A Personal Perspective on the Early,Early History of In Vivo (DNA-Based) Gene Therapy

A lthough I didn't know it at the time, my irreversible path to gene therapy actually was set in 1967. As a newly anointed physician in 1963, I was committed to pursuing science in human subjects. Indeed, my special interest in gout very early in my career was driven, among other reasons, by the fact that this “Disease of Kings and King of Diseases” is exclusive to the human species. Hence, for those wishing to do research on gout, there was no choice but to focus on the human subject. Accordingly, it is perhaps obvious why the best studies of purine metabolism in gouty subjects prior to the mid-1960s, emanating primarily from the laboratories of Wyngaarden (Howell et al., 1961) and Seegmiller (Seegmiller et al., 1955), were complex and highly sophisticated models of outstanding clinical research involving the use of radioactive or heavy isotopes and limited to human subjects. However, essentially all that was really known at that time about regulation of these relevant purine metabolic pathways was extrapolated from enzymology carried out in bacterial or avian systems.

As a resident in Medicine at Parkland Memorial Hospital and the University of Texas Southwestern Medical School (UTSW) in the fall of 1963, I was considering options for further training upon completion of the first 2 years of the program. The Chairman of the Department at UTSW, Dr. Donald Seldin, encouraged me to apply to the National Institutes of Health (NIH), and he agreed to provide strong support for my application. With deep appreciation, I followed his advice. Hence, you can only imagine my excitement when I was selected to go to the NIH, which led in turn to an opportunity to work in the Seegmiller laboratory. From 1965 to 1967, I was to serve as a Commissioned Officer of the Public Health Service with the rank of Lieutenant Commander and my official title was to be Clinical Associate at the NIH. This putative assignment was even further enriched by the fact that, as it turned out, essentially the only real alternative available for me in 1965 (given the persistence of the physician draft) was a 2-year commitment to serve in the armed forces, which would have included, almost certainly, at least 1 year serving as a general medical officer in the middle of the Vietnam War.

When I began my appointment at the NIH, Dr. Jay Seegmiller had just returned from a sabbatical during which he had learned the then-new technology of culturing human cells (initially only skin fibroblasts) derived from specific patients, as well as normal volunteers. He had also just been appointed to serve as Chief of the new Section on Human Biochemical Genetics within the Arthritis and Rheumatism Branch of what was then the National Institute of Arthritis and Metabolic Diseases at the NIH in Bethesda, Maryland. This new scientific tool promised to allow us to study human pathways and their regulation in cells from both normal and diseased subjects. What a breakthrough! I was actually going to be able to use this new technology to study my favorite disease. This opportunity also established for me a pathway in science that I was going to pursue for my entire scientific career. That is, while remaining focused on the human subject, to use the best and most elegant tools available in the biological sciences (and soon to be molecular biology) to apply as soon as possible to the understanding and treatment of human disease.

The first project to which several of us were assigned in Seegmiller's laboratory was to try to understand the basic metabolic abnormalities in a newly recognized (but at that time still controversial) disorder known as the Lesch Nyhan Syndrome (Lesch and Nyhan, 1964). This was a disorder affecting male subjects who had a bizarre form of self-mutilation, mental retardation, choreoathetosis, and the most striking degree of purine overproduction ever described with hyperuricemia and a high incidence of uric acid stone formation. It was this latter metabolic disorder of purine overproduction that attracted our attention as a human model of altered purine metabolism. With an intensive team effort, we discovered in 1967 that these patients had a complete deficiency of the enzyme hypoxanthine guanine phosphoribysltransferase (or HPRT) as the cause of their disease (Seegmiller et al., 1967). Shortly thereafter, we discovered that patients with a partial deficiency of the same enzyme had a severe form of gout, often with uric acid kidney stones, but without the neurologic disorder characteristic of the Lesch Nyhan syndrome (Kelley et al., 1967, 1969). This disease was later to be known as the Kelley Seegmiller syndrome.

I now knew the “Joy of Discovery,” and I knew this is what I wanted to do in my professional life. This decision to pursue a career as a physician-scientist was much to the disappointment of my father who was in the private practice of medicine in Palm Beach, Florida, and was holding a place open for me in his office so that I would be able to join him in practice upon completion of my training. For the rest of his life, whenever he introduced me to his friends (or I introduced him to mine), he always pointed out that he was the real Dr. Kelley.

