Overview of Gene Transfer Gene transfer involves the introduction of a novel transgene into a host genome with the goal of inducing the host cells to produce a desired gene product. Depending on the purpose of the transfer, the transgene may incorporate into the host genome (a longer term option) or it may simply exist as a separate piece of DNA (a shorter term option). Th ere are two distinct types of transfer: germline and somatic. Th e former involves the manipulation of germline cells, leading to heritable for aff ect off spring; the latter deals with the insertion of functional genes into somatic cells, which impacts only the individual whose cells are altered. 1

Gene transfer uses many approaches and techniques for cellular manipulation. Cells can be altered ex vivo , where target cells are removed prior to transformation, or in vivo , where manipulation of target cells is conducted in the organism. Th e insertion of novel genetic information can be conducted through nonviral gene transfer, such as DNA injection, or through viral methods, such as inactivated retroviruses and adenoviruses. 2 Th e use of viral vectors is currently being pursued as a therapeutic technique, as tissue uptake is highly eff ective and as it is the only means of integrating the transgene into the target genome. Recombinant DNA (rDNA) technology, which is a method for combining two or more sequences of DNA using gene-splicing techniques, oft en plays a role in the creation of viral vectors for gene transfer. 3

The Path to Gene Transfer Technology Over the past 70 years, many seemingly distinct, basic laboratory techniques developed, together, have allowed gene transfer technology to fl ourish. In 1944, Avery, MacLeod, and McCarty made the initial discovery of gene transfer while studying an unencapsulated R-variant of a pneumococcus strain (Figure 1).In the course of their experiments, they transformed the strain into an encapsulated one using a “desoxyribonucleic acid fraction,” which they had isolated from encapsulated colonies. Upon breeding the colonies out further, they found these changes were hereditarily transmissible to daughter colonies. 4

In 1962, 9 years aft er the establishment of the structure of DNA, Sybalski and colleagues carried out some of the earliest transformations of mammalian cells using HPRT -deficient, auxotrophic cells in rodents. Th e team attempted to “rescue” these cells using a DNA fraction containing the wild type HPRT gene. Th eir work demonstrated that foreign DNA fragments could

be taken up by target cells and used by those cells to produce the missing gene product; however, because of the state of the techniques and technology at the time, the transformation eff ect was only transient. 5

Th e discovery of reverse transcriptase by Temin, Mitzutani, and Baltimore provided the next brick needed in building the foundation for gene transfer. They found its use in vectors (especially retroviral vectors) to be highly effi cient and capable of infecting nearly 100% of exposed mammalian cells. 6,7 A few years later, Sambrook et al. used the SV40 virus to show that viral DNA is capable of integrating into the host cell genome. Th is was accomplished by labeling viral DNA with radioactive phosphorous, infecting established cell lines, and monitoring where the radioactivity is localizing. 8 Soon aft er the publication of these results, Hill and Hillova published a very similar experiment using polyoma viruses in hen fi broblasts, achieving comparable results. 9

In 1973, 1 year aft er the publication of Hill’s results, Stanley Cohen and Herbert Boyer were credited with the development of rDNA technology—the fi nal tool necessary for gene transfer technology to flourish. 10 Soon after, a calcium phosphate-aided transfection method was developed, which allowed for increased effi ciency in foreign DNA uptake. 11 Th e earliest studies that employed this technology involved the characterization and cloning of human beta-globin into plasmid and viral host sequences. 12 With this last tool in the toolbox, gene transfer was fi nally able to take off .

The Early Days of Gene Transfer With all of the necessary techniques available, some investigators began to actively pursue gene transfer research. Th e fi rst attempt at in vitro application of gene transfer technology took place in 1973 by Rogers et al. In their experiment, they sought to “rescue” cells in lines derived from two argininemia patients by transforming them with the Shope rabbit papillomavirus, which contained an arginase-producing gene. 13 Th ese experiments ultimately failed, because of a lack of understanding of underlying biological mechanisms of viral DNA incorporation. 3

As gene transfer and rDNA research took off , the implications and power of the technology became increasingly clear to those working with it. Prominent scientists in the fi eld, including Paul Berg, put forth a voluntary moratorium on rDNA research until

Translating Gene Transfer: A Stalled Effort Alexandra J. Greenberg , B.A. 1 , Jennifer McCormick , Ph.D. 2 , Carmen J. Tapia , B.A. 1 , and Anthony J. Windebank , M.D. 3

Commentary

1 Mayo Clinic College of Medicine , Center for Translational Science Activities, Rochester, Minnesota, USA; 2 Division of Health Care Policy Research, Mayo Clinic, Rochester, Minnesota, USA; 3 Department of Neurology, Mayo Clinic College of Medicine, Rochester, Minnesota, USA.

