Gene therapy for the fetus: is there a future?

Best Practice & Research Clinical Obstetrics and Gynaecology Vol. 22, No. 1, pp. 203–218, 2008 doi:10.1016/j.bpobgyn.2007.08.008 available online at http://www.sciencedirect.com

13 Gene therapy for the fetus: is there a future? Anna L. David *

PhD, MRCOG

Clinical Lecturer in Obstetrics & Gynaecology

Donald Peebles

MD, MRCOG

Reader in Obstetrics & Gynaecology Department of Obstetrics & Gynaecology, Royal Free & University College London Medical School, 86–96 Chenies Mews, London, WC1E 6HX, UK

Gene therapy uses the intracellular delivery of genetic material for the treatment of disease. A wide range of diseases – including cancer, vascular and neurodegenerative disorders and inherited genetic diseases – are being considered as targets for this therapy in adults. There are particular reasons why fetal application might prove better than application in the adult for treatment, or even prevention of early-onset genetic disorders such as cystic fibrosis and Duchenne muscular dystrophy. Research shows that gene transfer to the developing fetus targets rapidly expanding populations of stem cells, which are inaccessible after birth, and indicates that the use of integrating vector systems results in permanent gene transfer. In animal models of congenital disease such as haemophilia, studies show that the functionally immature fetal immune system does not respond to the product of the introduced gene, and therefore immune tolerance can be induced. This means that treatment could be repeated after birth, if that was necessary to continue to correct the disease. For clinicians and parents, fetal gene therapy would give a third choice following prenatal diagnosis of inherited disease, where termination of pregnancy or acceptance of an affected child are currently the only options. Application of this therapy in the fetus must be safe, reliable and cost-effective. Recent developments in the understanding of genetic disease, vector design, and minimally invasive delivery techniques have brought fetal gene therapy closer to clinical practice. However more research needs to be done in before it can be introduced as a therapy. Key words: congenital disease; fetus; gene therapy; gene transfer.

* Corresponding author. Tel.: þ44 20 7679 6651; Fax: þ44 20 7383 7429. E-mail address: [email protected] (A.L. David). 1521-6934/$ - see front matter ª 2007 Elsevier Ltd. All rights reserved.

204 A. L. David and D. Peebles

INTRODUCTION Gene therapy burst onto the scene in the 1980s and the first human gene therapy trials began over 10 years ago.1 But in spite of continuous technological progress, most clinical results have been disappointing. The reasons for this are many and include difficulty targeting the appropriate organ, a robust immune response to the therapy in adults and low level expression of the therapeutic gene product. Many of these difficulties may be avoidable by applying the therapy to the fetus. This chapter examines the evidence for fetal gene therapy and discusses if there is a future for such a treatment. WHAT IS GENE THERAPY? Gene therapy delivers genetic material to the cell to generate a therapeutic effect by correcting an existing abnormality or providing cells with a new function. To do this, a vector is used to deliver the genes into the appropriate cell. Genes can be inserted into somatic cells or into germ cells, although germ-line gene therapy is considered to be ethically unacceptable.2 IS THERE A NEED FOR FETAL GENE THERAPY? Congenital disease places a huge burden on the community and the health service. A study of paediatric inpatient admissions in 1996 in a US children’s hospital found that wholly genetic conditions accounted for one-third of hospital admissions and for 50% of the total hospital charges for that year.3 Thus, a preventative strategy such as fetal gene therapy could have an important social and economic impact. A criticism levelled at fetal gene therapy is that gene transfer to an individual after birth might be as effective as, and probably safer than, prenatal treatment. For many genetic diseases treatment is palliative rather than curative, resulting in patients living longer but with a reduced quality of life. This has particularly been the case in cystic fibrosis, in which life expectancy has risen from school age in 1955 to the mid-thirties today. To achieve this, however, patients require daily chest physiotherapy, antibiotic treatment, dietary supplementation, insulin for diabetes mellitus and – in many cases – lung transplants, which require additional immunosuppressive therapy. Effective treatment in utero might cure genetic disease, or at least provide partial correction that could have a huge impact on disease progression. WHAT ARE THE ADVANTAGES OF FETAL APPLICATION? Fetal gene therapy might offer particular benefits in certain early onset genetic disorders in which irreversible pathological damage to organs occurs before or shortly after birth.4 For many such diseases, the organ can be difficult to target after birth, for example the lung in cystic fibrosis, the brain in urea-cycle disorders, or the skin in epidermolysis bullosa. Fetal treatment can take advantage of developmental changes to access organs that are inaccessible after birth. Gene transfer to the developing fetus targets rapidly expanding stem-cell populations, providing a large population of transduced cells to provide a therapeutic effect. For example, after intravascular administration of lentivirus vectors to fetal mice, expression of a marker gene appeared to be

