Fetal and neonatal gene therapy: benefits and pitfalls - Nature

Gene Therapy (2004) 11, S92–S97 & 2004 Nature Publishing Group All rights reserved 0969-7128/04 $30.00 www.nature.com/gt

REVIEW

Fetal and neonatal gene therapy: benefits and pitfalls SN Waddington1, NL Kennea2, SMK Buckley1, LG Gregory1, M Themis1 and C Coutelle1 1

Imperial College London, Gene Therapy Research Group, Section of Cell and Molecular Biology, Division of Biomedical Sciences, Sir Alexander Fleming Building, Imperial College Road, London, UK; and 2Weston Laboratory, Imperial College London, Institute of Reproductive and Developmental Biology, Division of Paediatrics, Obstetrics and Gynaecology, London, UK

The current approaches to gene therapy of monogenetic diseases into mature organisms are confronted with several problems including the following: (1) the underlying genetic defect may have already caused irreversible pathological changes; (2) the level of sufficient protein expression to ameliorate or prevent the disease requires prohibitively large amounts of gene delivery vector; (3) adult tissues may be poorly infected by conventional vector systems dependent upon cellular proliferation for optimal infection, for example, oncoretrovirus vectors; (4) immune responses, either preexisting or developing following vector delivery, may rapidly eliminate transgenic protein expression and prevent future effective intervention. Early gene transfer, in the neonatal or even fetal period, may overcome some or all of these obstacles. The mammalian fetus enjoys a uniquely protected

environment in the womb, bathed in a biochemically and physically supportive fluid devoid of myriad extra-uterine pathogens. Strong physical and chemical barriers to infection might, perhaps, impede the frenetic cell division. The physical support and the biochemical support provided by the fetal–maternal placental interface may, therefore, minimize the onset of genetic diseases manifest early in life. The fetal organism must prepare itself for birth, but lacking a mature adaptive immune system may depend upon more primordial immune defences. It is the nature of these defences, and the vulnerabilities they protect, that are poorly understood in the context of gene therapy and might provide useful information for approaches to gene therapy in the young, as well as perhaps the mature organism. Gene Therapy (2004) 11, S92–S97. doi:10.1038/sj.gt.3302375

Keywords: In utero gene therapy; neonatal; monogenetic disorders

Introduction Transgene delivery and expression in the fetal or neonatal period is a useful tool for studying human models. One day, it may even be used therapeutically alongside adult gene therapy as a means to prevent or ameliorate monogenetic diseases. These encouraging studies, which have benefited from the recent improvements in vector technology and optimization of administration routes to appropriate disease models, have reported long-term phenotypic correction after fetal or neonatal application. These include glycogen storage disease type Ia,1 mucopolysaccharidosis type VII,2–6 bilirubin-UDP-glucuronosyltransferase deficiency (Crigler–Najjar syndrome),7,8 haemophilias A9 and B10–12 and congenital blindness (Leber congenital amaurosis).13 To fully understand the basis of these successful experiments in order to move towards clinical application, several key factors concerning early gene transfer must be closely examined. The four factors, frequently cited as the major advantages of fetal and neonatal gene therapy, are (1) Restitution of gene expression may avoid irreversible pathological processes; prevention is better than healing. (2) The earlier in life the vector is administered, the higher is the ratio of vector particles to cells, reducing the Correspondence: Dr SN Waddington, Imperial College London, Gene Therapy Research Group, Section of Cell and Molecular Biology, Division of Biomedical Sciences, Sir Alexander Fleming Building, Imperial College Road, London SW7 2AZ, UK

amount of vector required. (3) An ideal environment for infection of abundant stem cells and other progenitors may be provided; integrating vectors could, therefore, ‘hitch a ride’ with the subsequent cell divisions. (4) Immune mechanisms used by adults to defend against pathogens may be limited or absent: ‘the age of innocence’.

