"Presumably, if we can create a category of releasable data by regulation, as, for example, we could attempt to do conceivably in the case

of safety and efficacy data, we would then be challenged in court. There would ultimately be a judicial judgment about the intent of Congress in that matter. That is one way to go. The other way to go is to change it." --Donald Kennedy, FDA Commissioner, 1977

Introduction There are certain problems in the regulations of gene therapy enacted and framed by the U.S. Congress, which restrict the disclosure of gene therapy clinical trial data. Certain attempts were made by Donald Kennedy, FDA Commissioner, to create the reasonable category of data regulations. The law governing the confidentiality of safety and effectiveness data submitted to the Food and Drug Administration (FDA) during premarket clinical trials has not changed substantially between 1977 and today. But between 1999 and 2001, FDA and the National Institutes of Health (NIH) tested the road not taken by Commissioner Kennedy, each seeking to achieve by administrative action the disclosure of clinical safety data from privately funded human gene therapy research. The biotechnology industry objected vigorously, and laid the groundwork for what could have been a lawsuit of the sort prophesized by Commissioner Kennedy. This reports presents an outline of various laws in U.S. which regulate gene therapy. Further this report also discusses various methods of gene transfer as a part of gene therapy. The theoretical possibility of applying gene transfer methodologies to the human germline is explored. Transgenic methods for genetically manipulating embryos may in principle be applied to humans. In particular, microinjection of retroviral vector appears to hold the greatest promise, with transgenic primates already obtained from this approach. Sperm-mediated gene transfer offers potentially the easiest route to the human germline, however the requisite methodology is presently underdeveloped. Nuclear transfer (cloning) offers an alternative approach to germline genetic modification, however there are major health concerns associated with current nuclear transfer methods. It is concluded that human germline

gene therapy remains for all practical purposes a future possibility that must await significant and important advances in gene transfer technology.1 Human germline genetic modification is theoretically possible: the technologies of animal transgenesis (pronuclear microinjection, sperm-mediated gene transfer, nuclear transfer, etc) could in principle be applied to humans. The purpose of this paper is to consider the potential for applying the available genetic modification (GM) technologies to the goal of achieving human germline gene therapy. If germline gene therapy does become a technically viable proposition, one crucial question must be asked: Why do it? There is in effect a golden rule applying to disorders potentially amenable to germline gene therapy: in any disorder with enough molecular knowledge available to allow the prospect of germline gene therapy, that same knowledge should also be sufficient to allow detection of the disease-causing sequences via embryo pre-screening. Given the low transfer efficiencies and safety risks available at present (i.e. extrapolating from animal transgenesis), candidate disorders would have to be severe and unavoidable by pre-screening. However, it is conceptually possible that gene transfer technologies improve to the point at which it becomes easier and safer to perform germline gene therapy than to carry out embryo pre-screening. In this futuristic scenario of expanded genetic knowledge coupled with effective gene transfer technology, germline GM might become the preferred therapeutic route. The possibility of human germline genetic modification raises several important and vexing bioethical issues, including questions of responsibility towards future generations, difficulties of distinction between gene therapy and genetic enhancement, and the spectre of eugenics2. Thus, human germline genetic modification is far more ethically contentious than somatic gene therapy. However, such bioethical matters are beyond the scope of the present discussion. Instead, this paper focuses solely upon scientific issues related to human germline gene therapy.

1 2

Int. J. Med. Sci. 2004 1(2): 76-91 Smith KR. Gene therapy: Theoretical and bioethical concepts. Arch Med Res. 2003; 34(4):247-268; Resnik DB, Langer PJ. Human germline gene therapy reconsidered. Hum Gene Ther. 2001; 12(11):14491458; McDonough PG. The ethics of somatic and germline gene therapy. Adolescent Gynecology and Endocrinology- Basic and Clinical Aspects, 1997; 816:378-382.

Touchstones of appraising pertinence to humans. An ideal gene transfer system in the context of human germline gene therapy would have the following features: (a) the ability to deliver transgenes in a highly efficient manner; (b) non-prohibitive cost and expertise requirements; (c) minimal risk of causing insertional DNA damage; (d) low rate of mosaicism; (e) high DNA carrying capacity; (f) the ability to permit adequate and controlled transgene expression; and (g) the ability to target transgenes to precise genomic loci. Unfortunately, no single system amongst the presently available systems is able to provide all of features (a-g) above. Indeed, some gene transfer systems are so thoroughly unsuited to human germline gene therapy that they are not considered here. Of the systems that offer some positive features, in every case major drawbacks exist. In each case, particular scientific advances are required before the methods would be suitable for use in human germline gene therapy. In this respect, methods that require a relatively small degree of scientific research should be seen as more plausible than methods requiring many years of progress towards distant (possibly un obtainable) goals.

A Brief History of the Regulation of Gene Therapy. NIH Oversite Formation of the NIH Guidelines In 1971, scientist Paul Berg announced a plan to join a tumor virus gene with a bacterial DNA sequence for insertion into E. Coli.3 At a 1973 conference, several other scientists recommended that the scientific community refrain from combining animal viruses with bacterial DNA until the National Academy of Sciences (NAS) establish a "study committee" to evaluate the safety of such experiments and craft guidelines to minimize risk.4 The NAS pursued this recommendation, convening a panel chaired by Paul Berg in 1974. The panel recommended the suspension of also experiments deemed to pose an environmental hazard.5 The NAS panel recommended that the Director of the NIH establish a panel to evaluate recombinant DNA safety risks and to propose appropriate safety measures.6 Finally, the panel recommended that an international meeting be convened to "review scientific progress in [the] area [of recombinant DNA technology] and to further discuss appropriate ways to deal with the potential biohazards of
3

See Judith P. Swazey et al., Risks and Benefits, Rights and Responsibilities: A History of the Recombinant DNA Research Controversy, 51 S. CAL. L. REV. 1019, 1022 (1978); Joseph M. Rainsbury, Biotechnology on the RAC--FDAINIH Regulation of Human Gene Therapy, 55 FOOD & DRUG L.J. 575, 575- 576 (2000).
4

See Maxine Singer & Dieter Soll, Letter, Guidelines for Hybrid DNA Molecules, 181 SCIENCE 1114 (1973); see also Decision of the Director, National Institutes of Health to Release Guidelines for Research on Recombinant DNA Molecules, 41 Fed. Reg. 27,902, 27,902-27,903 (July 7, 1976); Swazey et al., supra note 2, at 1023.
5

See Paul Berg et al., Letter, Potential Hazards of Recombinant DNA Molecules, 185 SCIENCE 303 (1974); 41 Fed. Reg. at 27,903. The experiments of concern were those involving 1) the construction of certain DNA sequences that have the potential to introduce new toxins or antibiotic-resistance capabilities into strains of bacteria; and 2) the insertion of cancer-causing viral genes into DNA sequences that have the potential to replicate in bacteria, and thereby be transmitted from such bacteria into humans. See id.
6

See Berg. et al., supra note 4 ("[T]he director of the National Institutes of Health is requested to give immediate consideration to establishing an advisory committee charged with i) overseeing an experimental program to evaluate the potential biological and ecological hazards of the above types of recombinant DNA molecules; ii) developing procedures which will minimize the spread of such molecules within human and other populations; and (iii) devising guidelines to be followed by investigators working with potentially hazardous recombinant DNA molecules.").

recombinant DNA molecules."7 In October 1974, the Department of Health, Education and Welfare (HEW) responded to the call of the NAS by forming a Recombinant DNA Advisory Committee (RAC) to the Director of NIH.8 The initial purposes of the RAC were: 1) "to investigate the current state of knowledge regarding DNA recombinants, their survival in nature and transferability to other organisms"; 2) "to recommend programs of research to assess the possibility of spread of specific DNA recombinants and the possible hazards to public health and to the environment"; and 3) "to recommend guidelines on the basis of the research results."9 As the NAS panel proposed, scientists convened an international research meeting at the Asilomar Conference Center in February 1975 (Asilomar Conference).10 The scientists at the Asilomar Conference made recommendations for safety measures to be taken with recombinant DNA experiments, and concluded that some categories of experiments should be entirely avoided until the safety of such experiments could be more conclusively resolved.11 The RAC held its first meeting immediately after the Asilomar Conference, and agreed to adopt the Asilomar recommendations until it had the opportunity to fully develop its own guidance.12 After a series of RAC meetings, in December 1975 the RAC developed a proposed set of guidelines for review by the
7

Id.

See 39 Fed. Reg. 39,306 (Nov. 6, 1974); see also 41 Fed. Reg. at 27,903; Rainsbury, supra note 2, at 576. HEW created the RAC under the authority of PHSA 222, 42 U.S.C. 217a (1970), which enabled the Secretary of HEW to "appoint such advisory councils or committees ... for such periods of time, as he deems desirable ... for the purpose of advising him in connection with any of his functions." 39 Fed. Reg. at 39,306. In this case, the relevant "function" was the Secretary's authority to "conduct research, investigations, experiments, demonstrations and studies relating to the causes, diagnosis, treatment, control and prevention of the physical diseases and impairments of man." Id. (quoting PHSA 301, 42 U.S.C. 241(a) (1970)).
9

39 Fed. Reg. at 39,306 See 41 Fed. Reg. at 27,903; Swazey et al., supra note 2, at 1031. See 41 Fed. Reg. at 27,903; Swazey et al., supra note 2, at 1031- 1033. See 41 Fed. Reg. at 27,903.

10

11

12

Director of the NIH. After a final exchange between the Director (aided by an Advisory Committee to the Director) and the members of the RAC, the first official iteration of the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) was published in June 1976.13 The initial NIH Guidelines were focused almost exclusively on the problem of establishing adequate containment procedures to prevent the release of potentially harmful recombinant viruses or microorganisms.14 The NIH Guidelines required institutions at which NIH-funded recombinant DNA research was conducted to adhere to a system of containment procedures that were tiered by level of risk. The NIH Guidelines also required these institutions to form Institutional Biosafety Committees (IBCs) that would oversee the institutions' adherence to the requirements of the NIH Guidelines and would act as a liaison between the RAC and the investigators conducting recombinant DNA research at the institutions.15 The NIH Guidelines further mandated that investigators subject to its provisions engage in the detailed containment procedures enumerated in the Guidelines, and to engage in experimental practices in accordance with the requirements and limitations also provided therein.16 In December 1978, a clarification to the NIH Guidelines made it evident that the Guidelines applied to all "research conducted at ... [an] Institution [receiving NIH funding for recombinant DNA research], irrespective of the source of funding ...." 17 RAC's Involvement in Gene Therapy and the Points to Consider

13

See id. at 27,903.

14

See 41 Fed. Reg. at 27,904 (noting that while "[r]ecombinant DNA research offers great promise," it also poses "a potential risk--that microorganisms with transplanted genes may prove hazardous to man or other forms of life," and concluding that "special provisions are necessary for their containment").
15

See id. at 27,920. Initially, the 1BC acronym stood for "Institutional Biohazards Committee," see id., and the name was changed to its current form several years later, see 43 Fed. Reg. 60,080, 60,093 (Dec. 22, 1978).
16

See 41 Fed. Reg. at 27,912-920. See 43 Fed. Reg. 60108, 60128 (Dec. 22, 1978).