Upon completion of my 2 years of service as an officer in the Public Health Service assigned to the NIH (and to the Seegmiller lab) and an additional year of Residency in Medicine at the Massachusetts General Hospital, I accepted a position as Assistant Professor of Medicine at Duke under the leadership of another of my heroes, Dr. Jim Wyngaarden, who was Chair of the Department of Medicine. Thus, in the summer of 1968, I was able to begin my independent career in science, and I was truly blessed. Enriched with a constant flow of truly outstanding students, fellows, and postdoctoral trainees, as well as ample funding and superb research space immediately contiguous to the General Clinical Research Unit, I truly had the opportunity of a lifetime to pursue research focused on understanding and treating human disorders of purine metabolism using the emerging advances in basic science developed by others to fuel the enterprise. Today, we would call that translational research.

Over the next 16 years (1968 to 1984), by incorporating many of the wonderful advances taking place in protein chemistry and in molecular biology, we were able to make substantial progress in advancing our understanding of human disorders of purine metabolism. Academically, during the period from 1968 to 1974, I had progressed at Duke to become a tenured Professor and a Clinical Division Chief. From 1974 to 1975, I had the opportunity to spend a year on sabbatical at the Sir William Dunn School of Pathology at Oxford University, learning the most modern techniques of cell fusion and cell hybridization. In 1975, I moved to the University of Michigan to become Professor and Chairman of the Department of Internal Medicine. Fortunately, I was able to attract key scientists to move with me in the transition, and I also was able to recruit outstanding trainees to work with me in this new environment. In the latter group, key among those joining me in the lab shortly after my arrival were Jim Wilson as the first University of Michigan MD, PhD student funded thru the Medical Scientist Training Program (MSTP), Dr. Bev Mitchell as a postdoctoral fellow and Bev Davidson as a graduate student seeking her PhD degree.

In October 1984, Dr. French Anderson published a paper in Science entitled “Prospects for Human Gene Therapy” (Anderson, 1984). This was a largely theoretical dissertation describing what might be possible in the future as it related to the potential of somatic cell gene therapy. He described initial examples of inserting genes into cells in culture (ex vivo) using RNA viruses as the vector for this purpose. Once the gene integrated into the host DNA and the cell began expressing the gene of interest, the modified cells would be added to the organism (and presumably, someday, to the patient). Others had also used DNA vectors for gene transfer (Hamer et al., 1979; Mulligan et al., 1979; Sarver et al., 1981; Gluzman, 1982; Van Doren and Gluzman, 1984; Van Doren et al., 1984; Karlsson et al., 1985, 1986), but based on their publications, apparently only in the context of cell modification in vitro or for use ex vivo. The most striking impact on me at the time was that the diseases he thought most likely to be early candidates for gene therapy in the future were all diseases of human purine metabolism on which we were not only working, but arguably, for each of these diseases, our work was among the best in the world. This included HPRT deficiency, adenosine deaminase deficiency, and purine nucleoside phosphorylase deficiency. Given our long-term commitment to development of therapy for each of these diseases and our expertise in the laboratory, we needed to give serious thought to whether gene therapy was a plausible approach for us to pursue.

Clearly, the disease of most interest to me among those mentioned by Anderson was the Lesch Nyhan syndrome for which we not only were the first to describe the enzyme defect some 17 years earlier, but for which we had made considerable progress in terms of understanding the nature of the normal and mutant genes and proteins as well as the nature of the metabolic aberrations. We were also comfortable that we could control the consequences of the metabolic abnormality in these patients, but this had had no discernible effect in improving the seriously disabling neurologic and behavioral disorder. We also knew that even a small amount of enzyme function, as was found in patients with the Kelley Seegmiller syndrome, might be enough to ameliorate the symptoms since patients with this latter syndrome had little, if any, neurologic disease. Some had also suggested that the central nervous system (CNS) abnormalities in patients with the Lesch Nyhan syndrome were largely, if not entirely, functional since no pathologic finding had been demonstrated suggesting that the disorder might be reversible to some degree. Finally, their CNS disease was so devastating clinically that very aggressive therapy would be an appropriate consideration. However, we had overwhelming evidence that the Lesch Nyhan syndrome was a disease of neuronal cells in the CNS. How would we ever be able to take their cells out of the brain, infect them in vitro with either an RNA or DNA virus carrying the HPRT gene, and then put those cells back in the brain in an appropriate place in order to correct the disorder?

