Current Event

Nearly a decade after it permitted NIH intramural researchers to pioneer this country’s first two human gene therapy trials—and some 350 diverse human gene therapy protocols later—the NIH Recombinant DNA Advisory Committee (RAC) held a three-day assembly centered on the first reported death of a gene therapy research patient attributed to the therapy itself.

The death last September of Jesse Gelsinger, an 18-year-old patient with partial ornithine transcarbamylase (OTC) deficiency, injected a sobering pause in a field rushing toward the 21st century’s promise of an avalanche of gene therapy applications inspired by the completion of the Human Genome Project and related discoveries.

The death also precipitated an FDA freeze on the clinical trial in which Gelsinger was enrolled—a safety and biological efficacy study of the recombinant adenoviral vector—OTC gene delivered to the liver via the intrahepatic artery. Gelsinger was the scheduled penultimate patient in what was to have been an 18-patient study and the second one to receive the highest protocol-defined dose in this dose-escalation trial that was begun in 1997 at the University of Pennsylvania in Philadelphia.

The pivotal speakers at the RAC proceeding, held December 8 through 10, 1999, on the NIH campus and under bright media lights, were the OTC study investigators: veteran gene therapy researcher James Wilson, director of the Institute for Human Gene Therapy, and surgeon Steven Raper, both of UPenn, and pediatrician Mark Batshaw, chief academic officer at the Children’s National Medical Center in Washington. All three apologized for communication gaps with oversight agencies and lapses in complying with adverse event reporting requirements, which, had they been adhered to, might have led to a protocol modification or a reevaluation of Gelsinger’s candidacy for trial participation. Gelsinger’s precipitous and ultimately irreversible response to the experimental therapy included signs of disseminated intravascular coagulation (DIC), massive cytokine release, adult respiratory distress syndrome (ARDS), and multiple organ failure.

The purpose of the meeting, federal officials and RAC officers emphasized, was largely educational: to pool information on the biology and toxicity of adenoviral vectors; to scrutinize the conduct of the OTC trial with a view toward identifying red flags and improving gene therapy trial design in general; and to revisit federal reporting requirements, interagency communications, and the role of the RAC in gene therapy oversight. Patient advocates, however, voiced worries that gene therapy research might be stopped or curtailed; industry representatives cautioned against increased public scrutiny that might bare trade secrets or misleading information; and reporters wanted to know the specifics of protocol violations and any other problems in the OTC study, as well as steps planned by overseers to enforce compliance with scientific and ethical guidelines in the conduct of human gene therapy research.

RAC chair Claudia Mickelson, a bio-safety officer at the Massachusetts Institute of Technology in Cambridge, and molecular biologist and former RAC chair Inder Verma, of the Salk Institute for Biological Studies Laboratory of Genetics in San Diego, served as moderators.

Adenoviral Vector Experience

According to Amy Patterson, director of the NIH Office of Biotechnology Activities (OBA, formerly the Office of Recombinant DNA Activities), about 4,000 patients have participated in about 350 gene therapy trials in the past decade; adenoviral vectors have been used in about one-quarter. Immediately after OTC investigators notified OBA of Gelsinger’s death, Patterson said, OBA notified the more than 70 other researchers currently using similar methods in their own gene transfer studies.

After Gelsinger’s death, the FDA conducted a database search of all gene therapy protocols with adenovirus vector and found no similar ARDS, aplastic anemia, or extensive DIC findings, an FDA official said.

A refresher course in adenovirus basics was presented by experts summoned to the RAC proceedings. Among the relevant facts noted were that adenovirus can cause fatal hepatitis in immunocompromised individuals; and that adenovirus attaches to the coxsackie-adenovirus receptor (CAR) in the liver, a receptor whose function and level of expression in different tissues are not well known and whose abundance in the liver varies among humans and animal models. To render adenovirus suitable as a gene vector, viral genes required for replication are deleted; thus enfeebled, adenovirus generally serves well as a vector. There are, however, some concerns regarding viral infection: In simian species, adenoviral replication is enhanced in the context of co-infection with another virus, such as SV40; and adenovirus can also function as a helper virus, for instance, potentiating the growth of parvovirus. Still to be understood is the ability of certain integrins that facilitate adenoviral integration into target cells to induce cytotoxic and apoptotic responses.

Verma recounted his own lab’s adaptation of adenovirus for use as a gene vector, with the deletion of the E1a, E1b, and E4 adenoviral genes to blunt its immunogenicity and the summoning of cytotoxic T cell lymphocytes. The ease of disarming these replication components and of growing billions of virus particles quickly, as well as adenovirus infectivity of both dividing and nondividing cells, he said, make it an "attractive" vector.

