Gene Therapy (2008) 15, 329337 & 2008 Nature Publishing Group All rights reserved 0969-7128/08 $30.

00 www.nature.com/gt

REVIEW

Progress and prospects: gene therapy for performance and appearance enhancement
M Kiuru and RG Crystal
Department of Genetic Medicine, Weill Medical College of Cornell University, New York, NY, USA

While medical therapies aim at reversing, reducing or eliminating diseases, the goal of enhancements is to improve performance or appearance beyond normal levels. Distinction between the two interventions is not always clear as they often present as a continuum. Gene therapy typically aims at treating or preventing disease, but the technology can theoretically be employed for enhancement. Some of the gene therapy enhancement strategies include improving Keywords: enhancement; performance; appearance; ethics

performance by increasing muscle mass, endurance, memory, and cognition and bettering appearance by controlling weight, height and hair growth. In addition to the technical challenges of making enhancement strategies safe and effective, genetic enhancement presents signicant ethical / societal questions that must be addressed. Gene Therapy (2008) 15, 329337; doi:10.1038/sj.gt.3303100; published online 24 January 2008

In brief Progress  Treatment strategies are applied to enhancement of performance and appearance.  Gene transfer is used to enhance endurance and muscle strength.  Erythropoietin is a key regulator of erythrocyte production.  Vascular endothelial growth factor promotes blood vessel formation.  Insulin-like growth factor 1 enhances muscle strength.  Growth hormone leads to muscle buildup.  Various other candidate genes to increase muscle strength have also been studied.  Intracranial gene delivery is used to improve memory and learning.  Gene therapy provides a means to control weight, height and hair growth.  Modulation of leptin expression results in weight loss.  Growth hormone and estrogen are essential for controlling height.  Hair follicle is a target for modulation of hair growth and color.  Development of genetic enhancement strategies requires addressing ethical, societal and safety issues. Prospects  Enhancements are likely becoming more common and acceptable in the future.  Increasing numbers of genetic enhancement strategies will be investigated in different animal models.  Efcacy and safety of gene therapy technologies will be improved with the aim of developing clinically applicable approaches.  Strategies to detect illegal use of genetic enhancement, such as gene doping, will be developed.  Evaluation of ethical, societal and regulatory issues regarding genetic enhancement will be imperative before genetic enhancement can become a reality.

Treatment strategies are applied to enhancement of performance and appearance

Correspondence: Dr RG Crystal, Department of Genetic Medicine, Weill Medical College of Cornell University, 1300 York Avenue, Box 96, New York, NY, USA. E-mail: geneticmedicine@med.cornell.edu Received 24 September 2007; revised 8 December 2007; accepted 11 December 2007; published online 24 January 2008

The desire to improve physical performance, mental capacity and appearance is an inherent human trait.1 As a reection of this, society places high values on certain physical and mental traits and shuns others.2 Individuals with highly valued societal characteristics are rewarded

Enhancement gene therapy M Kiuru and RG Crystal

330

with position, respect and nancial gain, and often have a variety of societal advantages. With such high value placed on performance and appearance, it is not surprising that society accepts a variety of strategies to improve both. As an example, the use of botulinum toxin type A for facial enhancement is the most common cosmetic procedure currently undertaken in the United States.3 Other examples of widely accepted approaches for appearance enhancement include use of drugs and hair follicle transplantation for hair loss and surgical procedures to reverse the appearance of aging or unwanted body fat.3,4 Coffee drinking, the most common form of mental enhancement, is essentially a drug delivery system for caffeine, as is cigarette smoking to deliver nicotine to the central nervous system.1 Despite this common acceptance of physical and mental enhancement strategies, society draws the line at some strategies, either because the risk seems to be too high for what is considered a minor benet, and/or the strategy per se is not acceptable. For example, erythropoietin (EPO) is acceptable for treatment of chronic anemia but is not accepted for improvement of endurance in sports.5 Similarly, growth hormone (GH) is an appropriate treatment for short stature due to GH deciency or Turners syndrome, but not for use to become taller or stronger for social, esthetic or athletic reasons.6 The challenge is where to delineate the line between disease and enhancement, an issue that is often difcult to resolve. For many ethicists, genetic therapies are of signicant concern, in that they represent a slippery ethical slope ending in the acceptance of germ line therapies.4,7,8 Gene therapy was originally designed to compensate for and/or treat genetic or acquired diseases such as cystic brosis, bleeding disorders, muscular dystrophies and cancer,9 but gene therapy can also be employed for nontherapeutic purposes as an enhancement intervention.1,4 In the context that gene therapy is basically a delivery system for proteins, it is understandable that the technology of gene therapy is of concern regarding its use as enhancement therapy. In this review, we focus on recent advances in the eld and rst discuss examples of gene therapy that can be used for performance enhancement, such as augmentation of oxygen carrying capacity and muscle mass or enhancing memory and cognition (Tables 1 and 2). We then review gene therapy strategies that can be employed for appearance enhancement, such as improving weight control and hair growth (Table 3). Finally, we discuss the ethical and societal as well as safety issues involved in enhancement interventions.