17

The RAC began its involvement in the regulation of human gene therapy research during a period marked by a swell in public concern over the safety and ethics of human gene therapy technology. In 1981, a clinician from UCLA named Dr. Martin Cline rebelliously conducted a gene therapy experiment outside the United States, while failing to seek approval from his University Human Subjects Committee and misleading Israeli and Italian regulators.18 In 1982, a bioethics commission initiated by President Carter, the President's Commission for the Study of Ethical Problems in Medicine and Biomedical and Behavioral Research (President's Commission), put out a report entitled "Splicing Life," which discussed in detail the ethical and regulatory issues raised by the prospect of human gene therapy technology.19 The President's Commission urged the involvement of the RAC in the regulation of human gene therapy research, citing its institutional capacity and past success with recombinant DNA technology generally.20 In December 1983, the Working Group on Social and Ethical Issues (Social/Ethical Working Group) explicitly modified the NIH Guidelines to require investigators and institutions governed by the NIH Guidelines to seek RAC review, publication for public comment, and NIH approval of proposals for experiments that involve human gene therapy.21 The Social/Ethical Working Group recommended revision of the NIH
18

See Nelson A. Wivel & W. French Anderson, Human Gene Therapy: Public Policy and Regulatory Issues, in The Development of Human Gene Therapy 671, 672 (Theodore Friedmann, ed. 1999); Rainsbury, supra note 2, at 578. The treatment was for the genetic disease B-thalassemia, and involved the insertion of naked DNA though a calcium phosphate transfection method into an ex vivo culture of the patients' autologous bone marrow cells and the transplantation of those cells into the patients. See Wivel & Anderson, supra, at 673. Dr. Cline received harsh sanctions for his behavior, including censure by the NIH and withdrawal of his NIH grants. See id.; Rainsbury, supra note 2, at 578.
19

PRESIDENT'S COMM'N FOR THE STUDY OF ETHICAL PROBLEMS IN MED. AND BIOMEDICAL AND BEHAVIORAL RES., SPLICING LIFE (1982) [hereinafter SPLICING LIFE]. The Commission was formed in reaction to a letter to President Carter from several religious associations, expressing their concerns over the prospect of human gene therapy. See id. at 7-8.
20

See id. at 84-86. Soon after the Splicing Life report arrived, the House of Representatives held hearings on the prospect of human genetic engineering. See Hearings on Human Genetic Engineering Before the Subcomm. on Investigations and Oversight of the H. Comm. on Science and Technology, 97th Cong., 2d. Sess. 441 (1982); see also Rainsbury, supra note 2, at 579. These hearings did not result in any legislation of lasting import, however. See Rainsbury, supra note 2, at 579.
21

See 49 Fed. Reg. 696, 699 (Jan. 5, 1984) (proposing the addition of a new section III-A-4 to the NIH Guidelines); see also 49 Fed. Reg. 46,266, 46,268 (Nov. 23, 1984) (adopting section III-A-4). Section IIIA-4 defined human gene transfer research as the "[d]eliberate transfer of recombinant DNA or DNA derived from recombinant DNA into human subjects." Id. In conjunction with section IV-C-1-B of the NIH

Guidelines as they relate to human genetic experimentation, in line with three premises: "1) [t]here is currently no other national body that deals with ethical issues in the biomedical field; 2) the RAC's expertise would be supplemented by adding experts in the ethical issues of using human subjects; and 3) the RAC would review proposals on a case-by-case basis in response to investigator research."22 The Social/Ethics Working Group proposed a working group to "conduct initial review of proposals submitted to the RAC [involving genetic engineering in humans] ...."23 This recommendation was readily accepted, and by October 1984 the RAC Working Group on Human Gene Therapy (HGT Working Group) had its first meeting. 24 The initial HGT Working Group was comprised of "three scientists, three clinicians, three ethicists, three lawyers, two specialists in public policy, and a representative of the public." 25 In January 1985, the HGT Working Group published its first proposed "Points to Consider in the Design and Submission of Human Somatic-Cell Gene Therapy Protocols" (Points to Consider) document in the Federal Register for public comment.26

Guidelines, the NIH Director was only allowed to approve protocols for human gene transfer experiments that presented "no significant risk to health or the environment." See 49 Fed. Reg. at 46,271; see also 50 Fed. Reg. at 2942.
22

50 Fed. Reg. 2940, 2940 (Jan. 22, 1985). See 49 Fed. Reg. at 699. See 50 Fed. Reg. at 2941.

23

24

25

Id. The HGT Working Group later took the form of the Human Gene Therapy Subcommittee (HGTS). See 55 Fed. Reg. 7438, 7440 (Mar. 1, 1990); Rainsbury, supra note 2, at 580. The HGTS was eventually disbanded, leaving the RAC as the sole NIH body responsible for case-by-case human gene therapy protocol review. See 57 Fed. Reg. 14,774, 14,776-777 (Apr. 22, 1992).
26

See id. at 2942-2944. Note that in 1991 and again in 1996, FDA published technical guidances on the manufacture of human cell and gene therapy products and the conduct of clinical trials, which are also commonly referred to by the name Points to Consider. See CBER, FDA, POINTS TO CONSIDER IN HUMAN SOMATIC CELL THERAPY AND GENE THERAPY (DRAFT) (1991); CBER, FDA, ADDENDUM TO THE POINTS TO CONSIDER IN HUMAN SOMATIC CELL AND GENE THERAPY (1996). For the purposes of this Paper, "Points to Consider" refers to the document within the NIH Guidelines, unless otherwise noted. In 1994, the NIH formally annexed the Points to Consider to the NIH Guidlines as "Appendix M. "See 59 Fed. Reg. 34,496, 34,528 (Jul. 5, 1994). This article will refer to both Appendix M and the pre-1994 standalone documents as "Points to Consider."

In its initial form, the Points to Consider was primarily a list of questions posed to the gene therapy investigator, the answers to which would be considered during RAC review and NIH approval of the investigator's research protocol.27 One set of questions, labeled "Description of Proposal," posed requests for information such as the objectives and rationale of the proposed research, preclinical data regarding risks and benefits, proposed clinical procedures, patient selection and monitoring, and the proposed informed consent disclosures.28 Two more questions, labeled "Special Issues," concerned "the free flow of information about clinical trials of gene therapy."29 The "Special Issues" questions asked about the degree to which the investigator would allow for public access to "accurate information" about the gene therapy trial subject to review, and whether the investigator and his "funding source" would assert protection under "patent or trade secret laws" for "products" or "procedures" under the proposed study.30 The Points to Consider also required certain other submissions to the RAC, including scientific and nontechnical abstracts of the gene therapy protocol and relevant Institutional Review Board (IRB) and IBC minutes.31 Finally, the August 1985 iteration of the Points to Consider introduced a requirement that investigators conducting human gene therapy research subject to the NIH Guidelines immediately report "serious adverse effects of treatment" to the local IRB and the Office for Protection from Research Risks (OPRR) and file a written report of the adverse event
27

See 50 Fed. Reg. 33,462, 33,463 (Aug. 19, 1985) (describing the Points to Consider as a document "intended to provide guidance in preparing [human gene therapy] proposals for NIH consideration under Section III-A-4 of the NIH Guidelines ...").
28

See id. at 33,463-33,466. These questions asked about a great deal of information that reasonably could be considered trade secret or commercial confidential information. See infra Part III.B (describing Exemption 4 of the FOIA). For example, the Points to Consider asked for "a full description of the methods and reagents to be employed for gene delivery and the rationale for their use," including the structure of the cloned DNA (e.g., gene source, regulatory elements and description of the vector), and the preparation, structure, and composition of the materials that will be given to the patient or used to treat the patient's cells. See 50 Fed. Reg. at 33,464-33,465. The original Points to Consider questions regarding preclinical data were particularly probing, requiring the disclosure of information such as the efficiency of the delivery system, the stability of the viral vector, and toxicity, immunogenicity and oncogenicity data, as well as steps taken to reduce viral vector pathogenicity. See id. at 33,465.
29

See id. at 33,463. See id. at 33,463, 33,466. See id. at 33,466.

30

31

with those bodies as well as with the Office of Recombinant DNA Activities (ORDA).32 This version of the Points to Consider also introduced a requirement for biannual written reports on the "general progress of patients" to be filed with the IRB and ORDA.33 The rationale for the Points to Consider was most fully articulated by a revision to the NIH Guidelines that the HGT Working Group proposed in June 1986. According to this revision, patient safety was a significant motivation for the RAC to review protocols for human somatic-cell gene therapy research intended to treat "life-threatening" or "severely disabling conditions."34 Similarly, the August 1985 Points to Consider noted that the "Description of Proposal" questions principally addressed "[t]he questions usually discussed by IRBs in their review of any proposed research involving human subjects ...," and that these questions "deal[] with the short-term risks and benefits of the proposed research to the patient and to other people ...."35 Thus unlike the original NIH Guidelines, environmental containment was a negligible concern of the human gene therapy trials that the RAC was to review under the Points to Consider.36 The first human gene transfer protocol, a gene marking study, was approved by HGTS and the RAC under the Points to Consider in late 1988.37 A technology policy
32

See 50 Fed. Reg. at 33,466. OPRR was an office within the NIH charged with overseeing the ethical conduct of federally funded human subject research. In 2000, OPRR was elevated from the NIH to sit within the Office of the Secretary of HHS, as the newly named Office of Human Research Protections (OHRP). See 65 Fed. Reg. 37,136 (June 13, 2000). ORDA was an NIH office created along with the NIH Guidelines, to "serve as a focal point for information on recombinant DNA activities and provide advice to all within and outside NIH ...." See 43 Fed. Reg. 60,108, 60,127 (Dec. 22, 1978).
33

See id. at 33,466-33,467. See 51 Fed. Reg. 23,210, 23,210-23,211 (June 25, 1986). 50 Fed. Reg. at 33,464 (emphasis in original).

34

35

36

See 50 Fed. Reg. at 33,463 ("In general, it is expected that somatic-cell gene therapy protocols will not present a risk to the environment as the recombinant DNA is expected to be confined to the human subject. Nevertheless ... the 'Points to Consider' document asks the researchers to address specifically [sic] this point.").
37

See Wivel & Anderson, supra note 17, at 675-676 (Theodore Freidmann ed., 1999); Judith Areen & Patricia King, Legal Regulation of Human Gene Therapy, 1 HUMAN GENE THERAPY 151, 155 (1990).

organization, the Foundation on Economic Trends, delayed the start of this study by bringing litigation to challenge the propriety of the RAC's administrative procedures, but the suit eventually settled, with the Secretary of the Department of Health and Human Services (HHS) agreeing to hold all future RAC deliberations and votes in open public session.38 The following year, the HGTS and RAC approved the first attempt at therapeutic human gene transfer, for the treatment of adenosine deaminase-deficient severe combined immunodeficiency, an immune compromising disease.39 Changes in the Scope of RAC Authority and the NIH Guidelines In June 1994, the RAC revised the NIH Guidelines to limit the RAC's public review process to only those protocols with novel characteristics (such as a novel target disease or vector), that represent an uncertain risk to human health or the environment, or that the RAC otherwise determined would particularly merit public review.40 Several weeks later, a National AIDS Task Force on Drug Development (AIDS Task Force) met to identify ways to expedite research and development for AIDS therapies. In the course of this process, the AIDS Task Force turned its scrutiny toward the RAC, which it viewed as a duplicative body at odds with FDA authority.41 The Task Force suggested a "consolidated review" process would require gene therapy protocols to be submitted solely through FDA.42 The RAC refused at that time to adopt the recommendations of the AIDS Task Force,43 but ultimately agreed to a compromise proposal that would somewhat harmonize
38

See Rainsbury, supra note 2, at 584; Areen & King, supra note 36, at 155. See Wivel & Anderson, supra note 17, at 675-676. See 59 Fed. Reg. 34,472, 34,476 (July 5, 1994) (amending section III-A-2 of the NIH Guidelines).

39

40

41

See 59 Fed. Reg. 43,426, 43,427 (Aug. 23, 1994); ORDA, NIH, ENVIRONMENTAL ASSESSMENT AND FINDING OF NO SIGNIFICANT IMPACT CONCERNING A PROPOSED MODIFICATION OF THE NATIONAL INSTITUTES OF HEALTH GUIDELINES FOR RESEARCH INVOLVING RECOMBINANT DNA 3 (1997) [hereinafter OCTOBER 1997 ENVIRONMENTAL ASSESSMENT].
42

See 59 Fed. Reg. at 43,427. Under consolidated review, the RAC would only review protocols in special circumstances, and then only as an advisory committee to FDA, the latter which would retain ultimate approval authority. See id. Both FDA Commissioner David Kessler and NIH Director Harold Varmus told the AIDS Task Force that they were on board with the Task Force's recommendation. See id.
43

See Rainsbury, supra note 2, at 588.

protocol submissions between FDA and the RAC.44 In 1995, NIH Director Dr. Harold Varmus proposed the formation of two ad hoc "external review committees." One committee, to be chaired by Dr. Inder Verma (Verma Ad Hoc Committee), was to evaluate the RAC processes regarding approval of gene therapy protocols. The other committee was given a "broader mandate to examine research activities, especially those funded by NIH, in the gene therapy area."45 The Verma Ad Hoc Committee counseled against dissolving the RAC or reconstituting it within FDA, but echoed the concerns that caused the RAC to eliminate public review of non-novel protocols.46 The Verma Ad Hoc Committee lobbied for a change in FDA regulations that would "exempt the broad area of gene therapy from many of the proprietary restraints [against public disclosure of clinical trial data] reserved for ordinary therapeutic drug products and biologics that come under FDA review."47 According to the Ad Hoc Committee, the purpose of such an exemption would be to "expedite efforts to monitor and evaluate gene transfer protocols," and thus to "accelerate progress in the clinical application of gene therapy."48

44

See 60 Fed. Reg. 7630, 7639-7649 (Feb. 8, 1995); 60 Fed. Reg. 20,726, 20,727-737 (Apr. 27, 1995) (adoption by the NIH Director of the compromise proposal). The compromise proposal was offered by Philip Noguchi, then Director of the Division of Cell and Gene Therapy (DCGT) within the Office of Therapeutics Research and Review (OTRR) at CBER. See Rainsbury, supra note 2, at 588.
45

Harold Varmus, Letter, NIH Review of Gene Therapy Protocols, 267 SCIENCE 1877, 1889 (1995). The latter committee, co-chaired by Dr. Stuart Orkin and Dr. Arno Motulsky (Orkin-Motulsky Panel), found that few of the gene therapy protocols thus far had been "designed to yield useful basic information," and that investigators and sponsors from academia, government and industry had "overs[old] the results of laboratory and clinical studies," and thereby created a "mistaken and widespread perception that gene therapy is further developed and more successful than it actually is." See Stuart H. Orkin & Arno G. Motulsky, Report and Recommendations of the Panel to Assess the NIH Investment In Research on Gene Therapy 1-2 (1995), available at http:// www4.od.nih.gov/oba/rac/panelrep.pdf. The Orkin-Motulsky Panel thus recommended "a greater focus on the basic aspects of gene transfer," such as issues of vector design, delivery, and regulated gene expression. See id. at 2; Friedmann, supra note 17, at 10.
46

See AD HOC REVIEW COMM., RAC, NIH, EXECUTIVE SUMMARY OF FINDINGS AND RECOMMENDATIONS 1-2 (1995), available at http://www4.od.nih.gov/oba/rac/adhoc-re.pdf.
47

Id. at 2. Id.