Having been stimulated by the Anderson paper to think about how to approach gene therapy in patients with the Lesch Nyhan syndrome, and assisted by multiple “curb-side consults,” the “aha” moment struck. Could one insert the normal human HPRT gene into a recombinant DNA virus that was capable of entering the CNS in vivo? Could infection of the host with this vector carry the HPRT gene into the neuronal cells of the brain? Could the gene be expressed and produce measurable mRNA and protein? Could we construct a DNA vector that would persist in the nucleus of the neuronal cell, perhaps as an episomal element, continuously producing the normal human HPRT protein after even a single infection? The lack of integration into the host DNA would avoid the possibility of insertional mutagenesis, a concern with RNA vectors, which later proved to be a serious reality (Hacein-Bey-Abina et al., 2003). In addition, could we modify the virus in a way so that it would infect the neuronal cell without harming it? It seemed to us that this in vivo method was feasible as an approach to consider for somatic cell gene therapy in this clinical setting and perhaps would be preferable to the ex vivo method discussed by Anderson and others.

It was during the next few months, after the publication of the Anderson paper, that we began to gear up to tackle this approach. By now, Dr. Tom Palella had joined the faculty and the lab as an extremely creative and well-trained physician-scientist who would be key to this project. Dr. Jim Wilson, whose work in our lab with both the HPRT protein and the gene had been outstanding by every measure, had made this approach possible; but Jim was now moving through his clinical training and he was not available. Dr. Myron Levine, an expert in HSV-1, the DNA virus that we planned to use as the neurotropic vector, agreed that this approach made sense, and he was willing to lend his expertise. We also were fortunate to have Dr. Yugi Hidaka join us as a postdoctoral fellow. He was a superbly trained molecular biologist and problem solver who was to focus most of his effort on this project. For the first time in my research career, we would now need to turn to animal models to pursue our objectives.

Progress was slow and tedious, but eventually highly rewarding. As with much of the progress in science, in some ways, it took a village. However, from my perspective, we would not have been successful without the absolutely critical roles served by Drs. Palella and Hidaka.

In December 1987, we submitted a patent thru the University of Michigan on the use of a DNA vector for gene therapy. Our first definitive paper demonstrating the correction of HPRT deficiency in neuronal cells in culture was published in January 1988 (Palella et al., 1988) and the paper demonstrating proof-of-principle with expression of the HPRT gene in the CNS of mice in vivo using the same HSV-1 vector was presented at a plenary session of the Association of American Physicians in May and published in August 1989 (Palella et al., 1989). After a decade of hard work by all parties, a very broad patent was issued by the U.S. Patent Office in September 1997 (U.S. Patent: Viral-Mediated Gene Transfer System, Patent #5,672,344; Chartrand, 1997). This patent covers any DNA vector (or piece thereof), carrying any human gene, inserted into any tissue or organ, of any mammalian species including man. Based on our own knowledge of the literature and the 10-year review conducted by the U.S. Patent Office, we believe this was the first documentation of the successful use of a DNA vector for somatic cell gene therapy in vivo. We were delighted to have introduced the concept of, and established proof-of-principle in an experimental animal for, an entirely new approach to the treatment of human disease, but we were well aware that this work provided merely the initial foundation upon which would be built over future decades, a therapeutic modality with the potential to substantially advance the practice of medicine.

As our research was moving along well in the late 1980s, I began to realize that the successful development of gene therapy for the widespread application to the treatment, and perhaps even a functional cure, of some human diseases was truly going to require many years and literally thousands of scientists and clinicians around the world. There were a multitude of problems to be solved and questions to be answered, not the least of which would relate to the proper choice of diseases and patients, the development of more effective vectors, the achievement of appropriate levels of gene expression in relevant cells and tissues, and control or prevention of any adverse immune response. Even within a given institution, a serious commitment would be required to be successful. This would include a critical mass of outstanding scientists with multiple projects, a major supporting core facility, considerable expertise in related areas, substantial resources, and a long-term commitment despite the obvious high risks of this endeavor. I personally began to believe that the best use of my time and effort in order to advance this exciting and challenging field, once we had completed this research to the point of proof-of-principle in animals, would be to try to encourage, organize, and support expanded programs in gene therapy at a higher institutional level rather than trying to continue with a significant personal time commitment in the research laboratory. In anticipation of this opportunity and the massive requirements, I attempted in 1988 and 1989 to develop such a program as a multi-school initiative (perhaps an Institute or a Center) based at the University of Michigan.

