the cline affair
Authors: Ernest Beutler
Journal: Molecular Therapy (2001)

"Cline has done this experiment in the mouse and, as I understand it, it didn't work. He has made a great conceptual leap from the failure in the model system to trying it in humans. He is saying, 'It didn't work in mice, so I'm going to try it in man'" [quoted in 1Wade N. UCLA gene therapy racked by friendly fire.Science. 1980; 210: 509-511Crossref PubMed Scopus (25) Google Scholar]. This was but one of the numerous criticisms voiced when the news media reported that Martin Cline of UCLA had attempted to treat two patients with severe β-thalassemia by transfecting marrow cells with a recombinant human globin gene with a selectable marker. Probably as a result of the furor that arose, not all of the details of the experiment were published. However, relying on his published animal experiments and accounts of the clinical study as they appeared both in the scientific press and in a review written by Cline in 1985 [2Cline M.J. Perspectives for gene therapy: inserting new genetic information into mammalian cells by physical techniques and viral vectors.Pharmacol. Ther. 1985; 29: 69-92Crossref PubMed Scopus (20) Google Scholar], I shall try to address the following questions. Did this system work in mice? Was the extensive criticism that was leveled at Cline justified? What impact, if any, did this event have on the subsequent development of human gene therapy? No wild-eyed renegade, Martin Cline was a highly respected biomedical scientist. Head of the hematology division at UCLA, he had a distinguished and productive career in performing research at the interface of clinical medicine and basic science. He began to work with DNA long before most other clinical investigators. Having initiated one of the early bone marrow transplantation programs at UCLA, his understanding of hematopoietic stem cells and of gene transfer technology qualified him ideally to perform gene transfer studies in humans with genetic diseases of hematopoiesis. The experiment took place in July, 1980 [3Sun M. Cline loses two NIH grants.Science. 1981; 214: 1220Crossref PubMed Scopus (11) Google Scholar]. Two patients with severe β-thalassemia were the subjects of the study. Marrow cells were transfected with a recombinant genomic β-globin and herpes virus thymidine kinase clone as a selectable marker. About 109 cells were then infused into each patient after a segment of femur had been irradiated with 300 rads [2Cline M.J. Perspectives for gene therapy: inserting new genetic information into mammalian cells by physical techniques and viral vectors.Pharmacol. Ther. 1985; 29: 69-92Crossref PubMed Scopus (20) Google Scholar]. There was no clinical benefit to the patients 1It is difficult to accept the claim that viral sequences were detected in the peripheral blood of the patients for up to 10 weeks after the infusion of 109 marrow cells. A 50-kg patient is estimated [4Abboud, C. N., and Lichtman, M. A.2001. Structure of the marrow and the hematopoietic microenvironment. In Williams Hematology E. Beutler, M. A. Lichtman, B. S. Coller, T. J. Kipps, and U. Seligsohn, Eds., McGraw-Hill, New York.Google Scholar] to have approximately 1012 nucleated marrow cells. If the transfection efficiency is estimated to be of the order of 1:100,000 (10−5; [5Mercola K.E. Cline M.J. Sounding boards. The potentials of inserting new genetic information.N. Engl. J. Med. 1980; 303: 1297-1300Crossref PubMed Scopus (29) Google Scholar, 6Cline M.J. Gene therapy.Mol. Cell. Biochem. 1984; 59: 3-10Crossref PubMed Scopus (3) Google Scholar]), 104 of the infused cells would contain the viral DNA, which would be approximately one cell per hundred million marrow cells. No effort to select with methotrexate was made and, even with incorporation of multiple copies of the gene, it is difficult to conceive that Southern blot analysis could detect such a low-abundance gene. Indeed, Cline [7Cline M.J. Genetic engineering of mammalian cells: its potential application to genetic diseases of man.J. Lab. Clin. Med. 1982; 99: 299-308PubMed Google Scholar] estimated that, in what he termed an optimistic analysis, an innoculum of 109 marrow cells would contain one pluripotent stem cell. , and they were not harmed, but it developed that Cline had not received permission from UCLA's institutional review board. Although permission had been obtained from the institutions in Jerusalem and Naples where the studies had been performed, it seems that the protocol was modified after permission had been obtained. Had this approach been validated with animal studies? Indeed, Cline had published two papers in major journals, one in Nature in which mouse marrow cells had been transfected with dihydrofolate reductase (DHFR) and one in Science in which the herpes simplex thymidine kinase gene had been used to induce methotrexate resistance. The studies were elegantly designed. Marrow cells from a mouse strain with a marker chromosome (T6T6) were transfected and mixed with sham-transfected cells from the wild-type strain. The ratio of injected cells was compared with the ratio as determined by karyotype studies of the irradiated, reconstituted mouse. All appropriate controls were done. But in neither case was the recombinant construct that was used in the clinical trials studied. The investigations of DHFR are not particularly relevant to the subsequent human studies, because this selectable