Correspondence: AJ Greenberg (greenberg.alexandra@mayo.edu)

DOI: 10.1111/j.1752-8062.2011.00297.x

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Abstract The journey of gene transfer from laboratory to clinic has been slow and fraught with many challenges and barriers. Despite the development of the initial technology in the early 1970s, a standard clinical treatment involving “gene therapy” remains to be seen. Furthermore, much was written about the technology in the early 1990s, but since then, not much has been written about the journey of gene transfer. The translational path of gene transfer thus far, both pitfalls and successes, can serve as a study not only in navigating ethical and safety concerns, but also in the importance of scientist–public interactions. Here, we examine the translational progress of gene transfer and what can be gleaned from its history. Clin Trans Sci 2011; Volume 4: 279–281

Keywords: gene therapy , viruses , commentary

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Greenberg et al. � T ranslating G ene T ransfer

policy and ethical issues surrounding gene transfer could be worked out. A few months later, the National Academy of Science offi cially embraced the moratorium. Soon aft er this development, the National Institutes of Health formed the Recombinant Advisory Committee, primarily as a means of preemptively addressing public concerns about rDNA technology. 14

In 1975, Paul Berg and colleagues organized the Conference on Recombinant DNA Technology at Asilomar, the primary aim of which was to disseminate the moratorium internationally. 15 The conference produced a series of recommendations for experiments based on risk level and containment of vectors and transformed species. Additionally, a series of regulations on specifi c experiments were issued; these included experiments with highly pathogenic organisms, toxin genes, and anything that could pose a potentially serious biohazard. 16 With these guidelines in place, rDNA and gene transfer research resumed. 14

Martin Cline and his team were some of the fi rst to attempt translation of gene transfer into humans. Cline reported that the insertion of human globin gene into mouse bone marrow cells was able to aid in the regeneration of bone marrow in irradiated mice. He fi led a request for review to the UCLA human subjects committee, but went forth with his human experiments before the proposal was reviewed. Cline collected bone marrow from two international patients, treated it with his human globin gene vector, irradiated some of the patients’ cells in vivo , and replaced the altered cells. Th is incident, along with the results of Asilomar, spurred the National Institutes of Health to form a subcommittee of the Recombinant DNA Advisory Committee devoted solely to gene transfer issues, including issues of human subjects approval and administration. 17

Th e fi rst hint of translation and team science emerging as part of gene transfer occurred in 1983 at the Banbury meeting. Th is conference is hailed as the fi rst “truly productive” meeting of researchers and clinicians, in part because, at this point in time, researchers were more actively working on rDNA and gene transfer technology. With a more well-rounded perspective of what was needed from those working at both bench and bedside, participants left able to pursue clinically relevant experiments. 3

One product of these experiments was gene transfer for individuals with adenosine deaminase-defi cient severe combined

immunodeficiency disease (ADA-SCID). Seven years aft er the Banbury meeting, Ashanti de Silva became one of the fi rst two individuals to undergo gene transfer for this condition. Autologus T-lymphocytes were removed from patients, transformed with a corrected human ADA gene, cultured, and reintroduced. Treatment lasted only 2 years, yet the corrected gene persisted in the two patients even aft er the treatment had concluded. de Silva’s story was widely publicized in the popular press, and raised hopes for the future of “gene therapy.” 18 She remains the greatest success story of applied gene transfer to date.

Lessons from Gene Transfer Translation (Thus Far) Despite the success of de Silva’s treatment, progress in the translation of gene therapy beyond initial clinical trials has been slow. For many treatments and discoveries, the average time to translation is around

17 years; gene transfer may be greater than this average already. 19 Th e development of basic laboratory techniques crucial to gene transfer technology happened in quick succession over 20 years. Th e subsequent deceleration of transfer of “gene therapy” to the clinic can be attributed to several situations, including widely publicized tragedies, poor communication between researchers and patients, and omission of steps crucial in research to ensure the safety and effi cacy of novel treatments.

The Death of Jesse Gelsinger In 1999, Jesse Gelsigner, aged 18, was enrolled in a clinical trial for “gene therapy” aimed at treating ornithine transcarbamylase defi ciency (OTC) in infants. Th e study was being conducted at the University of Pennsylvania under the direction of Dr. James M. Wilson. Gelsigner was injected with a corrected viral vector; he died 4 days later of severe adverse complications, including a severe immune response, multiple organ failure, and brain death. Investigation revealed many problems with the informed consent process, including the omission of information regarding Jesse’s true eligibility, serious side eff ects in two prior patients, deaths of several lab animals (including nonhuman primates), and fi nancial confl icts of both researcher and university. 2

While there were many issues with the study that led to this tragedy, there are two particularly pertinent lessons to remember as we move forth with translation of any treatment. Th e fi rst lesson is that safe and eff ective translation requires patience—a fact that seems counterintuitive given the scientifi c community’s goal of “rapid translation.” As with the Cline experiments, steps in the regulatory processes were overlooked in the Gelsigner case. Gelsigner was enrolled in a trial targeted those with a severe, fatal form of OTC; his particular form of the disease was clinically manageable. Additionally, the severe side eff ects and results of the animal studies should have been added as an addendum to the informed consent. While some may feel that regulatory bodies slow down the progress of research and, in particular, translation, restraint needs to be used to ensure that harm to subjects is minimized and that any problems are fully disclosed.