Erythropoietin is a key regulator of erythrocyte production

EPO is an acidic glycoprotein hormone, produced by the kidneys, with a molecular mass of 34 kDa.50,51 It is a key regulator of erythrocyte production with major functions in promoting erythroid differentiation and initiating hemoglobin synthesis.55 Therapeutically, EPO is used to treat patients with severe anemia, such as patients with kidney failure, undergoing chemotherapy or with hereditary hemoglobin defects.55 As has been widely reported in the media in conjunction with endurance bicycle racing, EPO protein has been extensively used to enhance performance.5,4954 An example of natural enhancement of physical performance by enhanced effect of EPO is the winner of two gold medals in cross-country skiing in 1964 Olympic Games, who had a naturally-occurring activating mutation in the EPOR gene leading to an increase in the amount of erythrocytes, and therefore, enhancement of endurance.5 Gene transfer with EPO aims at delivering an additional copy of the EPO gene into the body to achieve stable protein expression in vivo, and therefore, eliminating frequent injections and the need to manufacture recombinant protein, exemplifying one of the main advantages of gene therapy.12 EPO gene therapy has been conducted in several species including mice, rats and monkeys by systemic or local delivery by using vectors derived from adenovirus,13 adeno-associated virus (AAV)1016 or lentivirus,17,18 by using plasmids alone or with adjunctive delivery systems1921 or by transplanting cells or tissues transduced with the EPO gene ex vivo.2225 In experimental animals, depending on the delivery strategy, gene transfer resulted in increased levels of EPO and an increased hematocrit (percent of erythrocytes in blood) for an extended period of time varying from 2 weeks to 6 years.1025 In a human study in patients with chronic renal failure, EPO delivery was conducted by transplanting autologous dermal tissue transduced ex vivo by adenovirus encoding EPO resulting in increased EPO expression and reticulocyte levels.23 Increased EPO expression and hematocrit lasted for 14 days in these patients. The decrease in EPO levels was associated with a dermal inltrate of cytotoxic T cells and anti-EPO antibodies were detected 90 days after implantation. While achieving stable expression is benecial in many ways, adverse effects may result from sustained overexpression of EPO leading to excessive erythrocytosis and hepatic, renal, neuronal and muscular degeneration.56 To circumvent these effects, controlled gene expression by using tetracycline or rapamycin-regulated AAV vectors and plasmids have been introduced.10,12,14,16 Detection of EPO misuse typically involves direct pharmacological approaches and indirect markers of erythropoiesis.51,55 While human recombinant protein produced in nonhuman cells can be detected based on the difference in isoelectric proles as a result of speciesspecic post-translational modications, means to detect EPO gene doping are limited.55 A study describing detection of EPO in serum by isoelectric prole after delivery of AAV encoding EPO into skeletal muscle argues that genetic doping can be detected, at least in serum, and is likely due to cell-type dependent differences in post-translational modications.55

Gene transfer is used to enhance endurance and muscle strength

The World Anti-Doping Agency denes gene and cell doping as the nontherapeutic use of genes, cells and genetic elements to enhance athletic performance.48 Gene doping is especially challenging, because of limitations in detection methods as the products of the transferred and endogenous genes are close to identical.5,4954 The areas involved in gene doping include enhancement of endurance and muscle strength (Table 1).
Gene Therapy

Enhancement gene therapy M Kiuru and RG Crystal

331
Table 1 Gene therapy for enhancement of physical performance Gene EPO Vector AAV, inducible AAV AAV, inducible AAV; Ad AAV, inducible AAV, with copolymer AAV, inducible Lentivirus Lentivirus Route Retinal Intramuscular Intramuscular Submandibular gland Retinal Adipose tissue Salivary gland Intramuscular Intramuscular Species/model Macaque Feline Rhesus monkey Mouse Macaque Mouse Mouse Rat (partially nephrectomized, model of uremia) Rat (naive or treated with lentivirus encoding for granulocyte colonystimulating factor) Mouse, rabbit, cynomolgus monkey Mouse Rat (induced anemia), rhesus monkey Human microdermis transplanted in mouse (SCID) Human (chronic renal failure) Mouse Mouse (partially nephrectomized) Rat (glucocorticoidinduced muscle atrophy) Mouse (dystrophindecient mdx mice) Rat (myocardial ischemia) Zebrash (embryo) Mouse (muscular dystrophy) Outcome Increased EPO levels in the eye; inducible (rapamycin) for 2.5 years Increased serum EPO levels and hematocrit; erythrocyte aplasia in one animal Increased serum EPO levels and hematocrit; inducible (rapamycin) for 6 years Increased serum EPO levels for 2 years (AAV) and 2 weeks (Ad) Increased EPO levels in the eye; repeatedly inducible (doxycyclin) for 2.5 years Increased EPO levels; return to normal levels after adipose tissue removal Increased EPO levels and hematocrit; inducible (rapamycin) Increased serum EPO levels and hematocrit for 20 weeks Increased EPO levels and hematocrit References
10

11

12

13

14

15

16

17

18

Plasmid, modied leader sequence Plasmid, inducible Plasmid Transduced microdermis, Ad or AAV Transduced microdermis, Ad EPO-secreting myoblasts in microcapsule Transduced MSCs, collagen matrix, retrovirus Plasmid AAV

Intramuscular Intramuscular Intravenous (limb) Subcutaneous

Increased serum EPO levels and hematocrit for 6 months (mice) and for 41 days (monkeys) Increased hematocrit for 42 days; inducible (tetracyclin) Correction of anemia (rats); long-term EPO expression (monkeys) Increased serum EPO levels and hematocrit for 115 days (AAV) and for 5060 days (Ad) Increased serum EPO levels for 2 weeks, increased reticulocytes Increased serum hematocrit for over 100 days Increased hematocrit for 19 weeks; increased swimming exercise capacity Prevention of glucorticoid-induced atrophy; increased muscle content, mass and cross-sectional area Increased muscle mass and strength; reduced myober degeneration; increased protection against contractioninduced injury Improved cardiac function; attenuated ventricle pathology; increased expression of angiogenic factors Increased body mass due to hyperplasia and hypertrophy Increase in muscle mass and absolute force

19

20

21

22

Subcutaneous Subcutaneous, peritoneum Subcutaneous

23

24

25

IGF1 IGF1 and microdystrophin GH Myostatin Myostatin, mutated propetide

Intramuscular Intramuscular

26

27

AAV Double-stranded RNA AAV

Intramyocardial Microinjection Intramuscular

28

29

30

Abbreviations: AAV, adeno-associated virus; Ad, adenovirus; EPO, erythropoietin; GH, growth hormone; IGF1, insulin-like growth factor 1; MSCs, mesenchymal stem cells.