gene was not used. However, the effect of DHFR was modest at best: with methotrexate treatment of the animals, a selective effect was seen in five independent experiments involving 19 mice, and in two experiments no effect was seen. In the case of the herpes simplex thymidine kinase, the data were even more sparse. Results are reported in only two animals in which the percentage of cells with the T6T6 karyotype were 74% and 84%, respectively, when an absence of any effect would have given a 40% value. Two years later additional data were published on 93 mice that received a mixture of herpes virus thymidine kinase transformed and mock transformed marrow. Perhaps these data were available to the investigators at the time of the clinical trial, but the results were not impressive. Only "33 percent of animals demonstrated at least transient predominance of the karyotype of the transformed marrow population; however, with all tk vectors, karyotype predominance was stable for more than two months in only 13 percent of animals" [8Mercola K.E. Bar-Eli M. Stang H.D. Slamon D.J. Cline M.J. Insertion of new genetic information into bone marrow cells of mice: comparison of two selectable genes.Ann. NY Acad. Sci. 1982; 397: 272-280Crossref PubMed Scopus (7) Google Scholar]. As we are not told what percentage of the mice showed at least transient predominance of the mock transformed cells, and there must have been some such animals, it is not clear that any effect, even the transient one, was actually demonstrated in this larger group. It is evident, then, that clearly positive results were not obtained in mice. However, success in animal studies should not be made a prerequisite to all human clinical investigations; negative animal studies do not preclude success. They merely make success much less likely. Nonetheless, the success or failure of investigations in animal models must have a role in making a risk/benefit assessment. Even had the murine data shown that there was considerable selection in favor of the transformed cells the chances for success of the human studies would have been vanishingly small. It would be less than fair, however, to suggest that as knowledgeable and insightful an investigator as Cline was unaware of this fact. Indeed, Cline states that "the patients were told that the likelihood of it working was very small." It is obvious that his intent was to test the technology [2Cline M.J. Perspectives for gene therapy: inserting new genetic information into mammalian cells by physical techniques and viral vectors.Pharmacol. Ther. 1985; 29: 69-92Crossref PubMed Scopus (20) Google Scholar]. To perform such investigations is a standard practice in the testing of drugs. The principal purpose of "phase 1" studies is to establish a tolerated dose. If a therapeutic effect is achieved, well and good, but therapeutic success is not a primary goal. Of course the risk/benefit ratio must be taken into account, and this becomes increasingly difficult as both the risk and the potential benefit approach zero. It must be remembered that in the time during which these studies were performed there was anxiety bordering on hysteria concerning the risks of recombinant DNA. Fostered in part by the molecular biology community itself, this unreasonable fear was nonetheless a fact of life, and probably contributed, at least in part, to the harsh reaction to Cline's studies. In actuality the 300 rads of ionizing irradiation delivered to these patients probably constituted more of a risk, especially because they were clearly not terminally ill, as suggested by one of the news reports [3Sun M. Cline loses two NIH grants.Science. 1981; 214: 1220Crossref PubMed Scopus (11) Google Scholar]. Nearly five years after the experiment they were stated to be in essentially unchanged condition [2Cline M.J. Perspectives for gene therapy: inserting new genetic information into mammalian cells by physical techniques and viral vectors.Pharmacol. Ther. 1985; 29: 69-92Crossref PubMed Scopus (20) Google Scholar]. Perhaps more important was the fact that Cline had violated the rules that had been established. Although he thought that consent of the institutions where the work was to be done was sufficient, this was not the case, and Cline himself later acknowledged that he had "exercised poor judgment in failing to halt the study and seek appropriate approval" [9Dickson D. NIH censure for Dr Martin Cline: tighter rules for future research plans.Nature. 1981; 291: 369Crossref PubMed Scopus (26) Google Scholar]. Review of the entire episode leads to the conclusion that the clinical studies undertaken by Cline and his colleagues in 1980 were premature. The vehemence of the reaction may well have been conditioned, at least in part, by personal considerations. This event may actually have had a somewhat favorable effect on the development of gene therapy. First, it put clinical investigators on notice that it was necessary to adhere scrupulously to the rules that had been established. Second, because no harm came to the patients, the studies may have helped to abate the unreasoned fear of the consequences of administering recombinant DNA molecules. Yet beyond its effect on the development of gene therapy, the episode is a tragic one because it interrupted the efforts of a highly talented, productive scientist who was in too much of a hurry to see patients benefit from the marvels of modern molecular biology.