The University of Michigan is a great university. For me, it had been a wonderful place to serve as a member of the faculty and, indeed, the leadership had been very supportive of many of my prior initiatives over more than a decade, but this effort was not to be successful. For this reason, among several others, I began to consider other professional opportunities.

The most attractive position to me, for a variety of reasons, was that of Executive Vice President of the University of Pennsylvania with responsibilities as CEO of the University of Pennsylvania Medical Center and Dean of the medical school. I accepted this position in the late summer of 1989. Prior to my departure from the University of Michigan, I was able to prepare for submission what was to be the first NIH program project approved for gene therapy. This was entitled “Experimental Models of Gene Therapy” and was funded by the NIH from January 1, 1990, to December 31, 1994. Dr. Francis Collins, at that time a member of the faculty at the University of Michigan, was chosen to serve as the initial PI, because, by October 1, 1989, I had departed for the University of Pennsylvania.

Once I assumed my new role at the University of Pennsylvania, this was the end of my career as an active physician-scientist. However, it was the beginning of a great opportunity to facilitate basic, translational, and clinical research and to apply my skills in a variety of ways to improving the delivery of health care and enhancing medical education. Upon my arrival at Penn, I learned that I had been recruited as “a turnaround agent” by university leadership and trustees who were deeply concerned about what they viewed as decades of decline in the image of the medical school as a leader in the biomedical sciences. Over the prior 14 years as Chairman of the Department of Internal Medicine, I had moved the largest department at the University of Michigan from a 42nd ranking to a 4th place ranking in NIH funding among more than 100 similar departments in the United States. This was achieved largely by identifying, recruiting, and supporting the best young faculty available to their first faculty position. Hence, I had a track record that I believe they found attractive. Accordingly, in those early years at Penn, I found an environment very supportive of change. In addition, I was impressed with not only the quality of the other University officers, but the immense stature, experience, and accomplishments of so many of the university trustees with whom I worked. Reflecting, in part, the rich heritage of the Wharton School and their alumni, this long list included many well-known names in the financial and business world. Among those who were particularly helpful to me, were the CEOs of three of the largest pharmaceutical and biotech companies of that era who served as members of the executive committee of the health system trustee board and with whom I worked very closely. This distinguished list included Dr. P. Roy Vagelos, Chairman and CEO, Merck & Co., Inc., Mr. James Vincent, CEO of Biogen, Inc., and Mr. Robert Cawthorn, CEO of Rhône-Poulenc-Rorer (now Sanofi-Aventis). Indeed, life was good, and the time was right to make progress.

It was during this period in the early 1990s that the field of gene therapy was beginning to expand. This included the first in vitro use of a second DNA virus, a recombinant adenovirus, as a proposed approach to in vivo gene therapy (Rosenfeld et al., 1991), followed by the first actual in vivo studies in human subjects from the Welsh/Genzyme collaboration (Zabner et al., 1993), the Boucher/Wilson collaboration (Boucher et al., 1994), and the Crystal lab (Crystal et al., 1994)—all focused on using this approach for the treatment of patients with cystic fibrosis.

It was also during this period of the early 1990s at the University of Pennsylvania that I was able to foster the development of several major programs, institutes, centers, and departments in areas such as bioethics, bioinformatics, biostatistics, clinical epidemiology, and, indeed, to establish a department of molecular and cellular engineering within the medical school. Among the accomplishments of which I am most proud, however, was the creation in 1992, with University of Pennsylvania approval, of the Institute for Human Gene Therapy.

I knew the best person for this position was my “old” friend, colleague, and former student, Dr. Jim Wilson. Fortunately, we were successful in recruiting Jim to the faculty of the University of Pennsylvania in the spring of 1993 as Director of the Institute for Human Gene Therapy and as Chairman of the Department of Molecular and Cellular Engineering in the medical school to implement this new initiative. It took several years to be able to provide the resources we had committed to make it possible for Jim to succeed. In addition to the formation of the new administrative entities described above, this also included leasing and renovating research space in a neighboring institution since no satisfactory space was available within the medical school at that time, all of which required university support and approval, as well as considerable financial support from the medical school and medical center. However, within 5 years after Jim's arrival, more than 200 faculty in the University were involved in one way or another in this new field of gene therapy.