Prevention is better than cure Many genetic mutations probably result in such profoundly adverse consequences that a viable embryo or fetus never develops. However, a minority of mutations have sufficiently little impact during gestation, while the fetus remains on the maternal life support machine, such that only after birth do the devastating consequences arise. An example of this is with some inborn errors of metabolism. Infants with urea cycle enzyme defects, such as ornithine transcarbamoylase deficiency, may rapidly develop acute metabolic crisis characterized by hyperammonemia, coma, brain damage and death after birth and separation from the placental circulation.14 Postnatal screening for phenylketonuria PKU can avoid severe brain damage due to metabolic intoxication but only at the price of, preferably lifelong, adherence to an unpalatable protein hydrolysate diet and fetal damage will occur if the diet is not strictly observed during pregnancy of PKU affected women (fetal PKU).15 Haemophilic neonates not

Fetal and neonatal gene therapy SN Waddington et al

infrequently suffer bleeding intracranial haemorrhage or bleeding beneath the scalp, at the site of venepuncture, at the umbilical stump or after circumcision.16 Although unchecked bleeding into the joints of neonates is uncommon, as the infant begins to crawl, unchecked hemarthrosis causes substantial and irreversible local damage. Therefore, a strong case for early prophylactic replacement of clotting factors, in the most severely affected, has been made.17 Even small increases in clotting factor concentrations lead to a profound amelioration in the bleeding tendency. However, repeated clotting factor injections into young infants is often only possible with central venous access which carries its own hazards. Lysosomal storage diseases such as the mucopolysaccaridoses, including Sly, Hunter and Hurler syndromes, Tay-Sachs disease and globoid cell leukodystrophy, demonstrate fetal pathology (discussed in Casal and Wolfe18); nevertheless, substantial therapeutic benefits have been observed in mouse and dog models following neonatal gene therapy.3,4,6 Both in humans with Duchenne muscular dystrophy and in the diaphragm of the mdx mouse model, repeated straining of the muscle causes myofibre damage and degeneration (Figure 1c). Eventually, muscular atrophy ensues as regeneration fails to compensate for ongoing necrosis. Again, both humans and animal models demonstrate pathological changes in the fetus and neonate.19 Only after birth, of course, are the respiratory muscles, particularly the diaphragm, and the muscles supporting posture and movement continually forced to work hard. For these and many other genetic diseases prophylactic gene therapy, even if only providing partial correction, may have a dramatic effect upon disease progression. Currently, the best vectors suffer the drawback of a relatively small payload size, which restricts the inclusion of finely regulated promoters and regulatory elements. The problem of unregulated gene expression was highlighted recently when supraphysiological expression of the low-density lipoprotein receptor in mice was found to cause the deposition of crystallized lipid and cholesterol in hepatocytes.20 The consequences of overexpression of transgenic protein during fetal and neonatal ontogeny are difficult to predict but must be assessed carefully for each disease gene.

Vector: cell ratio From birth to adulthood, body mass increases approximately 10-fold in guinea pigs, 20-fold in humans, and 60-fold in pigs; most other farm animals and pets fall in this range. Therefore, a relatively much lower amount of virus will infect a higher percentage of cells when introduced early rather than late in life (Figure 1d). The stoichiometric advantage increases as the age of intervention is reduced, and this may partially offset the apparent difficulty in scaling vector doses from small to large species based on mass alone. For example, compared with mice, disproportionately low concentrations of transgenic clotting factor were measured in the plasma of dogs despite their receipt of equivalent or higher doses of vector per kilogram of helper-dependent adenovirus21 or AAV2.22

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Figure 1 Consideration for gene delivery to the fetus or neonate. (a) Illustration of injection of the HIV lentivirus-based vector into the (b) fetal mouse circulation via the yolk sac vessels. (c) Muscle degeneration and repair evidenced by central nucleation of myofibres in the diaphragm of an 8-week-old mdx mouse. (d) Extensive infection of the conducting airways of the fetal mouse after intra-amniotic injection of lacZ adenovirus. (e) High levels of cellular proliferation in the fetal mouse kidney at 16 days gestation as detected by immunohistochemistry of bromo-deoxyuridine incorporation. Factors determining immunity to vector delivery include (f) adaptive immunity, including production of IgG (g) innate immunity such as retrocyclin expression (adapated from Cole et al54), (h) temporal variation in vector receptors, such as coxsackie adenovirus receptor (CAR), which may affect adenovirus infectivity, and (g) maternal influence including colonization of the neonatal gastrointestinal tract by bacteria in the maternal genito-urinary tract and milk.