Gene Therapy

Enhancement gene therapy M Kiuru and RG Crystal

332
Table 2 Gene ER/GC receptor Gene therapy for enhancement of mental performance Vector HSV-1 Route Intracranial (dentate gyrus) Species/model Rat (immobilization stress) Outcome Enhanced spatial memory performance; inhibited impairing effects of glucocorticoids on performance Enhanced learning rate and visual object discrimination References
31

Constitutively active PKC

HSV-1

Intracranial (postrhinal cortex)

Rat

32

Abbreviations: ER, estrogen; GC, glucocorticoid; HSV-1, herpes simplex virus 1; PKC, protein kinase C.

Table 3 Gene Leptin

Gene therapy for enhancement of appearance Vector AAV AAV AAV AAV AAV AAV AAV AAV AAV AAV Route Intracerebroventricular Intracerebroventricular Intracerebroventricular Intracerebroventricular Intracerebroventricular Intracerebroventricular Species/model Rat Rat (lean leptinresistant) Rat Rat Mouse (wild type, ob/ob, diabetic nonobese Akita) Mouse (ob/ob), rat Outcome Suppression of weight gain and adiposity for over 80 weeks Initial reduction of weight, then resistance to leptin Suppression of weight gain at reproduction, pregnancy and lactation; lower weight in the offspring Decreased energy intake and body weight Suppression of weight gain (wild type, ob/ob), decreased food intake (ob/ob) Suppression of weight gain during regular diet but not during high-fat diet Suppression of body weight and decrease in food intake Suppression of weight gain and hyperphagia Normalization of food intake and body weight; increase in lifespan Initial reduction of weight, then resistance to leptin; assessment of energy homeostasis and leptin signaling upon administration of leptin antagonist Reduction of weight and adiposity; improvement of glucose and fat metabolism Reduction of food intake and weight gain for over 42 days Increased diet-induced energy weight gain Reduction of weight and adiposity; decreased food intake and increased energy expenditure Stimulated hair growth; improved pharmacodistribution of ShhN compared to Shh References
33

34

35

36 37

38

Intracranial (dorsal vagal Rat complex) Intracerebroventricular Rat (ovariectomized) Intracranial (hypothalamus) Mouse (ob/ob) Intracerebroventricular Rat (lean leptinresistant)

39

40 41

42

POMC

AAV AAV

Intracranial (hypothalamus) Rat (aged obese) Intracranial (nucleus of solitary tract) Intracranial (hypothalamus) Intracranial (hypothalamus) Intracutaneous Rat (adult-onset obesity) Rat (lean) Rat (young and old) Mouse

43

44

GDNF Shh, Nterminal form

AAV AAV Ad

45 46

47

Abbreviations: AAV, adeno-associated virus; Ad, adenovirus; GDNF, glial cell line-derived neurotrophic factor; Ob, obese; POMC, proopiomelanocortin; Shh, Sonic hedgehog.

Vascular endothelial growth factor promotes blood vessel formation

Vascular endothelial growth factor (VEGF) is a homodimeric glycoprotein expressed as several isoforms functioning to promote formation of new blood vessels.57 As VEGF gene therapy has shown benecial effects on ischemic heart disease and peripheral arterial disease in animal models,57 it theoretically could also potentiate supply of oxygen and nutrients to muscles, heart and other tissues in athletes.5 As an example of performance enhancement, myocardial delivery of adenovirus
Gene Therapy

carrying a VEGF isoform 121 transgene into rats resulted in a signicant increase in exercise tolerance (Figure 1). In addition to Ad and AAV vectors encoding for VEGF, overexpression of VEGF gene has been demonstrated by using a zinc-nger DNA-binding transcription factor that increases the expression of all VEGF isoforms leading to enhancement of muscle regeneration and neovascularization.58 VEGF gene therapy clinical trials have been carried out for peripheral vascular disease and coronary artery disease.59 Although preclinical and clinical safety trials have been promising, the randomized controlled trials have not shown clinically relevant positive outcomes.59

Enhancement gene therapy M Kiuru and RG Crystal

AdVEGF121

AdNull

* *
5 10 15 20 Time (min) 25 30 35

PBS

Figure 1 Prolonged exercise tolerance in rats after AdVEGF121 intramyocardial gene delivery. Rats were administered intramyocardially with AdVEGF, AdNull or PBS solution (n 8 per group). Two weeks after treatment, the rats were exercised on a treadmill according to a standardized protocol and exhaustion was dened as the time point at which the rats sustained 10 falls per minute onto the electried grid at the end of each running track. Data presented as means.e. *Po0.006. Reprinted with permission from Schalch et al., J Thorac Cardiovasc Surg 2004; 127: 535.

Insulin-like growth factor 1 enhances muscle strength

Techniques to enhance muscle strength have stemmed from studies on degenerative muscle disorders, such as muscular dystrophies.60,61 Insulin-like growth factor 1 (IGF1) is a 70 amino-acid polypeptide critical in regulating the growth of muscle and other tissues and mediating many of the growth-promoting effects of GH.62 The anabolic effects of IGF1 result from stimulation of protein synthesis, proliferation and differentiation of muscle satellite cells whereas the anti-catabolic effects include inhibition of muscle proteolysis and apoptosis.26 IGF1 gene therapy by intramuscular injection of AAV27,61 or DNA electroporation26 resulted in increased muscle mass and strength in mice and rats.