48

Despite the expressed sentiment by the Verma Ad Hoc Committee that the RAC ought to retain its approval authority over novel protocols, NIH Director Varmus offered a proposal in late 1996 (Varmus Proposal) that moved back in the direction of the AIDS Task Force recommendations by suggesting that the RAC stop the review of protocols. 49 The Varmus Proposal would, among other things: 1) dissolve the RAC; 2) relinquish to FDA all NIH review and approval authority over individual protocols; 3) form an ORDA Advisory Committee (OAC), a leanly staffed body limited to studying field-wide concerns in safety and ethics; and 4) create periodic Gene Therapy Policy Conferences (GTPC) to discuss such field-wide issues.50 Notably, however, Dr. Varmus was only willing to support an arrangement that would adequately "maintain[] public access to information."51 Thus, Dr. Varmus proposed to "maintain the administration of gene therapy clinical trial data management functions through ORDA and in consultation with the OAC," including the proposed collection of adverse event data and the publication of such data in a "publicly available, comprehensive NIH database."52 The Varmus Proposal was quite controversial, and resulted in a significant outcry among scientists and some concerned members of the public.53 In November 1996, Dr. Varmus reached a compromise proposal, (Varmus Compromise) whereby the RAC would continue to exist and engage in review of individual novel protocols. The new RAC would be somewhat reduced in size, however, and would relinquish to FDA all authority to block gene therapy clinical trials.54 The Varmus Compromise was ultimately approved
49

See 61 Fed. Reg. 35,774 (July 6, 1996). See id; OCTOBER 1997 ENVIRONMENTAL ASSESSMENT, supra note 40, at 3-4. See 61 Fed. Reg. at 35,775.

50

51

52

Id. at 35,774, 33,776; see also id. at 33,776 ("Using current definitions, NIH will continue to capture incoming protocol information, ongoing data [including adverse and significant clinical events], and longterm follow-up data. In compliance with the NIH Guidelines, investigators will continue to be required to register human gene transfer experiments with ORDA to ensure continued public access to [a] comprehensive human gene transfer clinical trial database.").
53

See Rainsbury, supra note 2, at 591 (citing Meredith Wadman, Gene Panel Reprieved After Public Outcry, 384 NATURE 293, 297 (1996)).
54

See 61 Fed. Reg. 59,725 (Nov. 22, 1996).

by the RAC and finally adopted into the NIH Guidelines in October 1997. 55 Under the final compromise, the role of the RAC would be: 1) to review specific novel gene therapy protocols and to transmit recommendations to the NIH Director; 2) to recommend modifications to the Points to Consider to the NIH Director; 3) to recommend GTPC topics to the NIH Director; and 4) to publicly review human gene therapy clinical trial data submitted to ORDA under the reporting requirements of the Points to Consider.56 In the Varmus Compromise, Dr. Varmus remained committed to maintaining the public availability of adverse event data and other data relating to the progress of clinical trials regulated by the NIH Guidelines.57 In furtherance of this purpose, the Varmus Compromise urged the creation of a gene therapy database to pool such data (as annually reported by investigators under the Points to Consider) for convenient public access.58 FDA Oversight In the early days of human gene therapy research in the United States, FDA occupied a role that was narrower than that of the RAC, and certainly one that was less visible and salient to the public.59 However, the concerns at the heart of FDA regulation have over time become the central components in the regulation of human gene therapy: 1) the analysis of the safety and effectiveness of gene therapy; 2) the assurance of research
55

See 62 Fed. Reg. 59,032 (Oct. 31, 1997). See id. at 59,032. See supra note 51 and accompanying text.

56

57

58

See id. at 59,046; see also OCTOBER 1997 ENVIRONMENTAL ASSESSMENT, supra note 40, at 6 ("NIH will maintain and improve public access to human gene transfer information. The ORDA data management process will provide administrative details of protocol registration, annual status reports, and risk assessments. This database will improve public access to human gene transfer clinical trial information, including adverse event reporting.") (emphasis added).
59

Moreover, unlike the RAC, FDA was an institution in government that had long predated the invention of recombinant DNA technology and human gene therapy, and which will persist long after those technologies have become commonplace or are rejected. Thus, as described above, the institutional history of the RAC during the course of the development of human gene therapy technology was somewhat of a rollercoaster ride, whereas the FDA's institutional reactions during this period were far more subtle.

subject safety during the conduct of human gene therapy clinical trials; and 3) the assurance that research subjects give proper informed consent. Although in the early days of recombinant DNA regulation under the NIH Guidelines, FDA flirted with the idea of incorporating the Guidelines into FDA regulation, the FDA regulations ultimately remained rather aloof to the existence of the NIH Guidelines or the RAC. In the late 1970s, when the Guidelines still primarily regulated the microbial containment procedures at institutions conducting federally-funded recombinant DNA research, FDA Commissioner Donald Kennedy proposed adopting regulations that would require parties using recombinant DNA in the manufacture of FDA-regulated products to comply with the NIH Guidelines during the course of their use of recombinant DNA for such purposes.60 Such regulations were never promulgated, however, and compliance with the NIH Guidelines remained voluntary for those using recombinant DNA techniques in the manufacture of drugs outside NIH-funded institutions. In 1984, FDA asserted its intention to regulate the production of food additives, drugs and devices produced by or utilizing recombinant DNA technology.61 This statement was written on a high level of generality, with the bulk of the statement iterating the FDA's general authorities to regulate food, drugs, cosmetics and devices, and focused largely concerned with "traditional" biotechnology products such as recombinant therapeutic proteins.62 In two sentences, however, FDA alluded to human gene therapy: FDA announced its intention to subject "nucleic acids used for human gene therapy ... to the
60

See 43 Fed. Reg. 60,134 (Dec. 22, 1978) ("[T]he Commissioner intends to propose regulations to require that any firm seeking approval of a product requiring the use of recombinant DNA methods in its development or manufacture demonstrate the firm's compliance with the requirements of the NIH guidelines (in effect at the time work involving recombinant DNA is commenced) in connection with any work it has done or will do relating to that product.").
61

See Statement of Policy for Regulating Biotechnology Products, 49 Fed. Reg. 50,878 (Dec. 31, 1984).

62

See id. at 50,880 (speaking at length on the issue of manufacturing challenges for "pharmaceuticals" made with recombinant DNA technology). This is not to say that such biotechnology products were "traditional" by any means in 1984; but it is certainly the case today that while the class of recombinant erythropoietin drugs alone now accounts for $10 billion in annual sales, there has yet to be a single approved gene therapy product on the market. See Alex Berenson & Andrew Pollack, Doctors Reap Millions for Anemia Drugs, N.Y. TIMES, May 9, 2007.

same requirements as other biological drugs"; and remarked that "it is possible that there will be some redundancy" between scientific reviews of gene therapy products performed by FDA and the NIH. This brief mention did not provide the specific statutory authorities under which FDA would regulate human gene therapy, although from context it appears that FDA was relying on provisions such as the "new drug"63 and provisions of the Federal Food, Drug and Cosmetic Act of 1938 (FDCA), as well as the "biologics" 64 provision of the Public Health Service Act of 1944 (PHSA).65
63

See FDCA 505(b), 21 U.S.C. 355(b)(i) (1982). Today, section 505(a) of the FDCA precludes the introduction into interstate commerce of "new drugs" unless either an NDA or an Abbreviated NDA (ANDA) has been filed with FDA and approved. A "new drug" is "any [human] drug ... [that] is not generally recognized among experts ... as safe and effected for use under the conditions prescribed, recommended, or suggested in the labeling thereof ...," or "any [human] drug ... [that], as a result of investigations to determine its safety and effectiveness for use under such conditions, has become so recognized, but which has not, otherwise than in such investigations, been used to a material extent or for a material time under such conditions." Id. 321(p). Section 505(b) of the FDCA in turn defines the content of the NDA, including: reports of all scientific investigations made to show whether the drug is safe and effective for its intended use; a description of manufacturing methods and facilities, and quality controls for manufacturing, processing and packing; characterization of the composition and components of the drug; and the proposed drug labeling. Id. 355(b)(1). Under section 505(d) of the FDCA, the Secretary of HHS (or his delegate, the FDA Commissioner) can refuse to approve an NDA where, inter alia: the investigators failed to provide necessary scientific data to establish safety for the intended use; the evidence shows that the drug is unsafe for the intended use or is subject to degradation or loss of potency; or there is not substantial evidence of the drug's efficacy, as demonstrated by "adequate and well-controlled" clinical investigations. Id. 355(d). See generally 21 C.F.R. pt. 314 (2006) (containing the regulations that govern the NDA process). Section 505(i) of the FDCA is an exemption from the general rule of section 505(a), permitting the Secretary of HHS (or his delegate) to promulgate regulations allowing the delivery in interstate commerce of new drugs "intended solely for investigational use by experts qualified by scientific training and experience to investigate the safety and effectiveness of drugs." FDCA 505(i) 21 U.S.C. 355(i) (1982). The resulting regulations require the sponsor of a clinical trail with an investigational new drug to submit an Investigational New Drug Application (IND) to the FDA. See 21 C.F.R. 312.20(a) (2007). As the statutory provision outlines, the regulations governing INDs require the submission of preclinical data to FDA as well as a system of reporting between the sponsor of the IND and FDA during the course of the investigational clinical trial. See generally 21 C.F.R. pt. 312 (2006) (containing the regulations governing INDs).
64

42 U.S.C. 262(a) (1982). This statute was recodified from the Biologics Act of 1902, see PHSA, 58 Stat. 682, 702 (1944), and retains the scope of the original statute: no person shall "send, carry, or bring for sale, barter or exchange" in interstate or international commerce "any virus, therapeutic serum, toxin, antitoxin, vaccine, blood, blood component or derivative, allergenic product, or analogous product, or arsphenamine or its derivatives (or any other trivalent organic arsenic compound) applicable to the prevention, treatment, or cure of diseases or injuries of man," unless the product has been manufactured at a licensed facility, and meets standards of "safety, purity and potency." Id. 262(a), (d)(1).
65

See 49 Fed. Reg. at 50,878-50,880; see also OFFICE OF TECHNOLOGY ASSESSMENT, HUMAN GENE THERAPY: BACKGROUND PAPER 36 (1984) ("The FDA will become involved in human gene therapy if it involves products such as nucleic acids or genetically modified viruses that are subject to agency regulations (under authority of the Food Drug and Cosmetic Act and the Public Health Service Act).").

