapy makes a comeback with a cautious, supporting role," Wall Street Journal, May 30, 2002, p. D4.

8. J. Swirsky, "Gene therapy gives heart patients hope," New York Times, Feb. 17, 2002, Section 14LI, p. 2.

9. M. Healy, "Gene therapy rids 'bubble boy' disease," USA Today, April 18, 2002, p. D8.

10. S.A. Rosenberg et al., "Gene transfer into humans--immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction," New England Journal of Medicine, 323:570-8, Aug. 30, 1990.

11. L. Thompson, "Human gene therapy: harsh lessons, high hopes," FDA Consumer, September-October 2000 (

12. W.F. Anderson, "Gene therapy scores against cancer," Nature Medicine, 6:862-3, August 2000.

13. F.R. Khuri et al., "A controlled trial of intratumoral ONYX-015, a selectively-replicating adenovirus, in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer," Nature Medicine, 6:879-85, August 2000.
©2002, The Scientist Inc.
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