Growth hormone leads to muscle buildup

GH is a 191 amino-acid polypeptide functioning in the regulation of postnatal somatic growth.62 GH is a key regulator of a cascade leading to buildup of muscle and reducing fat.62 In addition to being used therapeutically, GH is misused for doping in sports. Recombinant GH protein constitutes a problem in monitoring against doping, as it is not easily detectable due to limited glycosylation, although it may be detected based on the absence of a mixture of variations and fragments of the protein typical of the endogenous GH.50 To avoid frequent injections and costly treatment by using recombinant GH protein, gene therapy for GH treatment is being developed. The same methodology may, however, be used for gene doping. The principle of AAV-mediated GH expression was demonstrated by myocardial gene delivery, which improved cardiac function and reduced pathologic remodeling post-myocardial infarction.28

proliferator-activated receptor-d (PPARd) and peroxisome proliferator-activated receptor-g co-activator-1 a and b (PGC-1 a and b). Myostatin belongs to the transforming growth factor b superfamily of signaling cytokines.63 It is synthesized as a 375 amino-acid precursor protein that is further processed giving rise to a 26 kDa mature protein. Myostatin is expressed almost exclusively in skeletal muscle cells and functions as a negative regulator of muscle growth. Targeted disruption of the myostatin gene in mice results in doubling of muscle mass.64 Similarly, a loss-of-function mutation in a human patient was reported to lead to muscle hypertrophy.64 Strategies to inhibit myostatin signaling include neutralizing antibodies against myostatin, blocking myostatin binding to its receptor either by follistatin, mutant activin type 2 receptor proteins, or myostatin propeptide, or silencing myostatin gene by RNAi. These approaches have resulted in amplication of muscle ber number and thickness leading to an increase in skeletal muscle mass or body mass.29,65 Recently, a gene therapy strategy for inhibition of myostatin was demonstrated by AAVmediated expression of mutated myostatin propeptide in animal models of muscular dystrophies leading to increased muscle mass and absolute force.30 PPARd plays an important role in adaptive response of skeletal muscle to environmental changes by controlling the myober type composition and driving the formation of functional type I muscle bers (oxidative and fatigueresistant slow bers).60,66 Transgenic mice with targeted expression of an activated form of PPARd showed increased running capacity and endurance as well as resistance to obesity.60 Corresponding results were obtained in wild-type mice with a PPARd agonist.60 Similar effects on myober composition and endurance have been reported in transgenic mice with overexpression of PGC-1 a and b.60,67 Interestingly, PPARd gene polymorphisms were recently found to be associated with cardiorespiratory tness and plasma lipid responses to endurance training in humans.68 Clinical trials aiming at increasing muscle strength have been conducted in patients affected with inherited muscle dystrophies. Intramuscular delivery of full-length dystrophin cDNA or AAV encoding partial dystrophin or intravenous delivery of antisense oligos that introduce skipping of exons harboring frameshift mutations in dystrophin gene have resulted in weak expression of dystrophin in muscle biopsies in these patients.59,69

333

Intracranial gene delivery is used to improve memory and learning

Gene therapy strategies have also been employed to enhance mental performance, such as memory and cognition (Table 2). Glucocorticoids, secreted by the adrenal gland during stress, may impair acquisition and retrieval of spatial memory and can either attenuate or improve consolidation.31 Estrogen, by contrast, enhances spatial memory performance and can inhibit the harmful effects of glucocorticoids.70 Using intracerebral delivery of a herpes simplex virus 1 vector encoding a chimeric receptor for estrogen and glucocorticoids, enhancement of spatial memory was observed in wildtype male rats.31
Gene Therapy

Various other candidate genes to increase muscle strength have also been studied
Other candidate molecules for increasing muscle strength by gene therapy include myostatin, peroxisome

Enhancement gene therapy M Kiuru and RG Crystal

334

Learning and memory have been postulated to be encoded by specic circuits within cortical areas.32 The neural network theories predict that activating small fraction of the neurons in a circuit can activate the circuit, and therefore, improve learning. This was achieved by herpes simplex virus 1-mediated delivery of protein kinase C into postrhinal cortex in rats, which resulted in transgene expression and improved learning rate and accuracy of visual object discrimination for 24 weeks.32

maturation of central dopaminergic neurons and when administered to the central nervous system, causes weight loss possibly due to increased locomotive activity.46 AAV-mediated GDNF expression in the hypothalamus of young and aged rats resulted in weight loss associated with decreased food intake and increased energy expenditure.46

Gene therapy provides a means to control weight, height and hair growth
As do cosmetic surgery and nonsurgical approaches to enhance skin and soft tissue and shape of the body, gene therapy strategies have provided means to enhance appearance. The main areas of enhancement by gene therapy include weight control and skin/hair enhancements (Table 3).

Growth hormone and estrogen are essential for controlling height

GH and estrogen are therapeutically used to control height.6,72 Recombinant human GH is used to accelerate growth and increase height as a treatment for short stature.6 Conditions approved for GH therapy include GH deciency as well as Turners syndrome, chronic renal insufciency, small for gestational age, Prader Willi syndrome or idiopathic short stature.6 In contrast, estrogen is an approved therapy for inhibiting further linear growth for patients with acromegaly (enlargement of extremities caused by excess GH production by the pituitary gland) or other causes of tall stature in girls.72 Compared to recombinant GH, alternative approaches by using gene therapy in vivo or ex vivo may provide signicant advantages for GH therapy by eliminating the need for frequent injections, reducing the costs of the treatment and possibly providing an approach for hormone delivery mimicking the natural process.73 Using strategies involving intravenous or intramuscular delivery of an adenovirus vector expressing GH, correction of growth deciency and body composition was demonstrated in GH-decient mice and an increase in body weight indicative of phenotype correction in dwarf mice, respectively.73 Successful treatment of hypophysectomized mice (a model of GH deciency) has been shown by intravenous administration of GH plasmid DNA.73