In 1986, FDA published a revised and "final" version of its 1984 statement on the regulation of biotechnology.66 FDA essentially repeated its position on human gene therapy from the 1984 statement, adding the possibility that viral components of gene therapy products would also come within the FDA's jurisdiction.67 Although the 1986 statement and the 1984 statement preceding it were masterpieces in the art of brevity,68 their ephemeral treatment of the subject belies the magnitude of responsibility that FDA would undertake in the exertion of such authority. FDA's relationship with the RAC and the NIH in the mid-1980s was far from frictionless. In August 1985, just as the NIH was seeking HHS approval of the first Points to Consider, FDA "exercised its new authority as the official 'lead agency' for biotechnology regulation," and asserted that a proposed interagency Biotechnology Science Board (BSB) should be the source of gene therapy guidance from then on.69 FDA published a memorandum expressing its opinion that the NIH Guidelines could not be published without BSB review, and that FDA's jurisdiction over clinical trials preempts any effort by the NIH to exert approval authority.70 In large part, the substance of this dispute was the difference between the RAC's system of public review of scientific data and FDA's general practice of maintaining IND sponsors' data as confidential.71 Eventually, NIH Director James Wyngaarden and FDA Commissioner Frank Young,
66

See 51 Fed. Reg. 23,309, 23,311 (June 26, 1986).

67

See id. Curiously, FDA dropped its characterization of possible simultaneous NIH review as a "redundancy." See id. ("It is possible that scientific reviews of these products will also be performed by the National Institutes of Health.").
68

That brevity may in part reflect the attitude of Henry Miller, the FDA's liaison to the RAC, who strongly believed that human somatic-cell gene therapy is not particularly different from any other drug technology regulated by the FDA. See Barbara J. Culliton, New Biotech Review Board Planned, 229 SCIENCE 376 (1985).
69

Tim Beardsley, NIH/FDA Dispute Likely to Delay Research, 316 NATURE 567 (1985). The proposed BSB would encompass the Department of Agriculture, the Environmental Protection Agency (EPA), FDA, the National Science Foundation (NSF), and the NIH. See Culliton, supra note 68.
70

See Culliton, supra note 68. See Beardsley, supra note 69.

71

were able to settle the matter and the Points to Consider proceeded to issue.72 Going forward, FDA fulfilled its promise to regulate clinical gene therapy research. In 1989, FDA exerted its authority in approving the first human gene therapy study.73 Dr. W. French Anderson, who along with Dr. Steven Rosenberg submitted this gene marking protocol, had discussed FDA's IND process with the HGTS as early as 1986 or 1987.74 In 1991, FDA issued its first Points to Consider document regarding matters of technical interest to FDA in the conduct of gene therapy INDs, such as preclinical safety data, the characterization and manufacture of the vector, and research subject monitoring protocols.75 In 1993, FDA finally published a statement of the statutory authorities under which it would regulate the use of human gene therapy products in clinical trials and ultimately the introduction of such products in interstate commerce.76 In this statement, FDA asserted that it could wholly rely on existing statutory authorities that were drafted for the regulation of drugs and biologics generally.77 FDA defined "gene therapy products" for the purposes of the statement as "products containing genetic material administered to modify or manipulate the expression of genetic material or to alter the biological

72

Id. See Rainsbury, supra note 2, at 583.

73

74

See Wivel & Anderson, supra note 17, at 674-665. Interestingly, in his own historical discussion of this seminal study, Dr. Anderson did not feel it necessary to call attention to his FDA IND submission. See id. at 675. This was perhaps less an oversight than a reflection of the struggle that Dr. Anderson faced with the HGTS and the RAC, versus the FDA's simple choice to let the study proceed without a clinical hold. See Rainsbury, supra note 2, at 583- 584.
75

See CBER, FDA, POINTS TO CONSIDER IN HUMAN SOMATIC CELL THERAPY AND GENE THERAPY (DRAFT) (1991).
76

See Application of Current Statutory Authorities to Human Somatic Cell Therapy Products and Gene Therapy Products, 58 Fed. Reg. 53,248 (Oct. 14, 1993).
77

See id. at 53,248 ("Existing FDA statutory authorities, although enacted prior to the advent of somatic cell and gene therapies, are sufficiently broad in scope to encompass these new products and require that areas such as quality control, safety, potency and efficacy be thoroughly addressed prior to marketing.").

properties of living cells,"78 and defined "gene therapy" broadly as "a medical intervention based on modification of the genetic material of living cells."79 The 1993 statement revealed that the concept of gene therapy stretches across a broad range of technologies, and thus can be subject to regulation under several distinct authorities. Notably, under this scheme, characterization as a "gene therapy product" need not imply that the product is to be regulated as a biologic. Certainly, gene therapy products containing viral vectors fall within the definition of biological products under section 351 of the PHSA. However, some "chemically synthesized" gene therapy products may meet the definition of "drug" under the FDCA, but would not fall within section 351 of the PHSA.80 FDA further reached the reasonable conclusion that where gene therapy is done ex vivo, it should be regulated as a gene therapy as well as a somatic cell therapy.81 In any event, whether a gene therapy product is a biologic drug, an ordinary drug, or even a somatic cell therapy, a clinical trial using such a product would be regulated in accordance with the ordinary IND provisions of 21 C.F.R. part 312. By 1998, FDA had published a final technical guidance on human somatic cell and gene therapy, and had fully occupied the authority that it first announced a decade and a half earlier.82 FDA machinery was primed, and laid in wait for an approvable gene therapy product.

78

Id. at 53,529. Id. at 53,251.

79

80

A "drug" under FDCA 201(g)(1) is an "article[] intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease in man or animals," or which otherwise is "an article (other than food) intended to affect the structure or any function of the body." 21 U.S.C. 321(g)(1) (2000).
81

See 58 Fed. Reg. at 53,251.

82

See CBER, FDA, GUIDANCE FOR INDUSTRY: GUIDANCE FOR HUMAN SOMATIC CELL THERAPY AND GENE THERAPY (1998) [hereinafter HUMAN SOMATIC CELL THERAPY AND GENE THERAPY].

Scientific perspective: Various Ways of Gene Transfer Gene Transfer to Human Embryos Most transgenic animals have been produced via the introduction of transgenes into embryos, and the associated technology and underpinning science is accordingly well developed. Thus, the human embryo is a potential candidate for human germline gene therapy. Unless dramatic improvements in the technologies are forthcoming, certain transfection methods are not at present a realistic proposition for gene transfer into human zygotes. Such methods include liposome-mediated gene transfer, electroporation, naked DNA uptake and many viral vectors. The problem is one of low transfection frequencies, coupled with fact that zygotes must be harvested (as opposed to grown in vitro). This

leaves pronuclear microinjection and retroviral transfer as the only contenders presently available that might be adapted for use with embryos in human germline gene therapy. Pronuclear Microinjection Jon Gordon in 1980 demonstrated that exogenous DNA could be introduced into the germline simply by the physical injection of a solution of cloned DNA into zygote pronulei83. Subsequently, pronuclear microinjection has become the most widely used method of germline gene transfer, despite the fact that it remains an intrinsically costly and laborious approach. The technique is most established with mice, however gene transfer via pronuclear microinjection has also been carried out with a wide range of other mammals including rats, rabbits, and farmyard animals. Accordingly, it is to be expected that the human zygote should in principle be similarly amenable to gene tranfer via pronuclear microinjection. The microinjection technique is intrinsically simple, although it requires expensive equipment and high levels of skill84. A fine glass needle is loaded with DNA solution. Under the microscope, the needle is guided through the cytoplasm towards one of the zygotes pronuclei. A nanolitre quantity of DNA solution is injected, bringing typically two hundred DNA molecules into the pronucleus. Pronuclear microinjection would be an obvious choice of transgene delivery method for human embryos. The technique is well established in animals, and is likely to be directly applicable to the human zygote85. Zygotes from various mammalian species have particular characteristics that necessitate amendments to the basic (murine) technique. For example, bovine and porcine zygotes are optically opaque, due to the presence of lipid granules in the cytoplasm; this necessitates centrifugation to displace the obscuring cytoplasmic material such that the pronuclei become visible. Similarly, the pronuclei in ovine zygotes are very difficult to visualise, due to sharing a very similar refractive index
83

Gordon JW, Scangos GA, Plotkin DJ, et al. Genetic transformation of mouse embryos by micro-injection of purified DNA. Proc Natl Acad Sci U S A. 1980; 77(12):7380-7384. 84 Hogan B, Beddington R, Constantin F, Lacy E. Manipulating the mouse embryo. New York: Cold Spring Harbor Laboratory, 1994. 85 Houdebine LM. The methods to generate transgenic animals and to control transgene expression. J Biotechnol. 2002; 98(2-3):145-160; Houdebine LM. Transgenesis and medical applications. Pathol Biol. 2002; 50(6):380-387; Wall RJ. Transgenic livestock: Progress and prospects for the future. Theriogenology 1996; 45(1):57-68.

with the cytoplasm; this necessitates the use of top-quality optics, such as differentiation interference contrast (DIC) microscopy, instead of standard phase contrast microscopy. Thus, empirical adjustments enable pronuclear microinjection to be employed with ygotes from essentially any mammal. It would be surprising and unfortunate if the human zygote proved to be an exception to this rule. Indeed, visualisation of the pronuclei in human fertilised eggs is not problematic. Although pronuclear microinjection would probably be usable with human zygotes, the major inherent problems of the method render it less than ideal for human germline gene therapy. A major problem is the relatively low rate of transgene integration: in mice, the overall efficiency of transgenesis (taking into account embryo loss in vitro and in vivo) is typically ca. 2%86. This level of efficiency is perfectly practicable for animal transgenesis, but it would be problematic for humans. Moreover, murine pronuclear microinjection transgene uptake values are several times higher than those achieved with other (nonrodent) species. Accordingly, even with hormonal induction of superovulation, the numbers of zygotes available per woman would be a strongly limiting factor in the potential use of pronuclear microinjection for human germline gene therapy. Embryo pre-screening (preimplantation genetic screening) might be one possible way around the problem of low transgene uptake efficiency. Using established techniques, one or two blastomeres could be taken from 8 cell stage embryos and analysed by PCR for the presence of transgene DNA. However, such pre-screening would not be 100% reliable, due to mosaicism within the early embryo. Following microinjection and successful integration of the transgene sequences, the transgene would be expected to be present in only 50% of the resulting blastomeres. Assuming that in humans, as with mice, 3 blastomeres are recruited to form the entire inner cell mass (ICM)87, then 1 in 8 of theresulting individuals would contain no transgene sequences, another 1 in 8 would contain transgene sequences in 100% of their
86

Bagis H, Papuccuoglu S. Studies on the production of transgenic mice. Turk J Vet Anim Sci. 1997; 21(4):287-292; Page RL, Canseco RS, Russell CG, et al. Transgene detection during early murine embryonic-development after pronuclear microinjection. Transgenic Res. 1995; 4(1):12-17; Hirabayashi M, Takahashi R, Ito K, et al. A comparative study on the integration of exogenous DNA into mouse, rat, rabbit, and pig genomes. Exp Anim. 2001; 50(2):125-131. 87 Bishop JO. Transgenic Mammals. Harlow: Pearson Education, 1999; Gilbert SF. Developmental Biology. Sunderland, Mass.: Sinauer Associates, 1997.