Th e second lesson we can draw from this case is the importance of the “civic scientist” and communication with the public. 20 As

Figure 1. Historical timeline of gene transfer technology. Public policy and publicized events are above; laboratory developments are below.

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to step into the public eye and frankly discuss implications of publicized fi ndings as a means of mediating the hype and hope that may be a result of a press release. If we can establish this connection, this trust, then we can ensure that “failures” in clinical trials will not instill the fear that it currently may in the population, as was seen in the case of the death of Jesse Gelsigner. Literally, translation requires that we translate our fi ndings into terms and concepts that communities can understand.

Finally, safe, rapid translation requires appropriate restraint. Many of the diffi cult issues that came about in the process of gene transfer’s translation arose through regulatory processes. While we need to strive to translate fi ndings to those who could benefi t more quickly, we need to ensure that we do not compromise patients’ health in the process.

translational researchers, it is of the utmost importance that we accurately convey to the public the underlying principles of our research. Management of these communications is key to ensuring that hopes are not raised in vain and to continue to establish trust between scientists and the broader population. 21 Aft er Gelsigner’s death, many articles were published in popular press indicating fear of the technology—that is, fear of what was going on in laboratories and clinics, unknown to the populace.

The SCID-X1 Trials Cavazzana-Calvo et al. published a report in Science in 2000 that they had developed a “defi nitive cure” for children with SCID-X1. 2,22 Eleven children were treated at the Necker Hospital for Sick Children in Paris with a murine leukemia virus containing a corrected γ-c chain cytokine receptor gene. Nine of the 11 children were “cured”; however, 3 years later, it was reported that two patients had developed a leukemia-like disorder. Investigation revealed that the transgene was inserting itself near the LMO2 oncogene, triggering its activation. Similar trials were immediately halted in order to investigate the mechanism by which these serious side eff ects developed. 23

Th e case of SCID-X1 trials is an excellent example of how open communication can facilitate the translation process. Compared with the Gelsigner case, there was a drastic improvement in communication of side eff ects to both patients involved in that trial shortly aft er they occurred. Additionally, the measures taken aft er these side eff ects occurred were in the spirit of translation—that is, researchers took these fi ndings from the bedside back to the bench, further optimize the treatment.

“Gene Therapy” for HIV patients? In February 2011, researchers announced the results of a Phase I Safety Trial at the Conference on Retroviruses and Opportunistic Infections in Boston, MA. Th e study involved six HIV-positive men, all on antiretroviral drugs, whose immune cell counts remained low. Th e study was conducted ex vivo , with researchers removing CD4+ T-cells from subjects, altering them via the insertion of a zinc fi nger enzyme that targets the CCR5 gene, and reinsertion of cells into their respective subjects. 24 With several headlines making statements about scientists fi nding a “cure for AIDS,” the reports released to the broader public on this fi nding were a prime example of the need for scientists to speak up in the media. Some scientists have successfully taken action, making statements to the press about remaining “cautiously optimis[tic].” 25 As with the previously described cases, we need to be wary of prematurely raising the public’s collective hope.

Conclusions Th e translation of gene transfer raises several important issues that translational researchers must grapple with. First, rapid translation requires a well-defi ned target—that is, a specifi c disease or condition of interest. Scientists are currently trying to address a vast number of illnesses using gene transfer. Unlike many other technologies and treatments that have been successfully and rapidly translated from fi rst clinical trials to widespread use, “gene therapy” stands to help those with myriad illnesses. A great diffi culty in quickly translating this technology is that eff orts are not concentrated on one condition; rather, they are spread across many diff erent ailments.

Second is the issue of mediating the public’s understanding and expectations of this technology. Th ere is a dire need for scientists

Acknowledgments The authors thank to Justin E. Juskewitch, B.A. and William S. Brimijoin, Ph.D., for their invaluable communications. Th is publication was supported by NIH/NCRR CTSA Grant Number TL1 RR024152. Its contents are solely the responsibility of the authors and do not necessarily represent the offi cial views of the NIH.

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