Modulation of leptin expression results in weight loss

Currently available approaches for treating obesity are characterized by poor compliance, transient effectiveness and undesirable side effects.71 There are several genes that have been linked to weight control and tested for therapeutic potential by gene therapy, including leptin,3342 proopiomelanocortin (POMC)4345 and glial cell line-derived neurotrophic factor (GDNF).46 The interplay between appetite-stimulating and appetite-inhibiting factors is crucial for the management of energy intake and expenditure.71 Leptin, produced by the adipose tissue and the hypothalamus, has a key role in restraining overeating by modulating the interaction between appetite-stimulating and appetite-inhibiting factors and by increasing thermogenic energy expenditure.71 By contrast, an opposing hormone ghrelin, produced by the stomach and hypothalamus, stimulates appetite. Mediators involved in the interplay include POMC, neuropeptide Y, g-aminobutyric acid and agoutirelated peptide. After initial reports on using adenovirus vectors for leptin gene therapy, AAV administration was shown to result in elevated plasma leptin levels and reduced body weight for extended periods of time.3342 In most of these studies, potential pleiotropic effects of leptin on other organs, including immune system, bone, blood vessels, kidneys and pancreas were circumvented by restricting AAV-mediated expression of leptin to the brain by using an intracerebroventricular route of administration.3342 AAV-mediated expression of leptin by intracerebroventricular administration inhibited fat accumulation and food intake for periods equivalent to the lifespan of a rodent in obese and wild-type mice, as well as in prepubertal and adult wild-type rats.33 Other candidates for weight control by gene therapy include POMC and GDNF.71 POMC is a prehormone of melanocortins that play a central role in the regulation of energy balance and glucose metabolism.71 AAVmediated expression of POMC by central nervous system delivery in rats resulted in a decrease in body weight and adiposity for a period of 40 days.43,44 GDNF is a neurotrophic factor that promotes the survival and
Gene Therapy

Hair follicle is a target for modulation of hair growth and color

By being readily accessible at the surface of the body, the hair follicle is an ideal target for gene therapy for hair follicle-associated diseases and for enhancement of hair growth.7476 Targeting of the hair follicle has been demonstrated by delivery of liposomeDNA complexes,74,75 plasmid-coated gold particles by using a gene gun (a 400-pounds per square inch helium pulse to accelerate plasmid-coated gold particles through cell membranes), and adenovirus-mediated transgene expression.47 In a report from 2004 by Yang et al., a change in hair color due to skin depigmentation in rats was achieved by delivery of agouti signaling protein cDNA into the skin by using the gene gun method. The change in depigmentation was the most prominent on day 14 and gradually decreased by day 28. Accelerated hair growth due to activation of hair follicle cycling was demonstrated by intradermal administration of an adenovirus encoding Sonic hedgehog (Shh), a developmental morphogen, and adenovirus-mediated Shh delivery was successfully used to treat chemotherapyinduced alopecia in mice. Greater efcacy was observed when adenovirus was used to deliver a modied form of

Enhancement gene therapy M Kiuru and RG Crystal

AdNull

AdShhN

Figure 2 Induction of hair growth by Sonic hedgehog (Shh) with intradermal administration of an adenovirus vector encoding a soluble form of Shh (AdShhN) by Lou et al.47 AdNull or AdShhN was administered into the dorsal skin of C57BL/6 mice. Preexisting dorsal hair was dyed blonde and new hair growth (in black) was evaluated 14 days after vector administration. (a) AdNull and (b) AdShhN. Reprinted with permission.

that create inequality among people are ethically concerning. One obvious example is when athletes who use doping compete against those who have not. Also, enhancements may generate inequality on an economic basis, if interventions are costly, and therefore, not available to underprivileged groups or societies. As in the case of any other technology, society will ultimately have the responsibility of deciding how and where the technology will be used. Because there are risks involved with gene therapy, risk/benet issues are highly relevant regarding enhancement gene therapy. All new treatments pose a some degree of risk, and gene therapy may induce additional risks due to the mode of delivery, often using viral vectors.7 The additional risks in gene therapy treatments relate to the fact that the drug (transgene) and the carrier (vector) have independent toxicities. The carrier toxicities may include inammatory or hematologic responses, whereas the drug toxicities may include inappropriate pharmacokinetics, insertional mutagenesis and immunological responses. One of the main goals of further research in the eld of gene therapy will be to minimize these risks in favor of benets. At present, the risks of gene therapy appear to be outweighed by the benets where the therapy is used to treat a disease. Regulatory agencies may set a different standard if enhancement rather than therapy is the goal.

335

Shh that lacks cholesterol leading to an expanded range of its diffusion (Figure 2).47 The proof-of-principle of skin-targeted gene therapy in humans was demonstrated in a patient with junctional epidermolysis bullosa, a blistering condition caused by a mutated laminin 5 gene.77,78 Ex vivo retroviral transduction of the patients keratinocytes with laminin 5 followed by transplantation resulted in normal levels of laminin 5 and correction of the skin phenotype within the graft for 1 year.77,78

Prospects
Enhancements are likely becoming more common and acceptable in the future. Already, there are several examples of gene therapy strategies being applied to not only disease treatment, but also enhancement of performance and appearance. Increasing numbers of genetic enhancement strategies will be investigated in different animal models. Concurrently, efcacy and safety of gene therapy technologies will be improved with the aim of developing clinically applicable strategies. Clinical gene therapy trials for treating diseases will precede genetic enhancement trials in humans. In addition, strategies to detect illegal use of genetic enhancement, such as gene doping, will be developed. Evaluation of ethical, societal and regulatory issues regarding genetic enhancement will be imperative before genetic enhancement can become a reality.

Development of genetic enhancement strategies requires addressing ethical, societal and safety issues
The use of gene therapy as an enhancement therapy involves multiple ethical issues related to both the goal and the mode of therapy. These include concerns regarding modifying the human genome, the distinction between therapy and enhancement, the possible ethical concerns related to enhancement itself, and the risks involved in conducting gene therapy treatments in practice.4,7,8 Given the potential to achieve enhancement of an individual through modication of somatic cells by gene therapy, there is a concern that the enhancement technologies used for genetic modication of somatic cells will be purposely applied to germline cells,4,8 leading to acceleration of changes in the human evolution. A second concern relates to the distinction between therapy and enhancement. While therapies are aimed at restoring normal functions, enhancements improve function beyond normal level. Current treatments have, however, changed what is thought to be normal as indicated, for example, by increasing lifespan.79,80 Furthermore, therapies and enhancements often present as a continuum and the distinction between the two interventions is not always clear. Finally, enhancements