University of California at San Francisco (UCSF) surgical oncologist Robert Warren offered observations from a trial involving adenovirally packaged p53 delivered via the intrahepatic artery to the liver of colorectal cancer patients with hepatocellular metastases. Postinfusion transaminase elevations invariably came down and were never dose limiting; hypotension, he said, was the dose-limiting toxicity, with liver toxicity "significantly less than expected." Commenting on questions later related to the possibility that high doses could mobilize proinflammatory cytokines and lead to intravascular coagulation, Warren said that duration of exposure to the vector in his trial may have been too short to observe adverse cytokine events. "We would expect adenovirus induction of cytokines in the liver," he said.

Margaret Rick, hematology chief in the NIH Clinical Center pathology department, noted that acute hepatic failure is among possible triggers of DIC. She suggested that patients be screened for hepatic vulnerabilities to tissue factor upregulation due to infection- or cytokine-induced endothelial cell damage.

RAC member Estuardo Aguilar-Cordova, of the Baylor College of Medicine in Houston, suggested that dose-related adenoviral toxicity may not be linear. "Perhaps there is a threshold effect at a certain point where only a slight increase in dose results in a great increase in toxicity," he said, noting that different species exhibit different levels of inflammatory response to the equivalent dose and, to make matters more complicated, titer and dose specifications are not standardized. "It’s not clear we are all even talking in the same language," he commented, a lament later answered by Savio Woo, president of the American Society for Gene Therapy, who offered his group’s services in developing standards for vector quantification.

Phase 1 adenoviral vector–gene therapy studies have been ongoing at the University of Pennsylvania for the past five years and have involved 95 patients, 18 of whom participated in the OTC study. Prior to his detailed account of that trial, Wilson summarized findings among the 77 other gene therapy patients enrolled in trials addressing cystic fibrosis and various types of malignancy. "In general," he said, "toxicities seem to be dose-dependent, time-limited, and confined to the target site." However, he added, there is a suggestion of "broader cytokine release" in the presence of fever.

Ron Crystal, chief of pulmonary and critical care medicine at New York’s Cornell Medical Center, summarized safety data from gene therapy studies involving E1/E3-deleted adenoviral vectors of three different transgenes. Among 90 patients with cystic fibrosis, metastatic colon cancer, or coronary or peripheral vascular conditions that would benefit from angiogenesis, there have been more than 140 gene administrations, more than 44,000 follow-up days, and 13 deaths–all related to the patients’ disease and unrelated to therapy dosage. One serious adverse event in a cystic fibrosis patient was linked to bronchoscopic delivery and has not recurred since a switch to aerosol spray, he said. Adverse pulmonary events were found to be dose related in animal studies conducted by the Genzyme Corporation (Cambridge, Mass.).

The OTC Trial

The prelude, conduct, and aftermath of the OTC trial were presented by Batshaw, Raper, and Wilson. Using the sparse fur mouse as a model, they delivered what would have been a fatal ammonia dose to animals pretreated with the adenoviral OTC gene vector. The ensuing rapid drops in glutamine and ammonia concentrations matched the speed the investigators sought to prevent the irreversible brain damage sustained by children who manage to survive coma longer than 72 hours. The adenoviral vector evolved from an E1-deleted to an E1-deleted/E2-mutated and, finally, to an E1/E4-deleted construct.

Although newborns with full-blown OTCD were the population for whom the gene therapy was ultimately targeted, it was decided that the study cohort for this first experiment to determine safety would be stable adults with partial disease who could give informed consent.

The investigators submitted their protocol application to the RAC in March 1994, when the RAC still had approval authority, along with the FDA, for all gene therapy protocols. In 1995, the RAC approved the protocol contingent on what members concluded would be a safer route of administration: intravenous, rather than intrahepatic. The FDA, however, later opted for intrahepatic delivery, in part to lessen the possibility of unintended delivery of the transgene to the gonads. The RAC was never informed of the protocol modification.

"We recognize that we probably should have gone back to the RAC to discuss this," Batshaw said, "but RAC’s responsibilities were changing. . . . We apologize." Wilson issued the same apology during his presentation later that day.

FDA approved the protocol—with the intrahepatic artery route—in 1996; the first patient was dosed in April 1997; and Jesse Gelsinger, the last patient, was dosed in September 1999.

All told, four men and 14 women participated in the trial. High fever post-infusion was frequent, as were backache and nausea. Drops in platelet counts and phosphates were dose related and increased over the course of the cohorts (the protocol called for six cohorts, with three patients in each—the first two women and the last a man—to receive doses escalating in half-log increments beginning with 2 times 9⁹ particles/kg and ending with 6 times 10¹¹). Similarly, transaminase elevations appeared to be dose related, but not consistently.