Vigorous proliferation The relative abundance of stem cells and vigorous proliferation of progenitor cells (Figure 1e) may make the fetal environment uniquely suited to gene therapy using some, but not all vectors. During the lifetime of an organism, depending upon age, some tissues have slow proliferation rates whereas others are undergoing rapid proliferation and, indeed, rapid cell turnover may be a property of some cell types throughout life. In highly proliferating cells, vector genomes that are not replicated will be inevitably diluted. However, integrating vectors will maintain their genomic presence established after infection and, after infection of appropriate progenitors, may provide restitution for a genetic deficiency in a large proportion of the adult tissue. Therefore, administration Gene Therapy


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of a relatively small amount of vector could result in much higher adult expression levels than what would be achieved by adult vector administration. Intravenous injection of Equine Infectious Anaemia-based lentivirus into the fetal mouse (illustrated in Figure 1a and b) resulted in permanent marker gene expression in multiple tissue types, particularly the liver and heart. In the liver, the presence of foci of gene expression suggests the presence of clonal expansion from single progenitors.23 The early cellular environment is ideal for vectors based on oncoretrovirus, such as murine moloney leukaemia virus, since these require cell division in order to integrate into the genome.24 This has indubitably contributed to the excellent efficacy of these vectors in treating animal models of mucopolysaccharidosis4 and haemophilia11 by neonatal administration. Although lentivirus integration is not constitutively dependent upon cell division, lentivirus-based vectors have been shown to infect actively dividing tissues more efficiently.25 In fact, 7-week-old mice showed 40-fold higher marker gene expression compared with 3.5-week-old mice following gene transfer using a lentivirus vector.26 However, the use of retrovirus vectors may be increasingly hazardous the earlier they are used. Moloney leukaemia virus vectors have been shown to preferentially integrate near actively transcribed genes.27 It is likely that many more growth- and cell-cycleassociated genes are actively transcribed in the fetal and neonatal compared with the adult organism. Therefore, the prospect of increased risk of insertional oncogenesis has to be considered.

The age of innocence Acquired immunity is one component of a battery of defences against pathogens and the ascendancy of these different components depends crucially upon the maturity of the organism. In the passage from fetal through neonatal to adult life, these may be roughly categorized as (i) adaptive immunity (ii) innate immune mechanisms and (iii) maternal influence, although in reality they form a continuum of closely interacting mechanisms. Whether the differential expression of (iv) receptors for vector attachment and entry throughout development can be considered an aspect of immunity depends upon one’s point of view.

Adaptive immunity For those gene therapists interested in the virtues of early intervention for monogenetic disease, there is ample data demonstrating that the fetal or neonatal immune system is much less likely than the adult’s to develop a vigorous immune response towards a transgenic protein. This may be due to the following: (i) the reduced number of immune cells in early life; (ii) the developmental immaturity of cells participating in the immune response; (iii) the deviance of the early immune response from that of the adult, with particular bias towards a TH2 rather than a TH1 response; and (iv) the absence of memory cells due to the naivety of the immune system (see Adkins et al28 and references cited therein). Selected studies illustrating such mechanisms are cited below. Neonatal mice have a delayed CD4-mediated inflammatory response to Pneumocystis carinii infection in the Gene Therapy