Conclusions
The desire to improve performance and appearance is an innate human trait as these qualities are often highly valued in the society. In addition to several conventional therapies currently approved to enhance performance and appearance, there are increasing numbers of studies by using gene therapy approaches not only to treat illnesses, but also to enhance endurance, muscle mass, memory, weight control, or hair and skin qualities. The combination of developing technology of genetic modication and the wish to improve performance and appearance pose important ethical, societal and regulatory issues that need to be acknowledged to maintain the principles of bioethicsautonomy, benecence, nonmalecence and justiceand need to be overcome before genetic enhancement can become a reality.
Gene Therapy

Enhancement gene therapy M Kiuru and RG Crystal

336

Acknowledgements
We thank N Mohamed for her help in preparing this paper. These studies were supported, in part, by U01 HL66952; U01 NS047458; P01 HL51746 and the Will Rogers Memorial Fund, Los Angeles, CA.
21

22

References
1 Miller H. Cat and mouse in regulating genetic enhancement. Nat Biotechnol 2005; 23: 171172. 2 Wenz P. Engineering genetic injustice. Bioethics 2005; 19: 111. 3 The American Society for Aesthetic Plastic Surgery. 11.5 Million Cosmetic Procedures in 2006. (http://www.surgery.org/press/ news-release.php?iid 465) last accessed 1/3/08. 4 Fuchs M. Gene therapy. An ethical prole of a new medical territory. J Gene Med 2006; 8: 13581362. 5 Haisma HJ, de HO. Gene doping. Int J Sports Med 2006; 27: 257266. 6 Allen DB. Growth hormone therapy for short stature: is the benet worth the burden? Pediatrics 2006; 118: 343348. 7 Kimmelman J. Recent developments in gene transfer: risk and ethics. BMJ 2005; 330: 7982. 8 Chan S, Harris J. The ethics of gene therapy. Curr Opin Mol Ther 2006; 8: 377383. 9 OConnor TP, Crystal RG. Genetic medicines: treatment strategies for hereditary disorders. Nat Rev Genet 2006; 7: 261276. 10 Lebherz C, Auricchio A, Maguire AM, Rivera VM, Tang W, Grant RL et al. Long-term inducible gene expression in the eye via adeno-associated virus gene transfer in nonhuman primates. Hum Gene Ther 2005; 16: 178186. 11 Walker MC, Mandell TC, Crawford PC, Simon GG, Cahill KS, Fernandes PJ et al. Expression of erythropoietin in cats treated with a recombinant adeno-associated viral vector. Am J Vet Res 2005; 66: 450456. 12 Rivera VM, Gao GP, Grant RL, Schnell MA, Zoltick PW, Rozamus LW et al. Long-term pharmacologically regulated expression of erythropoietin in primates following AAVmediated gene transfer. Blood 2005; 105: 14241430. 13 Voutetakis A, Bossis I, Kok MR, Zhang W, Wang J, Cotrim AP et al. Salivary glands as a potential gene transfer target for gene therapeutics of some monogenetic endocrine disorders. J Endocrinol 2005; 185: 363372. 14 Stieger K, Le MG, Lasne F, Weber M, Deschamps JY, Nivard D et al. Long-term doxycycline-regulated transgene expression in the retina of nonhuman primates following subretinal injection of recombinant AAV vectors. Mol Ther 2006; 13: 967975. 15 Mizukami H, Mimuro J, Ogura T, Okada T, Urabe M, Kume A et al. Adipose tissue as a novel target for in vivo gene transfer by adeno-associated viral vectors. Hum Gene Ther 2006; 17: 921928. 16 Wang J, Voutetakis A, Papa M, Rivera VM, Clackson T, Lodde BM et al. Rapamycin control of transgene expression from a single AAV vector in mouse salivary glands. Gene Therapy 2006; 13: 187190. 17 Oh TK, Quan GH, Kim HY, Park F, Kim ST. Correction of anemia in uremic rats by intramuscular injection of lentivirus carrying an erythropoietin gene. Am J Nephrol 2006; 26: 326334. 18 Brzezinski M, Yanay O, Waldron L, Barry SC, Osborne WR. G-CSF-lentivirus administration in rats provided sustained elevated neutrophil counts and subsequent EPO-lentivirus administration increased hematocrits. J Gene Med 2007; 9: 571578. 19 Fattori E, Cappelletti M, Zampaglione I, Mennuni C, Calvaruso F, Arcuri M et al. Gene electro-transfer of an improved erythropoietin plasmid in mice and non-human primates. J Gene Med 2005; 7: 228236. 20 Richard P, Pollard H, Lanctin C, Bello-Roufai M, Desigaux L, Escande D et al. Inducible production of erythropoietin using
Gene Therapy