An odd and surprising finding related to antibody response to the vector: adenovirus-naive patients developed an expected lymphoproliferative response (LPR), but those who had antibodies to begin with experienced a "transient LPR loss," a phenomenon some panelists viewed as a safety concern in the context of inadvertent exposure to adenovirus infection during LPR downtime. This loss of T cell ability to respond to adenovirus, Wilson said later that day, was not seen in any other clinical or animal trials.

In general, the team was concerned about platelet lowering and its relation to vector dose. In collaboration with FDA, the team began DIC monitoring with the second cohort, tracking platelet counts and fibrin split products before, during, and after infusion and keeping an eye out for the presence of vector in the systemic circulation.

But it was not until the fourth cohort that there were recurring problems that the investigators were required by protocol to report to FDA before proceeding with subsequent dosing. These involved marked elevations in liver function assays, two of which were reported in a timely manner and two of which were not. Raper acknowledged these lapses: "We should have called FDA before dosing patient 14, and we should have made another call before proceeding to the next dose level," he said.

Other irregularities mentioned by FDA officials during the meeting but not addressed publicly by the investigators included the investigators’ failure to get FDA permission to have a man (Jesse Gelsinger) replace a woman as the second patient in his cohort and that information in the consent form submitted to FDA—that primates had died on high doses in preclinical studies—was removed from the final consent document.

When Raper turned to the specifics of Jesse Gelsinger’s clinical course, he did not address a question raised during the meeting: whether the investigators should have proceeded to infuse Gelsinger in the face of abnormal liver function tests that required alternative pathway therapy prior to dosing. He later implied that patient status before infusion should be rethought.

Gelsinger’s Course

Gelsinger’s status eight hours postinfusion was not unlike that of other study participants. Raper said: he had a fever, normal liver function tests, and no evidence of DIC; he was given intravenous potassium phosphate for low phosphate concentrations. The morning after, however, nurses noticed an altered mental state; his ammonia concentration had risen, and he was jaundiced and tachycardic. A coagulation workup showed increased prothrombin time and fibrin split products and decreased platelets.

He became "progressively obtunded" and by the second evening he was comatose. Chest X-ray showed infiltrates suggestive of ARDS. Raper described a series of developments in Gelsinger and actions taken by medical teams to offset or reverse what eventually became what he called a "desperate lung situation." These measures did not save the patient.

Commenting generally on Gelsinger’s clinical course and the efforts to save his life, UCSF’s Warren later observed that "each intervention seemed logical and appropriate" but did not prevail against the "cascade of irreversible events."

With the Gelsinger family permission, Raper said, a postmortem examination was performed. There was little vector-induced hepatitis and no significant signs of DIC. Jesse Gelsinger died of intractable ARDS, with anoxia evident in the liver, kidney, brain, and spleen. The team speculated that ARDS was secondary to "secondary inflammatory response syndrome," referred to by Raper as "immune revolt."

Bone marrow yielded "perhaps the most unexpected findings: an absence of erythroid precursors and mature granulocytes, suggesting acute insult."

The team speculated that the anemia could be an idiosyncratic reaction to the medications or a sign of human parvovirus infection. One RAC consultant noted during discussions later that the one disease associated with such acute and striking bone marrow aplasia is parvovirus infection.

Questions

"We in no way expected to see what we saw in Jesse Gelsinger. Our animal studies never demonstrated these pulmonary events," said Wilson, who presented a chronology of the team’s vector manipulations and animal studies—including mice, newborn and adult rhesus monkeys, and baboons—and ruminated on the cytokine findings in Gelsinger’s case and the questions remaining to be answered.

Test animals, he said, had demonstrated self-limited, dose-dependent hepatitis, a transient decline in platelets, and transient transaminase elevations, with an apparently biphasic dose-toxicity relationship. Dose-limiting toxicities were liver damage and DIC at significantly higher doses, he said, than that received by Gelsinger. UCSF’s Warren commented: "We have not seen the pulmonary dysfunction in our 60 patients that you saw with Jesse Gelsinger."

Wilson elaborated on Gelsinger’s immune response to the vector, as well as vector biodistribution on autopsy. At the higher vector doses received by patients in the last two study cohorts, there were rapid, dramatic cytokine increases, specifically of interleukin-6 (IL-6) and IL-10. Recovery followed in all cases but Gelsinger’s, whose IL-6 trajectory never returned to base line. "Maybe we are activating some aspect of the immune system," Wilson said, suggesting that some "inciting event of acute cytokine release" from macrophages and monocytes had occurred. "What was different about this patient?" he asked—and listed hemodialysis, intubation, and external ventilation among the atypical procedures Gelsinger had undergone.