lungs, likely to be due to blunted TNFa production and the reduced migration ability of T cells.29 There is evidence that, although human cord blood B cells proliferate and upregulate MHC Class II and CD86 in response to unmethylated cytosine-phosphate-guanosine (CpG)-containing oligonucleotides, they also exhibit homing defects which would prevent them from efficiently entering the peripheral lymphoid organs.30 However, plasmacytoid dendritic cells from human cord blood produced much less IFN-a in response to CpG oligonucleotides than cells from adult blood.31 Although spontaneous in vitro proliferation of fetal and neonatal T lymphocytes was found to be greater than that of adults, they responded poorly when simulated with phytohaemagglutinin or allogeneic stimulator cells; IL-2, IL-4 and IFN-g secretion was only modest in neonatal T cells and entirely absent in those from fetuses.32 The consequences of this immune hypo-responsiveness were observed when a marker gene was delivered by an adenovirus vector to the airways of neonatal and adult cotton rats. Neonatal administration resulted in prolonged transgene expression without detectable antibody production. Harvested lymphocytes failed to demonstrate activation in response to vectors. In contrast, adult administration resulted in brief expression, antibody production and a robust in vitro response of lymphocytes to inactivated vectors.33 Tolerance to human factor IX has been demonstrated after in utero delivery using adenovirus34 and lentivirus12 vectors in mice, and following administration of oncoretroviral vectors in mice and dogs.11 There is some concern that immune tolerance to viral proteins might predispose the individual to unchecked infection by the wild-type pathogen postnatally. However, in utero administration did not result in tolerance to the virus,34 possibly because adenovirus induces vigorous stimulation of the innate immune system. The design of vector systems that do not transcribe viral proteins are likely to limit induction of immune tolerance, which is thought to depend largely upon continuous protein expression. Despite the wealth of data demonstrating early immune hypo-responsiveness, there is also evidence for a degree of early immune competency, which might, in theory, curtail efficacy of fetal and neonatal gene therapy regimes and conversely, should provide encouragement for those endeavouring to develop genetic vaccines for early application. For example, human neonatal T cells were found to be capable of developing an adult-like immune CD8 response to Trypanosoma cruzi. Following intrauterine antigen exposure, increased concentrations of TNFa, IL1b, IL6 and IL10 and CD45RO+ cells were detected in preterm infants.35 Additionally, although infants show a limited IFN-g response to hepatitis B surface antigen vaccination, they also show a greater immunological memory response. This might be specific to the particulate structure of the hepatitis B surface antigen since young infants produce relatively lower antibody responses after vaccination against diphtheria, pertussis, tetanus, Haemophilus influenzae type B and measles vaccines than adults.36 Human fetal B cells have reduced antibody repertoires due to reduced junctional diversity; however, this is not a limiting determinant of the quality of antibody response to viruses of infants beyond the neonatal period.37 It has also been observed that white blood cells from preterm


and term neonates show blunted IL-10 and TGFb production after lipopolysaccharide and phorbol myristoyl aceate/ionomycin stimulation, respectively. Furthermore, IL-10 was less effective in inhibiting IL1a, IL-6, IL-8 and TNFa produced in response to LPS.38 In conclusion, although the early adaptive immune system appears to be predisposed to tolerance to transgenic protein, it remains highly complex and poorly understood compared with the adult immune system. Therefore, careful choice and rigorous scrutiny of preclinical model systems would be essential prior to any clinical application.

Innate immune mechanisms Innate and acquired immunity interact closely in a multitude of ways, forming a defensive continuum. The innate antiviral activity of human leucocytes was shown to be lower in umbilical cord blood than blood from adults.39 However, in most other ways, it has been suggested that innate immunity is enhanced perinatally to compensate for the shortcomings of the acquired immune system. Innate immunity exists in the form of many barriers including immune cells, cell layers, extracellular matrices and antimicrobial compounds. For the fetus, the enclosing membranes are the first line of defence. When lacZ adenovirus was injected into the exocoelomic cavity between the amniotic and chorionic membranes, no transduction of fetal rat tissues was observed.40 The skin of the human fetus begins to stratify at week 9 and completes keratinization by week 14.41 The barrier function of the skin has been described as the ‘raison d’eˆtre’ of the epidermis.42 In mice, intra-amniotic injection of lacZ adenovirus resulted in skin expression, which decreased the later in gestation the injection was performed.43 Similarly, neonatal, but not adult muscle, was found to strongly express b-galactosidase after delivery by herpex simplex virus vector. Evidence was provided to suggest that the immaturity of the basal lamina of the neonatal muscle was the reason for this disparity.44 There is also evidence that the blood–brain barrier permeability decreases from the third trimester through to adult in sheep45 and rats.46 Respiratory mucus protects the airways from dehydration, and also from pathogens. Secretion begins in the fetal respiratory mucosae at 13 weeks of gestation, but the mucins expressed are quite different from those found in the adult and are thought to be involved in lung differentiation and maturation.47 Therefore, one might suppose that in utero the lung could be more vulnerable to infection by pathogens or vectors. The fetus is bathed in amniotic fluid, which not only provides physical support but also possesses antibacterial and antifungal properties.48 Lysozyme, which was identified in fractions of the amniotic fluid showing antimicrobial activity and is also found in the human placenta and fetal membranes, shows antiviral activity against HIV and ectromelia virus.49 Amniotic fluid was also found to inhibit in vitro infection of 3T3 cells by retroviral vectors.50 This and other antimicrobial compounds have also been found in cervical mucus plug and the vernix, a lipidrich deposit covering the skin of the baby at birth.48 The other compounds include the recently discovered antimicrobial peptides of which humans possess two major classes, defensins and cathelicidins. The defensin cryptidin was found in fetal mice as early as 17.5 days