The death of Jesse Gelsinger in 1999 during a gene therapy study at the Hospital of the University of Pennsylvania was an unfortunate event, but one from which we learned and progressed. Even though The Institute for Human Gene Therapy and the Department of Molecular and Cellular Engineering effectively ceased to exist on July 1, 2002, the gene therapy program was continued on a smaller scale under the direction of Jim Wilson.

Despite this setback to the field, and to Penn specifically, progress worldwide over the past decade has been substantial, with literally hundreds of laboratories contributing to a series of important advances in the field. The strategic importance we placed on gene therapy at Penn in the mid to late 1990s with its unique infrastructure and the recruitment and/or retention of a number of key faculty is clearly paying off as well. Jim Wilson continued to operate critical core laboratories and redirected his research toward the development of second generation vector technologies which are showing tremendous promise in the clinic (Gao et al., 2002). Dr. Jean Bennett was recruited as a junior investigator and doggedly pursued the development of gene therapy for retinal diseases. Dr. Sam Jacobson was brought in to help lead the clinical arm of this program. Jean and Sam reported, in separate clinical trials, the remarkable finding of reconstitution of sight in human subjects with an inherited form of blindness (Maguire et al., 2008; Cideciyan et al., 2009). Dr. Kathy High came to the University of Pennsylvania/Children's Hospital of Philadelphia with the goal of treating hemophilia using gene transfer. Her group was the first to test adeno-associated virus vectors in subjects with hemophilia B (Manno et al., 2006) which set the stage for a second generation phase I trial using vectors developed by Jim Wilson which showed unambiguous long-term benefit to subjects with hemophilia B (Nathwani et al., 2011; Ponder, 2011). Others who have emerged as leaders in gene therapy of inherited diseases include Dr. Dan Rader, in the area of hyperlipidemia, and Dr. Lee Sweeney and Dr. Hansell Stedman, in the area of muscular dystrophy. The strength of our programs have always been and continue to be in the area of gene therapy for inherited diseases with little presence in the area of cancer. This all changed with the recruitment of Dr. Carl June who was persuaded to come to Penn in 1999 by the joint efforts of Jim Wilson and Dr. Craig Thompson. Carl put together the quintessential translational program in cancer gene therapy and has pioneered the use of engineered T cells to block HIV infection and treat cancer (Varela-Rohena et al., 2008; Porter et al., 2011). The implications of his programs are enormous. The work of each of these scientists has been facilitated at Penn at one time or another by the gene therapy program and, I believe, that each of them either came to or remained at Penn over the past 15 years, at least in part, because of our institutional strength in the area.

For myself, I am immensely proud to have been associated with the early, early history of the field of in vivo somatic cell gene therapy and I am pleased to have helped pull together the people and resources to make the University of Pennsylvania one of the leading institutions in the world in the field. I believe, as I have said for more than two decades, that gene therapy and indeed, cell therapy, will be as important to improving human health in the 21st century as antibiotics were in the 20th century (and it will take that long).

I enjoyed immensely service as a member of the Recombinant DNA Advisory Committee and its human gene therapy subcommittee in the late 1980s and early 1990s. I had the honor of serving with French Anderson as the co-chair of the First International Symposium on Human Gene Therapy held at the NIH in December 1991. I also gave the opening plenary lecture at the First International Symposium on Gene Therapy held in Beijing, China, in October 1992. Finally, I was pleased to have provided the largest financial contribution to help start the American Society of Gene & Cell Therapy in the mid-1990s (Stamatoyannopoulos, 2010).

I thought it might be useful to provide this short essay relevant to the early, early history of this emerging field of in vivo gene therapy. For most of you in the field, you will wonder where this guy Kelley came from. That is an appropriate question since I have not been an active scientist for more than 20 years. I have tried to answer that question in this brief vignette. Upon hearing about our patent when it issued in 1997, the iconic Dr. Roy Vagelos, known best for his highly successful years as Chairman and CEO of Merck & Co., Inc. and, more recently, as nonexecutive Chairman of Regeneron Pharmaceuticals, Inc., remarked to me that I was the “Grandfather of the Field.” I like that notion.