The investigators used PCR to track the vector after hepatic artery infusion. The highest DNA concentration was found in the liver—first in the macrophages (Kupffer cells) and then the hepatocytes—with significant amounts also appearing in the spleen, lymph node, and bone marrow. Biodistribution, Wilson remarked, "was not as hepatospecific as I had hoped." RAC co-chair Verma later noted that animal models are generally not good enough to pinpoint biodistribution. He cited the fact that rhesus monkeys and humans do not have the same CAR distribution.

After the death, the team studied the specific vector lot Gelsinger had received, as well as the equivalent of the preceding lot, and tested them at the same doses in rhesus monkeys. "Clinically, the animals did fine," Wilson said.

Among issues to be investigated, he said, are what role, if any, Gelsinger’s underlying condition and medications played in his outcome; whether he had a genetic predisposition to such an outcome; why the vector distributed beyond the liver and whether this affected outcome; and what stimulated cytokine release and, again, whether this affected outcome. "We don’t know the roles of IL-6 and IL-10," he emphasized.

Asked what he would have done differently, Wilson replied, "Including not having done this at all, the trial could have been designed differently [with respect to] the half-log increments. The increments should be smaller." He noted that the dose-toxicity relationship appeared to be "elbow shaped" and that at the half-log increment the difference in dosage between the later cohorts is significantly greater than that between the earlier ones and "may be breaching that elbow." A RAC member agreed that there appeared to be a "narrow window between early and severe toxicity, requiring meticulous measures."

FDA’s investigation into the conduct of the UPenn trial was ongoing at Catalyst press time.

Gene Therapy Oversight   Overseers Answer the Press (left to right): Amy Patterson, director, NIH Office of Biotechnology Activities; Lana Skirboll, director, Office of NIH Science Policy; Phil Noguchi, director, FDA Division of Cellular and Gene Therapies; and Kathryn Zoon, director, FDA Center for Biologics Evaluation and Research, respond to questions during a news briefing after the first day of the RAC meeting. Nearly all questions centered on FDA's oversight role in the OTC study, protocol violations, and FDA's steps to uncover, punish, and prevent irregularities in gene therapy clinical trials. The rules and roles of federal agencies involved in overseeing gene therapy experiments are undergoing review and may be revised in the wake of Jesse Gelsinger’s death. Specifically, the role of the NIH Recombinant DNA Advisory Committee (RAC) is being revisited, and proposed amendments to the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) have been published. The harmony—or lack of it—between NIH and FDA adverse event reporting requirements in the conduct of clinical trials is also being scrutinized. Revisiting the RAC. Before vacating the office of NIH director, Harold Varmus announced the formation of a subcommittee of the director’s advisory group to recommend further actions NIH might take to minimize adverse events in gene therapy trials. Although the group’s charge also places the role of the RAC back on the table, Varmus stands by his actions in 1995 to recast the RAC from a quasi-regulatory body with approval authority over every gene therapy application.to a public policy forum on novel methods in gene therapy clinical trials and thorny issues related to gene therapy (see Harold Varmus interview, this issue). NIH Guidelines. NIH clarified the definition of adverse events and investigators’ reporting obligations in a proposed action to amend the NIH Guidelines, published in the Federal Register November 22, 1999. The proposal reaffirms that investigators must report serious adverse events immediately to NIH, so NIH may rapidly notify the RAC and others involved in gene transfer studies. "Immediately" is defined as no later than 15 days from the event. A "serious adverse event" is defined as any "expected or unexpected adverse event, related or unrelated to the intervention, occurring at any dose" that results in death, a life-threatening event, hospital admission or prolonged stay, or disability. It also rejects recent claims by some gene therapy investigators and sponsors that human gene transfer protocols and serious adverse event reports are trade secrets. Informed consent documents would reflect the necessarily public nature of RAC discussions of adverse events. The NIH Guidelines require the principal investigator to report serious adverse events to local review bodies and the FDA as well as to NIH and the federal Office of Protection from Research Risks. FDA reporting requirements require the study sponsor (who has presumably been informed by the investigator) to immediately report serious and unexpected adverse events to FDA. NIH proceedings are public; FDA proceedings are often closed. During the RAC proceedings, industry representatives accepted the need for immediate reporting only of "related and unexpected" serious adverse events, and some RAC members voiced skepticism that they could deal meaningfully with reports of all adverse events. –F.P.

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