cells, and the remaining 6 from 8 individuals would be mosaics, consisting of 1/3rd or 2/3rd transgene-containing cells. Accordingly, pre-screening would have a failure rate of more than 50%. The only feasible way that pre-screening might work at an acceptable level of efficiency would be to screen blastocyst-stage embryos. However, blastocyst biopsy techniques are in their infancy, and it remains to be seen whether such techniques could be applied to ICM cells (as opposed to trophoblast cells). Extrapolating from murine data, it would typically require ca. 50 zygotes to produce one genetically modified individual. Assuming 8 eggs per superovulation cycle, it would take approximately 6 months per woman to obtain 50 eggs. Pronuclear microinjection involving such a period of time, if coupled with effective blastocyst pre-screening to select for the small number of transgenecontaining embryos, might be a feasible means of performing human germline gene therapy. However, reported pronuclear microinjection efficiency values are significantly lower for most mammals other than mice. If human pronuclear microinjection turned out to have a similar efficiency as that obtained with sheep or pigs, then the time taken per genetically modified individual would be ca. 5-fold longer i.e. more than 2.5 years. And if the rate of transgenesis turned out to be similar to that obtained with cattle, the time would extend beyond 8 years. The efficiency of transgene uptake through pronuclear microinjection is simply not known for humans, nor can it be known a priori. Thus, a circular problem exists: only if the efficiency turned out to be fortuitously high (i.e. similar to murine rates) would there be any point in attempting the technique with humans but the necessary data on efficiency could only come from actual attempts with humans. Another problem associated with pronuclear microinjection concerns transgene expression. Only around 60% of pronuclear microinjection-derived mice show transgene expression. Furthermore, in the animals showing expression, there are frequently problems of low-level expression or inappropriate expression (e.g. non-tissue-specific, non-temporal). Accordingly, pronuclear microinjection as a means to human germline gene therapy requires improvements in transgene expression. It is the non-targeted nature of transgene integration associated with pronuclear microinjection that is the root cause of expression problems. Some improvements may come from advances in transgene design, such as the use of matrix attachment regions (MARs) or locus control

regions (LCRs): placed on either side of a gene within a transgene construct, these insulator sequences appear to allow the gene to occupy a separate chromosomal domain and thus avoid position-related expression problems88. However, the best solution would be to target transgenes to precise genomic loci, and at present this is not possible with pronuclear microinjection. Given the fact that even the best designs of targeting transgene undergo random integration more frequently than targeted integration, the only foreseeable way to achieve high efficiency gene targeting with pronuclear microinjection would be to stimulate homologous recombination (HR) by co-injecting appropriate recombinase enzymes with the transgene. However, elucidation of such enzymes is at an early stage, and it remains to be seem whether this approach could ever provide the quantum leap improvements in targeting efficiency that would be required in the case of human germline gene therapy. Random integration also raises the concern that an endogenous gene will be damaged by transgene insertion. The degree of risk for any one insertion event must approximate to the proportion of coding sequences (plus controlling elements) within the human genome, a figure of no more than 2%. Thus, endogenous gene damage may be expected to occur in around 1 in every 50 human zygotes integrating transgene DNA. In embryos sustaining such damage, there are several possible outcomes: (a) where a developmentally crucial gene is damaged, the result is likely to be embryo death, and the subsequent non-appearance of a genetically modified individual; (b) where one allele of an important gene is affected, haplosufficiency may permit the development of a normal or near-normal genetically modified individual; (c) where a non-essential gene is affected (such as an allele for hair colour, or a repeated gene), the resulting genetically modified individual may contain a phenotypic change that has no health implications; or (d) where an important gene is affected, debility is likely to occur in the resulting genetically modified individual. Outcomes (a-c), while not desirable, would not necessarily be highly problematic, and the occurrence of these outcomes means that the undesirable outcome (d) would occur at a frequency significantly lower than 1 in 50 genetically modified individuals. Nevertheless, such magnitude of risk implies that pronuclear microinjection in its present stage of development is not acceptable as a means to human germline gene therapy.
88

Smith KR. Gene transfer in higher animals: theoretical considerations and key concepts. J Biotechnol. 2002; 99(1):1-22.

Retroviral Transfer The genome of retroviruses can be manipulated to carry exogenous DNA. Retroviral vectors (RVVs) are one of the most frequently employed forms of gene delivery in somatic gene therapy89. Additionally, RVVs are able to deliver genes to the germline, as established in animal transgenesis90. Zygotes may be incubated in media containing high concentrations of the resultant retroviral vector. Alternatively, retroviral vector-producing cell monolayers may be used, upon which zygotes are co-cultivated. In either case, up to ca. 90% of (surviving) embryos will be infected. Following zygote transfer into pseudopregnant females, the infected embryos should give rise to transgenic offspring. Molecular genetic analysis of transgenics produced in this way usually show integration of a single proviral copy into a given chromosomal site. Rearrangements of the host genome are normally restricted to short direct repeats at the site of integration. In many embryos the germline cells contain viral integrants: thus, transmission of the transgene to the next generation will often occur. Methods have also been developed to allow infection into postimplantation embryos. In this context, virus uptake is effective for many somatic cell lines, however germline cells are infected at low frequency, due to a high level of mosaicism91. Retroviral transfer remains an alternative to pronuclear microinjection in the context of human germline gene therapy. Traditional RVVs would be of minimal potential use, due to the high levels of mosaicism associated with these

89

Braas G, Searle PF, Slater NKH, Lyddiatt A. Strategies for the isolation and purification of retroviral vectors for gene therapy. Bioseparation 1996; 6(4):211-228. 90 Morgan RA, French Anderson W. Human Gene Therapy. Annu Rev Biochem. 1993; 62:191;Hu WS, Pathak VK. Design of retroviral vectors and helper cells for gene therapy. Pharmacol Rev. 2000;52(4):493511; Cepko CL, Roberts BE, Mulligan RC. Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell 1984; 37(3):1053-1062.
91

Braas G, Searle PF, Slater NKH, Lyddiatt A. Strategies for the isolation and purification of retroviral vectors for gene therapy. Bioseparation 1996; 6(4):211-228; Morgan RA, French Anderson W. Human Gene Therapy. Annu Rev Biochem. 1993; 62:191.

vectors. However, the new generation of lentiviral vectors would avoid such problems92. These vectors have the additional advantage of high gene transfer rates (7080% of animals born are transgenic). Accordingly, lentiviral vectors represent plausible candidates for human germline gene therapy. However, the small insert capacity (9-10 kb) would preclude the transfer of many human genes. Additionally, control possibilities are less with RVV delivered transgenes compared with transgenes delivered by microinjection. The safety problems associated with RVVs (insertional oncogenesis, viral reactivation) would also be a major concern93. In principle, judicious genetic alteration of the lentivirus genome would ensure that the resultant vector would have a very high level of safety. However, given the critical context of human germline gene therapy, one would have to question whether our basic scientific understanding of retroviruses is sufficiently advanced to empower rational vector design. Somatic gene therapy provides a salutary lesson here. Human trials involving several hundred patients have been carried out for over a decade using RVVs. Despite the theoretical risks referred to above, a lack of reports of serious adverse affects has resulted in a growing acceptance of the practical safety of RVVs. However, it has been recently reported that two patients (both young children) being treated for X-linked severe combined immunodeficiency disease (SCIDX) using RVV-based vectors have developed leukaemia. In both patients, RVV had integrated into a gene (LMO2) known to cause leukaemia if activated inappropriately. It is not known why the same endogenous gene had been targeted by the RVV concerned. The full cause of leukaemia in these patients is still under investigation, however the fact that both patients share the same integration site, coupled with the fact that the patients were both from the same (10-patient) trial, strongly implicates the particular RVVs

92

Ikawa M, Tanaka N, Kao WWY, Verma IM. Generation of transgenic mice using lentiviral vectors: A novel preclinical assessment of lentiviral vectors for gene therapy. Mol Ther. 2003; 8(4):666-673; Lois C, Hong EJ, Pease S, et al. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 2002; 295(5556):868-872; Pfeifer A, Ikawa M, Dayn Y, Verma IM. Transgenesis by lentiviral vectors: Lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc Natl Acad Sci U S A. 2002; 99(4):2140-2145.
93

Cornetta K, Morgan RA, Anderson WF. Safety issues related to retroviral-mediated gene transfer in humans Human Gene Ther. 1991; 2(1):5-14; Gunter KC, Khan AS, Noguchi P. The safety of retroviral vectors. Human Gene Ther. 1993; 4(5):643; Temin HM. Safety considerations in somatic gene-therapy of human-disease with retrovirus vectors. Hum Gene Ther. 1990; 1(2):111-123.

employed in this trial94. Indeed, clinical trials involving this particular RVV-based therapy have been halted pending further investigations and pre-clinical trials [30]. It is to be hoped that enhanced RVV design will prevent any recurrence of iatrogenic leukaemia or similar serious adverse affects in somatic gene therapy. However, the occurrence of such adverse RVV effects lends weight to the argument that more basic virology is needed before any potential human germline gene therapy RVV could be deemed sufficiently safe. At the very least, extensive in vitro (cell culture) and in vivo (mammalian transgenesis) experimentation would be required in order to establish the safety of any proposed RVV (lentiviral-based or otherwise) for human germline gene therapy. Microinjection of Retroviral Vector A combination of microinjection with retroviral vectors has proved successful with bovines95 and primates96. In the primate case, microinjection was used to deliver a retroviral vector into the perivitelline space of 224 mature rhesus oocytes. (The oocytes were subsequently fertilized by intracytoplasmic sperm injection.) The retroviral vector particles had an envelope type known to recognise and bind to the membrane of all cell types. The retroviral vector was microinjected at a developmental stage at which the oocyte nuclear membrane was absent, thus permitting nuclear entry. From 20 embryo transfers, three animals were born, one of which was transgenic. Additionally, a miscarried pair of twins was transgenic. Although this combined method of gene transfer is laborious, it is the only approach that has permitted the generation of transgenic primates thus far. Given the success with primates of
94

Buckley RH. Gene therapy for SCID - a complication after remarkable progress. Lancet 2002; 360(9341):1185-1186; Kaiser J. Gene Therapy - Seeking the cause of induced leukemias in X-SCID trial. Science 2003; 299(5606):495-495; Gansbacher B, Danos O, Dickson G, et al. French group reports on the adverse event in a clinical trial of gene therapy for X-linked severe combined immune deficiency (X-SCID) - Position statement from the European Society of Gene Therapy (ESGT). J Gene Med. 2003; 5(1):82-84.
95

Fox JL. US authorities uphold suspension of SCID gene therapy. Nat Biotechnol. 2003; 21(3):217-217; Chan AWS, Homan EJ, Ballou LU, et al. Transgenic cattle produced by reverse-transcribed gene transfer in oocytes. Proc Natl Acad Sci U S A. 1998; 95(24):14028-14033.
96

Chan AWS, Chong KY, Martinovich C, et al. Transgenic monkeys produced by retroviral gene transfer into mature oocytes. Science 2001; 291(5502):309-312.

microinjection of RVV into oocytes, this approach is likely to be effective for human germline gene therapy. The drawbacks would be similar to those associated with (a) microinjection (i.e. embryo loss) and (b) RVVs (i.e. transgene size limitations, problems with control of expression, safety risks). The process would also be laborious, but it would be expected to avoid the problems of mosaicism associated with most RVVs. Additionally, this form of gene transfer might require fewer eggs than required for pronuclear microinjection. The reported overall rate of transgenesis with rhesus monkeys was 1.3%; although this compares unfavourably with murine efficiencies (up to ca. 6% transgenic), it is significantly better than the rates achieved for animals such as sheep, cows and pigs. Moreover, this combined technique is in its infancy, and its efficiency may well improve with use. Sperm-mediated Gene Transfer The scientific literature contains over forty reports of the successful in vitro uptake of exogene constructs (transgenes) by animal sperm cells97. A majority of these reports provide evidence of post-fertilisation transfer and maintenance of transgenes. Several of the studies report the subsequent generation of viable progeny animals, the cells of which contain transgene DNA sequences. While a minority of studies have used augmentation techniques (electroporation or liposomes) to force sperm to capture exogenes, the standard methodology is very straightforward: prior to in vitro fertilization (IVF) or artificial insemination (AI), washed sperm cells are simply incubated in a DNAcontaining solution. As a potential tool for genetically manipulating animals, spermmediated gene transfer (SMGT) has the advantages of simplicity and cost-effectiveness, in contrast with more established methods of transgenesis such as pronuclear micrinjection. However, despite the above successes and regardless of its potential utility, SMGT has not yet become established as a reliable form of genetic modification. Concerted attempts to utilise SMGT have often produced negative results. The most notable example of such a failure is to be found in the collated results of several independent research groups: of 890 mice analysed, not a single animal contained
97

Gandolfi F. Sperm-mediated transgenesis. Theriogenology 2000; 53(1):127-137; Smith KR. Sperm cell mediated transgenesis: A review. Anim Biotechnol. 1999; 10(1-2):1-13.

transgene DNA98. Indeed, some biologists have expressed scepticism of the fundamental basis for SMGT99. Such scepticism is posited on the assumption that major evolutionary chaos would result if sperm cells were able to act as exogene vectors. Given that the reproductive tracts contain free DNA molecules (originating from natural cell death and breakage), it seems reasonable to expect sperm cells to be highly resistant to the risk of picking up such molecules100. Nevertheless, there exists a fairly well established body of empirical data showing that sperm cells are able, at least under particular experimental circumstances, to interact with and carry exogenes101. Furthermore, isolated reports of the successful use of SMGT for genetic modification continue to be published. A notable recent example is the generation of several transgenic pigs following the artificial insemination of sows with sperm cells preincubated with transgene DNA102. There are two possible ways to make sense of the above experimental and theoretical considerations. The first possible explanation is that SMGT is fundamentally unattainable. If so, the empirical evidence in support of SMGT must be faulty. For example, perhaps sperm can associate with exogenous DNA but cannot convey the DNA into the oocyte; and transgene sequences may have been erroneously identified in tissue samples, perhaps due to DNA contamination affecting sensitive detection methods such as PCR. This scenario is certainly not impossible: scientific research contains several examples of theory being misled by mistaken data. Indeed, early reports of SMGT were compared with the (then contemporary) claims of
98