gestation (term at approximately 20 days) expressed in the suprabasal keratinocytes of fetal and neonatal but not adult mice.51 In vitro, defensins have been shown to inhibit replication of HIV-152 and infection of 293 cells with null adenovirus vector.53 Specifically, the thetadefensin, retrocyclin (Figure 1g), was found to inhibit early HIV infection, probably by preventing viral fusion or entry.54,55 Cathelicidins, which have been shown to be greatly upregulated in neonatal skin,56 have been shown to prevent infection of Vero76 cells by herpes simplex virus in vitro.57 Human intestinal defensin expression was found to be much less in the 24-week fetus compared with the adult and was posited as a contributory factor in the high incidence of necrotizing enterocolitis in preterm infants.58

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Vector receptors To gain entry to the cell, viruses utilize a wide range of cell surface receptors that generally serve critical purposes at some point or throughout life. However, as viruses and their hosts have coevolved over millennia, it is much easier to discuss the consequences than the reasons for differential receptor development throughout early ontogeny in the context of viral and, ergo, vector delivery. It was demonstrated that marker gene expression after adenovirus vector delivery was much greater in the neonate than the adult in almost all tissues and similar differential expression of av integrin receptor, known to mediate vector endocytosis, was observed.59 Similarly, it was found that skeletal muscle fibres were infected efficiently by adenoviral vectors in neonatal but less so in adult mice, and that this was likely to be due to the relatively high neonatal expression of coxsackie and adenovirus receptor (Figure 1h).60 Maternal influence Transplacental transport of maternal immunoglobulins to the developing fetus is important in the protection of the newborn from infection, since neonatal antibody production is delayed. Fcg receptors, which are believed to facilitate this transport, are expressed predominantly on fetal endothelial cells in the third trimester. This is thought to explain why preterm neonates have reduced levels of maternal immunoglobulin.61 In mice, transplacental passage of antibodies against herpes simplex virus were shown to protect the neonate against viral-induced mortality. After birth, mammals receive protective antibodies from maternal milk; humans receive primarily IgA and mice primarily IgG.62 Therefore, pre-existing maternal immunity to virus vector components might impede fetal or neonatal gene transfer by vector neutralization or antibody-dependent cellular cytotoxicity. In addition to antibodies, maternal milk supplies the neonate with other antimicrobial and antiviral compounds including lysozyme and lactoferrin, which shows antiviral activity against herpes simplex virus,63 HIV64 and adenovirus.65 At birth, the sterility of the fetal gut is lost rapidly as colonization by symbiotic bacteria begins (Figure 1i). This colonization is often strongly influenced by the composition of the flora of the maternal vagina and gastrointestinal tract but also the general environment, and is believed to enhance the mucosal protective barrier, modify the systemic immune system and exclude less desirable microbes by competition (for a review see Millar et al66 and Fanaro et al67). Gene Therapy


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Administration of the probiotic Lactobacillus rhamnosus has been shown to limit viral gastroenteritis and diarrhoea in infants receiving antibiotics.68 There can be no doubt that enteric flora would influence gene transfer to these tissues, something which might be considered to limit or prevent gut pathology in cystic fibrosis. However, these influences remain undefined. Recent studies in animal models illustrate the efficacy of early gene transfer. However, the mechanisms underlying these successes, and those impeding even better results, are still less well understood than in the adult. The best age for intervention will be dictated by many factors, including the nature and severity of the disease, the ease of its diagnosis, the safety and efficacy of the gene therapy, the practicality of the intervention and, ultimately the ethical, societal and financial implications of such an approach.

Acknowledgements Simon Waddington is salary funded by the Katharine Dormandy Trust for haemophilia and allied disorders.

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