Gene Therapy (2004) 11, S92–S97
& 2004 Nature Publishing Group All rights reserved 0969-7128/04 $30.00
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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 pre-
existing 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
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 mucopolysac-
charidosis type VII,2–6 bilirubin-UDP-glucuronosyltrans-
ferase 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 concern-
ing 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
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, mini-
mize 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
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 pro-
foundly 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
S93
infrequently suffer bleeding intracranial haemorrhage
or bleeding beneath the scalp, at the site of venepunc-
ture, 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 irrever-
sible 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 ther-
apy.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, mus-
cular 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 continu-
ally 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. Figure 1 Consideration for gene delivery to the fetus or neonate. (a)
Currently, the best vectors suffer the drawback of Illustration of injection of the HIV lentivirus-based vector into the (b) fetal
mouse circulation via the yolk sac vessels. (c) Muscle degeneration and
a relatively small payload size, which restricts the repair evidenced by central nucleation of myofibres in the diaphragm of an
inclusion of finely regulated promoters and regulatory 8-week-old mdx mouse. (d) Extensive infection of the conducting airways
elements. The problem of unregulated gene expression of the fetal mouse after intra-amniotic injection of lacZ adenovirus. (e)
was highlighted recently when supraphysiological ex- High levels of cellular proliferation in the fetal mouse kidney at 16 days
pression of the low-density lipoprotein receptor in mice gestation as detected by immunohistochemistry of bromo-deoxyuridine
was found to cause the deposition of crystallized lipid incorporation. Factors determining immunity to vector delivery include
(f) adaptive immunity, including production of IgG (g) innate immunity
and cholesterol in hepatocytes.20 The consequences of such as retrocyclin expression (adapated from Cole et al54), (h) temporal
overexpression of transgenic protein during fetal and variation in vector receptors, such as coxsackie adenovirus receptor (CAR),
neonatal ontogeny are difficult to predict but must be which may affect adenovirus infectivity, and (g) maternal influence
assessed carefully for each disease gene. including colonization of the neonatal gastrointestinal tract by bacteria in
the maternal genito-urinary tract and milk.

Vector: cell ratio

From birth to adulthood, body mass increases approxi-
mately 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 inter-
vention 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 concentra-
tions 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
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
 Fetal and neonatal gene therapy
SN Waddington et al
S94
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 multi-
ple 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 con-
tributed 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 effi-
ciently.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. Molo-
ney leukaemia virus vectors have been shown to
preferentially integrate near actively transcribed genes.27
It is likely that many more growth- and cell-cycle-
associated 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 ma-
turity 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 re-
sponse; (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 inflamma-
tory 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 phytohae-
magglutinin 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 adminis-
tration 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. How-
ever, in utero administration did not result in tolerance to
the virus,34 possibly because adenovirus induces vigor-
ous 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 influ-
enzae 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 myr-
istoyl aceate/ionomycin stimulation, respectively.
Furthermore, IL-10 was less effective in inhibiting IL-
1a, 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’être’ 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 dehy-
dration, 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 mem-
branes, shows antiviral activity against HIV and ectro-
melia 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 lipid-
rich deposit covering the skin of the baby at birth.48 The
other compounds include the recently discovered anti-
microbial peptides of which humans possess two major
classes, defensins and cathelicidins. The defensin crypti-
din was found in fetal mice as early as 17.5 days
Fetal and neonatal gene therapy
SN Waddington et al
S95
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 theta-
defensin, 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
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 expres-
sion 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, transpla-
cental passage of antibodies against herpes simplex virus
were shown to protect the neonate against viral-induced
mortality. After birth, mammals receive protective anti-
bodies 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 cytotoxi-
city. In addition to antibodies, maternal milk supplies the
neonate with other antimicrobial and antiviral com-
pounds 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 competi-
tion (for a review see Millar et al66 and Fanaro et al67).
Gene Therapy
 Fetal and neonatal gene therapy
SN Waddington et al
S96
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 under-
lying 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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