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

intramuscular injection of block copolymer/DNA formulation. J Gene Med 2005; 7: 8086. Sebestyen MG, Hegge JO, Noble MA, Lewis DL, Herweijer H, Wolff JA. Progress toward a nonviral gene therapy protocol for the treatment of anemia. Hum Gene Ther 2007; 18: 269285. Brill-Almon E, Stern B, Ak D, Kaye J, Langer N, Bellomo S et al. Ex vivo transduction of human dermal tissue structures for autologous implantation production and delivery of therapeutic proteins. Mol Ther 2005; 12: 274282. Lippin Y, Dranitzki-Elhalel M, Brill-Almon E, Mei-Zahav C, Mizrachi S, Liberman Y et al. Human erythropoietin gene therapy for patients with chronic renal failure. Blood 2005; 106: 22802286. Orive G, De CM, Ponce S, Hernandez RM, Gascon AR, Bosch M et al. Long-term expression of erythropoietin from myoblasts immobilized in biocompatible and neovascularized microcapsules. Mol Ther 2005; 12: 283289. Eliopoulos N, Gagnon RF, Francois M, Galipeau J. Erythropoietin delivery by genetically engineered bone marrow stromal cells for correction of anemia in mice with chronic renal failure. J Am Soc Nephrol 2006; 17: 15761584. Schakman O, Gilson H, de Coninck V, Lause P, Verniers J, Havaux X et al. Insulin-like growth factor-I gene transfer by electroporation prevents skeletal muscle atrophy in glucocorticoid-treated rats. Endocrinology 2005; 146: 17891797. Abmayr S, Gregorevic P, Allen JM, Chamberlain JS. Phenotypic improvement of dystrophic muscles by rAAV/microdystrophin vectors is augmented by Igf1 codelivery. Mol Ther 2005; 12: 441450. Kusano K, Tsutsumi Y, Dean J, Gavin M, Ma H, Silver M et al. Long-term stable expression of human growth hormone by rAAV promotes myocardial protection post-myocardial infarction. J Mol Cell Cardiol 2007; 42: 390399. Acosta J, Carpio Y, Borroto I, Gonzalez O, Estrada MP. Myostatin gene silenced by RNAi show a zebrash giant phenotype. J Biotechnol 2005; 119: 324331. Bartoli M, Poupiot J, Vulin A, Fougerousse F, Arandel L, Daniele N et al. AAV-mediated delivery of a mutated myostatin propeptide ameliorates calpain 3 but not alpha-sarcoglycan deciency. Gene Therapy 2007; 14: 733740. Nicholas A, Munhoz CD, Ferguson D, Campbell L, Sapolsky R. Enhancing cognition after stress with gene therapy. J Neurosci 2006; 26: 1163711643. Zhang GR, Wang X, Kong L, Lu XG, Lee B, Liu M et al. Genetic enhancement of visual learning by activation of protein kinase C pathways in small groups of rat cortical neurons. J Neurosci 2005; 25: 84688481. Boghossian S, Lecklin A, Torto R, Kalra PS, Kalra SP. Suppression of fat deposition for the life time with gene therapy. Peptides 2005; 26: 15121519. Scarpace PJ, Matheny M, Tumer N, Cheng KY, Zhang Y. Leptin resistance exacerbates diet-induced obesity and is associated with diminished maximal leptin signalling capacity in rats. Diabetologia 2005; 48: 10751083. Lecklin A, Dube MG, Torto RN, Kalra PS, Kalra SP. Perigestational suppression of weight gain with central leptin gene therapy results in lower weight F1 generation. Peptides 2005; 26: 11761187. Otukonyong EE, Dube MG, Torto R, Kalra PS, Kalra SP. Central leptin differentially modulates ultradian secretory patterns of insulin, leptin and ghrelin independent of effects on food intake and body weight. Peptides 2005; 26: 25592566. Ueno N, Inui A, Kalra PS, Kalra SP. Leptin transgene expression in the hypothalamus enforces euglycemia in diabetic, insulindecient nonobese Akita mice and leptin-decient obese ob/ob mice. Peptides 2006; 27: 23322342. Boghossian S, Dube MG, Torto R, Kalra PS, Kalra SP. Hypothalamic clamp on insulin release by leptin-transgene expression. Peptides 2006; 27: 32453254. Boghossian S, Lecklin A, Dube MG, Kalra PS, Kalra SP. Increased leptin expression in the dorsal vagal complex suppresses