Brinster RL, Sandgren EP, Behringer RR, Palmiter RD. No simple solution for making transgenic mice. Cell 1989; 59(2):239-241.
99

Birnstiel ML, Busslinger M. Dangerous Liaisons - Spermatozoa as natural vectors for foreign DNA. Cell 1989; 57(5):701-702; Chen TM, Chen YH. Transgenic sperm or deadly missiles? Fertil Steril. 1996; 66(1):167-167.
100

Smith KR. The role of sperm-mediated gene transfer in genome mutation and evolution. Med Hypotheses 2002; 59(4):433-437.
101

Zani M, Lavitrano M, French D, Lulli V. The mechanism of binding of exogenous DNA to sperm cells: factors controlling the DNA uptake. Exp Cell Res. 1995; 217(1):57; Maione B, Lavitrano M, Spadafora C, Kiessling AA. Sperm-mediated gene transfer in mice. Mol Reprod Dev. 1998; 50(4):406-409.
102

Lavitrano M, Stoppacciaro A, Bacci ML, et al. Human decay accelerating factor transgenic pigs for xenotransplantation obtained by sperm-mediated gene transfer. Transplant Proc. 1999; 31(1-2):972-974; Lazzereschi D, Forni M, Cappello F, et al. Efficiency of transgenesis using sperm-mediated gene transfer: Generation of hDAF transgenic pigs. Transplant Proc. 2000; 32(5):892-894.

cold fusion in physics103. By contrast, the second possible explanation is that SMGT is viable, and that the claims of experimental success were not made in error. In this case, the explanation for the successful results must be that certain favourable factors applied in the fortuitous cases in which transgenes were taken up and transferred by sperm. Accordingly, several researchers have made efforts to elucidate such hidden parameters. Underpinning such research into hidden factors has been the notion of the existence of inhibitory factors (IFs) associated with sperm cells. These IFs are envisaged to prevent exogenous DNA uptake so as to protect the genetic integrity of the conceptus. The corollary of this notion is that successful instances of sperm cells taking up exogenous DNA may be attributed to the fortuitous removal or inhibition of IF(s)104. Seminal fluid reportedly contains an inhibitory factor (IF-1) that appears to actively block the binding of exogenous DNA to sperm and to the above-mentioned proteins [44]. Additionally, three classes of proteins identified in sperm cells have been claimed to exhibit DNA-binding properties105. There is also some evidence that the binding of transgene DNA can trigger the activation of endogenous nucleases in sperm cells, which cleave both transgene and sperm chromosomal DNA106. The possible existence of IF(s) or other mechanisms against foreign DNA may explain the varied and often negative results obtained from attempts to use sperm to act as transgene vectors. A superficial binding of
103

Birnstiel ML, Busslinger M. Dangerous Liaisons - Spermatozoa as natural vectors for foreign DNA. Cell 1989; 57(5):701-702; Chen TM, Chen YH. Transgenic sperm or deadly missiles? Fertil Steril. 1996; 66(1):167-167; Smith KR. The role of sperm-mediated gene transfer in genome mutation and evolution. Med Hypotheses 2002; 59(4):433-437; Zani M, Lavitrano M, French D, Lulli V. The mechanism of binding of exogenous DNA to sperm cells: factors controlling the DNA uptake. Exp Cell Res. 1995; 217(1):57; Maione B, Lavitrano M, Spadafora C, Kiessling AA. Sperm-mediated gene transfer in mice. Mol Reprod Dev. 1998; 50(4):406-409.
104

Zani M, Lavitrano M, French D, et al. The mechanism of binding of exogenous DNA to sperm cells factors controlling the DNA uptake. Exp Cell Res. 1995; 217(1):57-64.
105

Lavitrano M, French D, Zani M, et al. The interaction between exogenous DNA and sperm cells. Mol Reprod Dev. 1992; 31(3):161-169; Lavitrano M, Maione B, Forte E, et al. The interaction of sperm cells with exogenous DNA: A role of CD4 and major histocompatibility complex class II molecules. Exp Cell Res. 1997; 233(1):56-62.
106

Sotolongo B, Lino E, Ward WS. Ability of hamster spermatozoa to digest their own DNA. Biol Reprod. 2003; 69(6):2029-2035; Spadafora C. Sperm cells and foreign DNA: a controversial relation. Bioessays 1998; 20(11):955-964; Szczygiel MA, Moisyadi S, Ward WS. Expression of foreign DNA is associated with paternal chromosome degradation in intracytoplasmic sperm injection-mediated transgenesis in the mouse. Biol Reprod. 2003; 68(5):1903-1910.

exogenous DNA to sperm cells would be very unlikely to result in successful transgenesis, given the rigours of fertilisation. Conceptually, therefore, it is necessary to envisage the exogenous DNA being actively taken up by the sperm cell. Ultrastructural autoradiographic studies have indicated that exogenous DNA becomes concentrated within the posterior part of the nuclear area of the head, the inference being that binding of DNA by the sperm is followed by internalisation107. One very interesting possibility is the combination of naked DNA autouptake with microinjection, a process that has been termed transgenICSI. In this recent approach, sperm exposed to naked transgene molecules are microinjected into oocytes. Success has been reported with mice, with approximately 20% of founder animals integrating and expressing the transgene108. Transgene uptake and expression following transgenICSI has also been reported in rhesus monkey embryos109 and porcine embryos110, although transgenic offspring did not result. The success of transgenICSI provides support for the notion that sperm are indeed able to act as transgene vectors. However, some caution is required in making such a conclusion. Firstly, the experiments conducted need to be repeated and built upon before it can be said with certainty that the effect is a real one. Secondly, it could be the case that the trangene molecules bound only weakly to the
107

Francolini M, Lavitrano M, Lamia CL, et al. Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells. Mol Reprod Dev. 1993; 34(2):133-139; Camaioni A, Russo MA, Odorisio T, et al. Uptake of exogenous DNA by mammalian spermatozoa specific localization of DNA on sperm heads. J Reprod Fertil. 1992; 96(1):203-212.
108

Szczygiel MA, Moisyadi S, Ward WS. Expression of foreign DNA is associated with paternal chromosome degradation in intracytoplasmic sperm injection-mediated transgenesis in the mouse. Biol Reprod. 2003; 68(5):1903-1910; Perry ACF, Wakayama T, Kishikawa H, et al. Mammalian transgenesis by intracytoplasmic sperm injection. Science 1999; 284(5417):1180-1183.
109

Chan AWS, Luetjens CM, Dominko T, et al. Foreign DNA transmission by ICSI: injection of spermatozoa bound with exogenous DNA results in embryonic GFP expression and live Rhesus monkey births. Mol Hum Reprod. 2000; 6(1):26-33; Chan AWS, Luetjens CM, Dominko T, et al. TransgenICSI reviewed: Foreign DNA transmission by intracytoplasmic sperm injection in rhesus monkey. Mol Reprod Dev. 2000; 56(2):325-328.
110

Lai LX, Sun QY, Wu GM, et al. Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes. Zygote 2001; 9(4):339-346; Nagashima H, Fujimura T, Takahagi Y, et al. Development of efficient strategies for the production of genetically modified pigs. Theriogenology 2003; 59(1):95-106.; Joris H, Nagy Z, Van de Velde H, et al. Intracytoplasmic sperm injection: laboratory set-up and injection procedure. Hum Reprod. 1998; 13:76-86.

sperm cells, such that only direct delivery (by ICSI) permitted the DNA to remain in place. If so, then this would not support the notion that SMGT can work when used with IVF or AI, because weakly bound or superficially located DNA might be stripped away and lost from the incoming pronucleus during fertilisation. If it were correct that ICSI is an indispensable part of the process, then SMGT would appear to have little advantage over pronuclear microinjection in terms of inherent technical difficulties and expense. However, the efficiency of the process does appear to be somewhat better than that of pronuclear microinjection. The available experimental data on standard human ICSI (i.e. not involving genetic modification) indicate that: (a) the majority (ca. 75%) of eggs are successfully fertilised; and (b) lysis following ICSI occurs at a relatively low rate (ca. 10%)111. For transgenICSI, the reported rates of success (i.e. transgenics per transfer) vary, but a figure of around ca.35% is fairly typical 112. Whereas it would be somewhat surprising if sperm cells have the inherent ability to easily capture and transfer naked transgene molecules such that the DNA remains in place during fertilisation, it remains conceptually possible to use transfection techniques to force sperm cells to capture (and thus transfer) exogenous DNA. Success has been claimed in this regard using electroporation and liposomemediated gene transfer. Since 1990, several reports claiming successful transgene uptake and/or transfer following electroporation of sperm cells have
111

Joris H, Nagy Z, Van de Velde H, et al. Intracytoplasmic sperm injection: laboratory set-up and injection procedure. Hum Reprod. 1998; 13:76-86; Van Steirteghem A, Nagy P, Joris H, et al. Results of intracytoplasmic sperm injection with ejaculated, fresh and frozen-thawed epididymal and testicular spermatozoa. Hum Reprod. 1998; 13:134-142; Mansour R. Intracytoplasmic sperm injection: a state of the art technique. Hum Reprod. 1998; 4(1):43-56; Huguet E, Esponda P. Foreign DNA introduced into the vas deferens is gained by mammalian spermatozoa. Mol Reprod Dev. 1998; 51(1):42-52.
112

Szczygiel MA, Moisyadi S, Ward WS. Expression of foreign DNA is associated with paternal chromosome degradation in intracytoplasmic sperm injection-mediated transgenesis in the mouse. Biol Reprod. 2003; 68(5):1903-1910; Perry ACF, Wakayama T, Kishikawa H, et al. Mammalian transgenesis by intracytoplasmic sperm injection. Science 1999; 284(5417):1180-1183. Chan AWS, Luetjens CM, Dominko T, et al. Foreign DNA transmission by ICSI: injection of spermatozoa bound with exogenous DNA results in embryonic GFP expression and live Rhesus monkey births. Mol Hum Reprod. 2000; 6(1):26-33. Chan AWS, Luetjens CM, Dominko T, et al. TransgenICSI reviewed: Foreign DNA transmission by intracytoplasmic sperm injection in rhesus monkey. Mol Reprod Dev. 2000; 56(2):325328; Lai LX, Sun QY, Wu GM, et al. Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes. Zygote 2001; 9(4):339-346; Nagashima H, Fujimura T, Takahagi Y, et al. Development of efficient strategies for the production of genetically modified pigs. Theriogenology 2003; 59(1):95-106; Joris H, Nagy Z, Van de Velde H, et al. Intracytoplasmic sperm injection: laboratory set-up and injection procedure. Hum Reprod. 1998; 13:76-86.

been published, and there have been a number of reports of sperm cells taking up liposome-encapsulated DNA [34]. More research is clearly needed to determine whether and to what extent transfection techniques such as liposome-mediated gene transfer or electroporation may be able to augment SMGT. Nevertheless, given that these gene transfer techniques have been shown to work with a wide range of somatic cell types, in vitro and in vivo, there is no reason to presume that sperm cells are inherently unable to be transfected using such methods. An alternative possibility could be to introduce the transgene into testicular (sperm) stem cells in vivo. This would in principle remove the need to collect, manipulate or transfer eggs, thus providing a major streamlining of germline GM. Preliminary results have been reported in mice, where transgene constructs were directly injected into the testis. For example, 60-70% of sperm were reported to carry the transgene following injection of naked DNA into the vas deferens [60], with a follow-up reportclaiming detection of the transgene in the cells of 7.5% of offspring animals produced following fertilisation with the transgene-bearing sperm [61]. Similar results were reported by Sato et al, using liposome-encapsulated transgene molecules injected close to the epididymis113. In vitro gene delivery into ex vivo spermatogonial stem cells of both adult and immature animals has recently been reported 114. Nagano et al obtained stable transgene integration and expression in up to 20% of murine spermatogonial stem cells following retroviral transgene delivery115. Genetically modified stem cells were transferred into the testes of infertile recipient mice, leading to transgeneity in ca. 4.5% of the resultant progeny, plus successful transmission to subsequent generations. Similar results were obtained by Orwig et al in rats 116. Although
113

Sato M, Ishikawa A, Kimura M. Direct injection of foreign DNA into mouse testis as a possible in vivo gene transfer system via epididymal spermatozoa. Mol Reprod Dev. 2002; 61(1):49-56; Sato M, Gotoh K, Kimura M. Sperm-mediated gene transfer by direct injection of foreign DNA into mouse testis. Transgenics 1999; 2(4):357-369; Sato M, Yabuki K, Watanabe T, Kimura M. Testis-mediated gene transfer (TMGT) in mice: Successful transmission of introduced DNA from F0 to F2 generations. Transgenics 1999; 3(1):11.
114

Brinster RL. Germline stem cell transplantation and transgenesis. Science 2002; 296(5576):2174-2176.