Enhancement gene therapy M Kiuru and RG Crystal

337
40 adiposity without affecting energy intake and metabolic hormones. Obesity (Silver Spring) 2006; 14: 10031009. Torto R, Boghossian S, Dube MG, Kalra PS, Kalra SP. Central leptin gene therapy blocks ovariectomy-induced adiposity. Obesity (Silver Spring) 2006; 14: 13121319. Boghossian S, Ueno N, Dube MG, Kalra P, Kalra S. Leptin gene transfer in the hypothalamus enhances longevity in adult monogenic mutant mice in the absence of circulating leptin. Neurobiol Aging 2007; 28: 15941604. Scarpace PJ, Matheny M, Zhang Y, Cheng KY, Tumer N. Leptin antagonist reveals an uncoupling between leptin receptor signal transducer and activator of transcription 3 signaling and metabolic responses with central leptin resistance. J Pharmacol Exp Ther 2007; 320: 706712. Li G, Zhang Y, Wilsey JT, Scarpace PJ. Hypothalamic proopiomelanocortin gene delivery ameliorates obesity and glucose intolerance in aged rats. Diabetologia 2005; 48: 23762385. Li G, Zhang Y, Rodrigues E, Zheng D, Matheny M, Cheng KY et al. Melanocortin activation of nucleus of the solitary tract avoids anorectic tachyphylaxis and induces prolonged weight loss. Am J Physiol Endocrinol Metab 2007; 293: E252E258. Li G, Zhang Y, Cheng KY, Scarpace PJ. Lean rats with hypothalamic pro-opiomelanocortin overexpression exhibit greater diet-induced obesity and impaired central melanocortin responsiveness. Diabetologia 2007; 50: 14901499. Tumer N, Scarpace PJ, Dogan MD, Broxson CS, Matheny M, Yurek DM et al. Hypothalamic rAAV-mediated GDNF gene delivery ameliorates age-related obesity. Neurobiol Aging 2006; 27: 459470. Lou H, Crystal RG, Leopold PL. Enhanced efcacy of cholesterol-minus sonic hedgehog in postnatal skin. Mol Ther 2005; 12: 575578. The World Anti-doping Code. The 2006 Prohibited List. (http://www.wada-ama.org/rtecontent/document/2006_LIST. pdf) last accessed 1/3/08. Gene Doping. Play TrueAn Ofcial Publication of the World Anti-Doping Agency, 2005. (http://www.wada-ama.org/ rtecontent/document/Play_True_01_2005_en.pdf) last accessed 1/3/08. Azzazy HM, Mansour MM, Christenson RH. Doping in the recombinant era: strategies and counterstrategies. Clin Biochem 2005; 38: 959965. Lippi G, Franchini M, Salvagno GL, Guidi GC. Biochemistry, physiology, and complications of blood doping: facts and speculation. Crit Rev Clin Lab Sci 2006; 43: 349391. Miah A. Rethinking enhancement in sport. Ann NY Acad Sci 2006; 1093: 301320. Trent RJ, Alexander IE. Gene therapy in sport. Br J Sports Med 2006; 40: 45. Baoutina A, Alexander IE, Rasko JE, Emslie KR. Potential use of gene transfer in athletic performance enhancement. Mol Ther 2007; 15: 17511766. Diamanti-Kandarakis E, Konstantinopoulos PA, Papailiou J, Kandarakis SA, Andreopoulos A, Sykiotis GP. Erythropoietin abuse and erythropoietin gene doping: detection strategies in the genomic era. Sports Med 2005; 35: 831840. Heinicke K, Baum O, Ogunshola OO, Vogel J, Stallmach T, Wolfer DP et al. Excessive erythrocytosis in adult mice overexpressing erythropoietin leads to hepatic, renal, neuronal, and muscular degeneration. Am J Physiol Regul Integr Comp Physiol 2006; 291: R947R956. Yla-Herttuala S, Rissanen TT, Vajanto I, Hartikainen J. Vascular endothelial growth factors: biology and current status of clinical applications in cardiovascular medicine. J Am Coll Cardiol 2007; 49: 10151026. Xie D, Li Y, Reed EA, Odronic SI, Kontos CD, Annex BH. An engineered vascular endothelial growth factor-activating transcription factor induces therapeutic angiogenesis in ApoE knockout mice with hindlimb ischemia. J Vasc Surg 2006; 44: 166175. Alton E, Ferrari S, Griesenbach U. Progress and prospects: gene therapy clinical trials (part 1). Gene Therapy 2007; 14: 14391447. Bassel-Duby R, Olson EN. Signaling pathways in skeletal muscle remodeling. Annu Rev Biochem 2006; 75: 1937. Schakman O, Thissen JP. Gene therapy with anabolic growth factors to prevent muscle atrophy. Curr Opin Clin Nutr Metab Care 2006; 9: 207213. Walenkamp MJ, Wit JM. Genetic disorders in the growth hormoneinsulin-like growth factor-I axis. Horm Res 2006; 66: 221230. Matsakas A, Diel P. The growth factor myostatin, a key regulator in skeletal muscle growth and homeostasis. Int J Sports Med 2005; 26: 8389. Joulia-Ekaza D, Cabello G. Myostatin regulation of muscle development: molecular basis, natural mutations, physiopathological aspects. Exp Cell Res 2006; 312: 24012414. Lee SJ, Reed LA, Davies MV, Girgenrath S, Goad ME, Tomkinson KN et al. Regulation of muscle growth by multiple ligands signaling through activin type II receptors. Proc Natl Acad Sci USA 2005; 102: 1811718122. Grimaldi PA. Regulatory role of peroxisome proliferator-activated receptor delta (PPAR delta) in muscle metabolism. A new target for metabolic syndrome treatment? Biochimie 2005; 87: 58. Arany Z, Lebrasseur N, Morris C, Smith E, Yang W, Ma Y et al. The transcriptional coactivator PGC-1beta drives the formation of oxidative type IIX bers in skeletal muscle. Cell Metab 2007; 5: 3546. Hautala AJ, Leon AS, Skinner JS, Rao DC, Bouchard C, Rankinen T. Peroxisome proliferator-activated receptor-delta polymorphisms are associated with physical performance and plasma lipids: the HERITAGE Family Study. Am J Physiol Heart Circ Physiol 2007; 292: H2498H2505. Takeshima Y, Yagi M, Wada H, Ishibashi K, Nishiyama A, Kakumoto M et al. Intravenous infusion of an antisense oligonucleotide results in exon skipping in muscle dystrophin mRNA of Duchenne muscular dystrophy. Pediatr Res 2006; 59: 690694. Daniel JM. Effects of oestrogen on cognition: what have we learned from basic research? J Neuroendocrinol 2006; 18: 787795. Kalra SP, Kalra PS. Gene-transfer technology: a preventive neurotherapy to curb obesity, ameliorate metabolic syndrome and extend life expectancy. Trends Pharmacol Sci 2005; 26: 488495. Lee JM, Howell JD. Tall girls: the social shaping of a medical therapy. Arch Pediatr Adolesc Med 2006; 160: 10351039. Peroni CN, Gout PW, Bartolini P. Animal models for growth hormone gene therapy. Curr Gene Ther 2005; 5: 493509. Vogt A, Mandt N, Lademann J, Schaefer H, Blume-Peytavi U. Follicular targetinga promising tool in selective dermatotherapy. J Investig Dermatol Symp Proc 2005; 10: 252255. Hoffman RM. The hair follicle and its stem cells as drug delivery targets. Expert Opin Drug Deliv 2006; 3: 437443. Sugiyama-Nakagiri Y, Akiyama M, Shimizu H. Hair follicle stem cell-targeted gene transfer and reconstitution system. Gene Therapy 2006; 13: 732737. Alton E, Ferrari S, Griesenbach U. Progress and prospects: gene therapy clinical trials (part 2). Gene Therapy 2007; 14: 15551563. Mavilio F, Pellegrini G, Ferrari S, Di NF, Di IE, Recchia A et al. Correction of junctional epidermolysis bullosa by transplantation of genetically modied epidermal stem cells. Nat Med 2006; 12: 13971402. Whitehouse PJ, Juengst ET. Antiaging medicine and mild cognitive impairment: practice and policy issues for geriatrics. J Am Geriatr Soc 2005; 53: 14171422. Marshall J. Life extension research: an analysis of contemporary biological theories and ethical issues. Med Health Care Philos 2006; 9: 8796.
Gene Therapy

59 60 61

41

42

62

63

43

64

44

65

45

66

46

67

68

47

48

69

49

70 71

50

51

72 73 74

52 53 54

75 76

55

77 78

56

57

79

80

58