115

Nagano M, Brinster CJ, Orwig KE, et al. Transgenic mice produced by retroviral transduction of male germline stem cells. Proc Natl Acad Sci U S A. 2001; 98(23):13090-13095.
116

Orwig KE, Avarbock MR, Brinster RL. Retrovirus-mediated modification of male germline stem cells in rats. Biol Reprod. 2002; 67(3):874-879.

this form of transgenesis is at an early stage of development, preliminary work with spermatogonial stem cells in other mammals such as pigs and goats suggests that the approach is likely to be widely applicable117. If human ex vivo spermatogonial stem cells are similarly able to pick up and transmit transgenes, an exciting potential route to germline gene therapy might emerge. If SMGT does indeed work as reported, or if it can be made to work, then this would have very profound implications for human germline gene therapy. Gene transfer into embryos (using pronuclear microinjection or RVVs) is inherently very costly and technically demanding, due in large part to the need to remove embryos from the female, to manipulate the embryos and finally to return the embryos to the reproductive tract. By contrast, SMGT coupled with AI would permit germline GM with minimum levels of expense and expertise, as would SMGT coupled with testicular injections. Thus, SMGT would in principle permit the widespread use of human germline gene therapy: relatively poor countries would be able to use the technique, and highly centralised facilities would not be required. Of course, such easily available human germline gene therapy would raise serious ethical concerns. However, even if SMGT were to prove effective as a means to gene transfer, it would be fundamentally limited in the context of human germline gene therapy due to its unsuitability as a means of gene targeting. This limitation is of course shared with the embryo-based gene transfer methods considered above. However, there are at least some glimmers of hope for future gene targeting possibilities in the case of embryo-based approaches: some (albeit very limited) success has been achieved with targeting RVVs118, and the low natural rate of HR in zygotes might conceivably be increased if appropriate recombinase enzymes were to be discovered and co-injected119. By contrast, there have been no reports of gene

117

Honaramooz A, Megee SO, Dobrinski I. Germ cell transplantation in pigs. Biol Reprod. 2002; 66(1):2128; Honaramooz A, Behboodi E, Blash S, et al. Germ cell transplantation in goats. Mol Reprod Dev. 2003; 64(4):422-428.
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Ellis J, Bernstein A. Gene targeting with retroviral vectors - recombination by gene conversion into regions of nonhomology. Mol Cell Biol. 1989; 9(4):1621-1627.
119

Smith K. Theoretical mechanisms in targeted and random integration of transgene DNA. Reprod Nutr Dev. 2001; 41(6):465-485.

targeting using SMGT, and it is difficult to envisage even in outline how this might ever be achieved. Episomal Vectors Various extrachromosomal plasmid vectors (episomes) have been used as transgenes120. Such vectors have been employed to produce transgenic animals, via a variety of routes including pronuclear microinjection and SMGT121. In the context of human germline gene therapy, such vectors offer the potential advantage of eliminating the threat to genome integrity associated with uncontrolled genomic integration. However, in transgenic animals, episomal plasmid vectors tend to behave in an unstable fashion122. During development, plasmid copy numbers fluctuate and plasmids are lost from some cells. Plasmid inheritance to subsequent generations of animals is similarly problematic. Moreover, worrying health problems (such as tumour formation) have been associated with some episomal vectors123. Of course, the behaviour of an episome must relate in large part to its genetic constitution, and therefore stability problems and safety
120

Colosimo A, Guida V, Palka G, Dallapiccola B. Extrachromosomal genes: a powerful tool in gene targeting approaches. Gene Ther. 2002; 9(11):679-682; 73. Stoll SM, Calos MP. Extrachromosomal plasmid vectors for gene therapy. Curr Opin Mol Ther. 2002; 4(4):299-305.
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Khoo HW, Ang LH, Lim HB, Wong KY. Sperm Cells as Vectors for Introducing Foreign DNA into Zebrafish. Aquaculture 1992; 107(1):1-19; Mannik A, Piirsoo M, Nordstrom K, et al. Effective generation of transgenic mice by Bovine papillomavirus type 1 based self-replicating plasmid that is maintained as extrachromosomal genetic element in three generations of animals. Plasmid 2003; 49(3):193-204; Celebi C, Auvray P, Benvegnu T, et al. Transient transmission of a transgene in mouse offspring following in vivo transfection of male germ cells. Mol Reprod Dev. 2002; 62(4):477-482.
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Mannik A, Runkorg K, Jaanson N, et al. Induction of the bovine papillomavirus origin "onion skin"type DNA replication at high E1 protein concentrations in vivo. J Virol. 2002; 76(11):5835-5845; Lacey M, Alpert S, Hanahan D. Bovine papillomavirus genome elicits skin tumors in transgenic mice. Nature 1986; 322(6080):609-612.
123

Celebi C, Auvray P, Benvegnu T, et al. Transient transmission of a transgene in mouse offspring following in vivo transfection of male germ cells. Mol Reprod Dev. 2002; 62(4):477-482; Mannik A, Runkorg K, Jaanson N, et al. Induction of the bovine papillomavirus origin "onion skin"-type DNA replication at high E1 protein concentrations in vivo. J Virol. 2002; 76(11):5835-5845; Lacey M, Alpert S, Hanahan D. Bovine papillomavirus genome elicits skin tumors in transgenic mice. Nature 1986; 322(6080):609-612; De Jong G, Telenius A, Vanderbyl S, et al. Efficient in-vitro transfer of a 60-Mb mammalian artificial chromosome into murine and hamster cells using cationic lipids and dendrimers. Chromosome Res. 2001; 9(6):475-485; Grimes B, Cooke H. Engineering mammalian chromosomes. Hum Mol Genet. 1998; 7(10):1635-1640.

limitations may in principle be surmounted by improved plasmid design. However, until such improvements are realised, episomal plasmid vectors could not be considered for human germline gene therapy. Autonomous artificial mini-chromosomes, (mammalian artificial chromosomes, MACs) have been constructed and successfully introduced into mammalian cells124. MACs comprise centromeres, telomeres and replication origins, and are maintained autonomously within the host cell. Structural genes, promoters and enhancers (etc) can be included in MACs. Preliminary research indicates that MACs can be used, via pronuclear microinjection, to create transgenic animals in which the MACs are maintained autonomously125. In the context of human germline gene therapy, these specialised constructs would be expected to give a number of benefits compared with integrated transgenes, including higher and more controllable expression. More speculatively, MACs may be able to function as genetic platforms for the safe subsequent receipt of incoming transgenes. Although this technology is in its infancy, MACs would appear to hold significant future potential for human germline gene therapy126. Totipotent Cells At present, gene targeting requires the use of in vitro selection in order to enrich for rare targeted cells amongst a majority of random integration cells. In vitro selection cannot be conducted on embryos or sperm cells. Consequently, gene targeting in the context of
124

Nature 1986; 322(6080):609-612; De Jong G, Telenius A, Vanderbyl S, et al. Efficient in-vitro transfer of a 60-Mb mammalian artificial chromosome into murine and hamster cells using cationic lipids and dendrimers. Chromosome Res. 2001; 9(6):475-485; Grimes B, Cooke H. Engineering mammalian chromosomes. Hum Mol Genet. 1998; 7(10):1635-1640.
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Co DO, Borowski AH, Leung JD, et al. Generation of transgenic mice and germline transmission of a mammalian artificial chromosome introduced into embryos by pronuclear microinjection. Chromosome Res. 2000; 8(3):183-191.
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Co DO, Borowski AH, Leung JD, et al. Generation of transgenic mice and germline transmission of a mammalian artificial chromosome introduced into embryos by pronuclear microinjection. Chromosome Res. 2000; 8(3):183-191; Hadlaczky G. Satellite DNA-based artificial chromosomes for use in gene therapy. Curr Opin Mol Ther 2001; 3(2):125-132; Choo KHA. Engineering human chromosomes for gene therapy studies. Trends in Molecular Medicine 2001; 7(6):235-237; Grimes BR, Warburton PE, Farr CJ. Chromosome engineering: prospects for gene therapy. Gene Ther. 2002; 9(11):713-718; Lipps HJ, Jenke ACW, Nehlsen K, et al. Chromosome-based vectors for gene therapy. Gene Ther. 2003; 304:23-33; Abbondanzo SJ, Gadi I, Stewart CL. Derivation of embryonic stem-cell lines. Guide to Techniques in Mouse Development, 1993; 225:803-823.

human germline gene therapy would require gene transfer to be carried out with some form of dividing cells in vitro. ESCs Inner cell mass (ICM) cells from the mouse blastocyst can be propagated in vitro as embryonic stem (ES) cells127. In contrast to other cultured cell lines, ESCs retain their normal karyotype even after many months in culture, during which time they remain totipotent. Furthermore, ESCs are capable of colonising the embryo. These unique properties allow ESCs to form chimeras when injected into blastocysts or aggregated with morulae. The resultant embryos can be transferred to the uterus of a pseudopregnant female mouse for gestation. In cases where an ESC has successfully contributed to the embryo, the resultant offspring will be chimeric (up to ca. 50% of animals). The ESC contribution to a mouse can high (up to ca. 80% of the cells), and will often include the germline cells. However, it should be noted that, with some transgenes, the production of chimeras can be problematic or even unattainable, especially when germline transmission is required to breed pure lines of heterozygous or homozygous animals128. It is during the in vitro culture stage that ESCs may be transgenically manipulated
129

Many gene delivery systems are effective with ESCs, including viral vectors, liposomes, and electroporation. The great advantage of ESCs is that they can be subjected to a range of selective agents in vitro, which allows the selection of particular transgenic
127

Abbondanzo SJ, Gadi I, Stewart CL. Derivation of embryonic stem-cell lines. Guide to Techniques in Mouse Development, 1993; 225:803-823; Brook FA, Gardner RL. The origin and efficient derivation of embryonic stem cells in the mouse. Proc Natl Acad Sci U S A. 1997; 94(11):5709-5712.
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Abbondanzo SJ, Gadi I, Stewart CL. Derivation of embryonic stem-cell lines. Guide to Techniques in Mouse Development, 1993; 225:803-823; Brook FA, Gardner RL. The origin and efficient derivation of embryonic stem cells in the mouse. Proc Natl Acad Sci U S A. 1997; 94(11):5709-5712. Robertson EJ. Embryo-derived stem cell lines. In: Robertson EJ, ed. Teratocarcinomas and embryonic stem cells, a practical approach. Oxford: IRL Press, 1987:71-112. Torres M. The use of embryonic stem cells for the genetic manipulation of the mouse. Cellular and Molecular Procedures in Developmental Biology, 1998;36:99-114. Pirity M, Hadjantonakis AK, Nagy A. Embryonic stem cells, creating transgenic animals. Methods Cell Biol.1998; 57:279-293.
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Pirity M, Hadjantonakis AK, Nagy A. Embryonic stem cells, creating transgenic animals. Methods Cell Biol.1998; 57:279-293. Torres M. The use of embryonic stem cells for the genetic manipulation of the mouse. Curr Top Dev Biol.1998; 36:99-114.

modifications. This ability makes ESCs extremely useful for gene targeting experiments and applications130. However, the use of ESCs is limited due to the fact that, to date, the mouse is the only animal from which ESC lines have been unequivocally established. It would be surprising if this limitation represents a fundamental biological barrier. However, further empirical work is needed before totipotent ESC lines become available for other species. Indeed, efforts to isolate non-murine ESCs have been ongoing for nearly two decades but to date no germline-competent ESCs have been isolated in other vertebrates131. In 1998, Thomson et al isolated ESCs from human blastocysts132. Subsequently many other researchers have also isolated human ESCs133, and a new field in biology has resulted. Furthermore, gene targeting has been achieved in human ESCs134. However, totipotency has not been demonstrated in any human ESC line. Unfortunately, this may prove to be a rather intractable situation: proof of totipotency could only (given current technology) come from the establishment of a chimeric human being. It is manifest that the necessary experiments required to pursue this goal would be ethically unacceptable. Thus, the ESC route presently remains firmly closed against human germline gene therapy.
130

Metzger D, Feil R. Engineering the mouse genome by site-specific recombination. Curr Opin Biotechnol.1999; 10(5):470-476. Muller U. Ten years of gene targeting: targeted mouse mutants, from vector design to phenotype analysis. Mech Dev. 1999; 82(1-2):3-21.
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Prelle K, Vassiliev IM, Vassilieva SG, et al. Establishment of pluripotent cell lines from vertebrate species -Present status and future prospects. Cells Tissues Organs 1999; 165(3-4):220-236.Wheeler MB, Walters EM, Clark SG. Transgenic animals in biomedicine and agriculture: outlook for the future. Anim Reprod Sci 2003; 79(3-4):265-289.
132

Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282(5391):1145-1147.
133

Amit M, Shariki C, Margulets V, Itskovitz-Eldor J. Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod. 2004; 70(3):837-845.Conley BJ, Trounson AO, Mollard R. Human embryonic stem cells form embryoid bodies containing visceral endoderm-like derivatives. Fetal Diagn Ther. 2004; 19(3):218-223. Park JH, Kim SJ, Oh EJ, et al. Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell line. Biol Reprod. 2003; 69(6):2007-2014. Reubinoff BE, Pera MF, Fong CY, et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol. 2000; 18(4):399-404.
134

Zwaka TP, Thomson JA. Homologous recombination in human embryonic stem cells. Nat Biotechnol. 2003; 21(3):319-321.

Nuclear Transfer The successful transfer of reprogrammed sheep donor nuclei has recently been achieved135. Unfertilised, metaphase-stage enucleated (universal recipient) eggs received the transferred nuclei. Donor nuclei originated from somatic cells that had been forced into a form of cell cycle stasis (by incubating the cells in a minimal nutrient medium), such that DNA replication and gene expression were halted (or virtually so). Nuclear transfer was conducted by depositing a donor cell under the zona pellucida of a universal recipient egg, and fusing the two cells by electrical stimulation. This process resulted (in some cases) in successful embryo development, the donor nuclei having been reprogrammed into totipotency. Offspring were produced following the transfer of such reconstructed embryos to recipient ewes. Subsequent molecular genetic testing showed that the lambs DNA had originated from the donor cells. In some of the experiments, the donor nuclei were obtained from embryo-derived cultured cell lines. Following these groundbreaking experiments, successful cloning from cultured cells of various mammals including cattle, goats and pigs has been reported136. Interestingly, a human ESC line has recently been derived from cloned human blastocysts produced by NT137, pointing to a possible new field of application for NT technology. The prospects for germline GM via NT are very significant: transgenes can be introduced to somatic donor cells in vitro, permitting germline genetic modifications. This has been achieved in animals138. Several

135

Campbell KHS, McWhir J, Ritchie WA, Wilmut I. Sheep cloned by nuclear transfer from a cultured cell line. Nature 1996:64-65. Schnieke AE, Kind AJ, Ritchie WA, et al. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 1997; 278(5346):2130-2133. Wilmut I. Viable offspring derived from fetal and adult mammalian cells. Societal, medical and ethical implications of cloning. London: Office for Official Publications of the European Communities, 1997:3-8.
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Tsunoda Y, Kato Y. The recent progress on nuclear transfer in mammals. Zoolog Sci. 2000; 17(9):11771184. Wolf E, Schernthaner W, Zakhartchenko V, et al. Transgenic technology in farm animals - progress and perspectives. Exp Physiol. 2000; 85(6):615-625.
137

Hwang WS, Ryu YJ, Park JH, et al. Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst. Science 2004; 303(5664):1669-1674.
138

Ibid.

gene delivery systems are applicable to NT, including liposomes and electroporation. Moreover, because selection can be applied to cultured donor cells, NT can be used to achieve germline gene-targeting. Gene targeted transgenic animals have been created in this way139. Thus, NT is potentially able to provide the same range of transgenic manipulations presently available in mice (via the ESC route) to all mammal species. Accordingly, it seems probable that the technique could in principle be readily applied to humans as a means to achieving germline modifications. However, in comparison with ESC transgenesis, NT has thus far proved to be relatively inefficient: only a small proportion of reconstructed embryos survive to become live animals. For example, McCreath et al produced live targeted sheep at an efficiency of less than 4% , and Lia et al produced live targeted pigs at an efficiency of less than 2%140. Such low efficiencies, presuming reconstructed human embryos to behave similarly, represent a potential problem for human NT-based germline gene therapy. Although embryo pre-selection could be used to ensure that only transgenecontaining embryos were allowed to gestate, the problem would remain that a large number of valuable donor eggs would be required for each GM attempt. The health status of NT-derived animals is also proving to be problematic141. Developmental abnormalities are very common, and frequently result in death (foetal or postnatal) or debility. For example, of fourteen live-born lambs, seven died within 30 hours of birth, and four died within twelve weeks142. Similarly, out of seven piglets, two piglets died shortly after birth, and one died at 17 days; only one appeared to be entirely free of developmental abnormalities143. Transgenesis and gene targeting are not of themselves implicated: the health problems are associated with NT per se. During the in vitro (cell culture) stage, the pattern of chromosomal imprinting
139

Clark AJ, Burl S, Denning C, Dickinson P. Gene targeting in livestock: a preview. Transgenic Res. 2000; 9(4-5):263-275. McCreath KJ, Howcroft J, Campbell KHS, et al. Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature 2000; 405(6790):1066-1069.
140

Ibid

141

Lai LX, Kolber-Simonds D, Park KW, et al. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer coning. Science 2002; 295(5557):1089-1092.
142

Supranote 58 ibid.

143

may change; there are indications that inappropriate expression of imprinted genes following such epigeneticalteration may be mainly responsible for the poor health of NTderived animals144. Research into epigenetic reprogramming in NT embryos is in progress, and it is to be hoped that developmental abnormalities arising from NT will eventually be eliminated or reduced in frequency. Meanwhile, it is anticipated that NTrelated health problems, to the extent that the basis for such is epigenetic, are unlikely to affect the offspring of surviving first-generation animals. However, until such time as the first-generation health problems are solved, NT appears to be too dangerous to consider for human germline gene therapy. This is unfortunate, because without NT (or human ESCs) the in vitro selection required for germline gene targeting cannot be conducted. Thus, germline GM in humans would be restricted to add-in alterations; gene knockout and gene repair germline alterations in humans are not a practical proposition with the technology available at present. Non-selective gene targeting In embryonic stem cells and in certain somatic cells in vitro, unusually high levels of gene targeting have been reported. Isogenic transgenes, derived from the same (syngenic) laboratory animal strain as the target animal, contain homology blocks that are genetically identical (or virtually identical) to the target homology regions. Riele et al 145 reported a 20-fold improvement in targeting efficiency when an isogenic transgene was used to target the retinoblastoma susceptibly gene (Rb) in murine ESCs, yielding a remarkably favourable ratio of random to targeted integration (approximately 1:4).

144

Smith LC, Bordignon V, Babkine M, et al. Benefits and problems with cloning animals. Can Vet J. 2000; 41(12):919-924. Renard JP, Zhou Q, LeBourhis D, et al. Nuclear transfer technologies: Between successes and doubts.Theriogenology 2002; 57(1):203-222. Kono T. Influence of epigenetic changes during oocyte growth on nuclear reprogramming after nuclear transfer. Reprod Fertil Dev. 1998; 10(78):593-598. Rideout WM, Eggan K, Jaenisch R. Nuclear cloning and epigenetic reprogramming of the genome. Science 2001; 293(5532):1093-1098. Wakayama T, Yanagimachi R. Mouse cloning with nucleus donor cells of different age and type. Mol Reprod Dev. 2001; 58(4):376-383.
145

Riele HT, Maandag ER, Berns A. Highly efficient gene targeting in embryonic stem-cells through homologous recombination with isogenic DNA constructs. Proc Natl Acad Sci U S A. 1992; 89(11):51285132.

Similar results were obtained from a systematic study by Vandeursen and Wieringa146, in which the creatine kinase M gene (CKM) in ESCs was targeted. More recently, adenoassociated virus (AAV) vectors have been used to gene target somatic cells at high frequencies. Hirata et al used AAV vectors to introduce transgenes into the hypoxanthine phosphoribosyl transferase (HPRT) and Type I collagen (COL1A1) loci in normal human fibroblasts147. The transgenes were targeted at high frequencies, such that the majority of transgene-containing cells had undergone gene targeting with an appropriately designed vector. AAV targeting frequencies have been further improved by selective creation of double-strand DNA breaks in the target site148. Most recently, adult human mesenchymal stem cells (MSCs) have also been targeted with high efficiency using AVV vectors149. The foregoing reports suggest that in ESCs and in certain somatic cells the efficiency of gene targeting can be sufficient to bypass the need for selection. Selection-based gene targeting places limits on transgene design, due to the need to engineer the requisite selective elements into the transgene. Therefore, the ability to conduct gene targeting in somatic cells without the need for selection would be a welcome addition to the armamentarium of gene transfer technologies that may in future permit human germline gene therapy. Conclusions If either the NIH or FDA had been taken to court by BIO over the legality of their respective disclosure rules, it is quite likely that BIO would have either emerged victorious, or that the court would have imposed such rigorous procedural requirements on the agency before it that the agency's disclosure policy would be dead in the water.
146

Vandeursen J, Wieringa B. Targeting of the creatine kinase-M gene in embryonic stem-cells using isogenic and nonisogenic vectors. Nucleic Acids Res. 1992; 20(15):3815-3820.
147

Hirata R, Chamberlain J, Dong R, Russell DW. Targeted transgene insertion into human chromosomes by adeno- associated virus vectors. Nat Biotechnol. 2002; 20(7):735-738.
148

Porteus MH, Cathomen T, Weitzman MD, Baltimore D. Efficient gene targeting mediated by adenoassociated virus and DNA double-strand breaks. Mol Cell Biol. 2003; 23(10):3558-3565. Miller DG, Petek LM, Russell DW. Human gene targeting by adeno-associated virus vectors is enhanced by DNA double-strand breaks. Mol Cell Biol. 2003; 23(10):3550-3557.
149

Chamberlain JR, Schwarze U, Wang PR, et al. Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science 2004; 303(5661):1198-1201.

That is not to say that the NIH and FDA did not have valid claims from a policy perspective. The doctrine of reverse-FOIA operates as a default presumption to preserve regulated entitles from the rash or ill-considered decisions of agencies to release information that could potentially cause substantial harm. The mechanism can thus appear unduly rigid and overinclusive, as it sweeps in both reckless disclosures and wellreasoned regulations within an agency's general statutory authority to regulate. The NIH and FDA disclosure rules very well may have been the proper policy choices to meet a challenging scientific, medical and ethical problem. It is not clear how comforted the agencies or the public should be that policy changes could always be sought from Congress of the U.S. As such there is no active steps in India regarding gene patenting. As far as the scientific development is concerned it is probable that the human germline could be readily manipulated using current transgenic techniques. To achieve this, pronuclear microinjection would probably be effective, as would retroviral transfer, particularly using lentivirus-based vectors. A combination of microinjection and RVVs would probably be most effective indeed such a combination has recently given rise to the first transgenic primates. SMGT may be effective also, at least in some forms of the approach, such as transgenICSI. However, AI-based SMGT is not yet an established method of transgenesis, therefore the prospects for this potentially very important form of gene transfer are less certain. Totipotent human ESCs have not been established for humans, thus ESC-based gene transfer remains despite its effectiveness in mice unavailable for human germline gene therapy. The lack of human ESCs leaves NT-based gene transfer as the only method that might be able to permit gene targeting in human germline gene therapy. NT could probably be readily applied to humans; however, the high level of health problems observed in first generation NT-derived animals render the approach in its present form unfeasible for human germline gene therapy. Table 1 summarises the key features of the major candidate methods that might serve to achieve human germline gene therapy. If human gene transfer technology is limited to adding-in gene functions via non-targeted

transgene integration, and if the process needs be performed on individual embryos isolated from the reproductive tract, it is likely that human germline gene therapy will remain insufficiently safe, excessively inefficient and of inadequate clinical value to permit its use. The widespread availability and applicability of safe and effective human germline gene therapy would require the development of gene transfer methods that would (a) permit gene targeting while (b) avoiding the need for ex vivo embryo isolation and manipulation. Unfortunately, at present these two requirements are mutually exclusive. Laborious manipulations involving large numbers of embryos would in principle best be avoided by the use of SMGT, either in vivo or ex vivo. However, highefficiency gene targeting is not available at present without the use of in vitro selection. Thus, the widespread use of human germline gene therapy does not appear likely to flow from incremental improvements in current GM methods. Rather, widespread human germline gene therapy would appear to be a future possibility that must await substantial scientific advancement. Naturally, it is impossible to predict when such improvements might be forthcoming.