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distributed in the liver in focal clusters, suggesting they may have arisen from individual progenitors.5,6 The fetus has a size advantage in a number of ways. In adult gene therapy studies of factor IX adenovirus vectors, larger animals (e.g. haemophiliac dogs) require far higher doses per kg body mass of viral vector to achieve the same levels of transgenic factor IX expression than the smaller mouse models, making it difficult to scale vector doses from small to large species based on body mass alone.7 Production of clinical-grade vector is time consuming and expensive, and the small size of the fetus could lead to increased vector biodistribution at the same vector dose as an adult. The fetus has a functionally immature immune system compared to an adult, which might be to its advantage. Worldwide, up to 50% of adults have pre-existing humoral immunity to adenovirus and adeno-associated virus serotypes from which commonly used gene therapy vectors are derived.8 Even in the absence of a pre-existing immune sensitivity, vector administration to adults often results in the development of an immune response that reduces the duration and level of transgene expression. For example, after intramuscular injection of adenovirus vector containing the dystrophin gene into adult Duchenne muscular dystrophy transgenic mice, antibodies to the dystrophin protein were detected.9 This complication is particularly important when gene therapy is aiming to correct a genetic disease in which complete absence of a gene product is observed. Immune tolerance to exogenous protein can be induced in the fetus if the protein is introduced before the immune system is competent. Tolerance also requires that the exogenous protein expression is maintained, albeit at low level, and so the ability of the vector to give long-term expression is vital. In newborn mice that are more susceptible to immune tolerization than human neonates, administration of adenovirus vectors achieved long-term correction of type VII mucopolysaccharidosis10, whereas use of this vector to correct genetic disease in adult mice gives only temporary expression of the transgene in most cases.11 In a mouse animal model of haemophilia B, one study showed that the functionally immature fetal immune system does not respond to the product of the introduced gene (see later), and therefore immune tolerance can be induced.12 This means that treatment could be repeated after birth, if a single fetal treatment was not sufficient to cure the individual of the disease. WHICH DISEASES COULD FETAL GENE THERAPY BE USED FOR? Fetal gene therapy has been proposed to be appropriate for life-threatening disorders, in which prenatal gene delivery maintains a clear advantage over cell transplantation or postnatal gene therapy and for which there are currently no satisfactory treatments available.13 Some of the diseases that may be suitable for fetal treatment are listed in Table 1. Preclinical studies are encouraging. Fetal application of gene therapy in mouse models of congenital disease such as haemophilia A14, haemophilia B12, congenital blindness15, Crigler–Najjar type 1 syndrome16 and Pompe disease (glycogen storage disease type II)17 have shown phenotypic correction of the condition. Progress in the treatment of one condition, haemophilia B, is discussed in detail here to illustrate recent progress. Deficiency in factor IX (FIX) and factor VIII (FVIII) proteins of the blood coagulation cascade, result in haemophilias A and B, respectively, and have a combined incidence of around 1 in 8000 people.18 Current treatment uses replacement therapy with human FVIII or FIX, which is expensive but effective. Beneficial effects occur after achieving

Disease

Therapeutic gene product

Cystic fibrosis Duchenne muscular dystrophy Spinal muscular atrophy Haemophilia b-thalassaemia Lysosomal storage disease, e.g. Gaucher Urea-cycle defects, e.g. ornithine carbamylase deficiency Severe combined immunodeficiency Epidermolysis bullosa, e.g. dystrophica Hypoxic ischaemic encephalopathy Severe intrauterine growth restriction

Target cells/organ

Age at onset

CF transmembrane conductance regulator Dystrophin

Airway and intestinal epithelial cells Myocytes

Third trimester

1:4000

Mid-thirties

2 years

1:4500

25 years

Survival motor neuron protein Human factor VIII or IX clotting factors Globin

Motor neurons

6 months (type 1)

1:10,000

2 years

Hepatocytes

1 year

1:6000

Erythrocyte precursors

<1 year

1:2700

Glucocerebrosidase

Hepatocytes

9.5 years

Ornithine transcarbamylase

Hepatocytes

2 days

1:9000 overall 1:59,000 1:30,000 overall 1:105,000

Adulthood with treatment <20 years in developing countries <2 years

gc cytokine receptor (X-linked SCID) Type VII collagen

Haematopoietic precursor cells Keratinocytes

Birth

1:1,000,000

Birth

1:40,000

<6 months if no bone marrow transplant Adulthood

Neurotrophic factors

Cortical neurons

birth

1:1000

Adulthood

Placental growth factors

Trophoblast

Fetus

1:500

Days

CF, cystic fibrosis; SCID, severe combined immunodeficiency.

Incidence

Life expectancy

2 days (severe neonatal onset)

206 A. L. David and D. Peebles

Table 1. Examples of candidate diseases for fetal gene therapy.

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only 1% of the normal levels of clotting factor. Unfortunately, a proportion of patients develop antibodies to therapy, resulting in ineffective treatment and occasional anaphylaxis.19 Indeed, the complications of haemophilia treatment, which include the major risk of HIV and hepatitis B infections, have in some cases been far worse than the diseases themselves, increasing their morbidity and mortality.20 The clotting proteins are required in the blood and can be secreted functionally from a variety of tissues, thus the actual site of production is not so important as long as therapeutic plasma levels are realized. Adult gene therapy strategies have concentrated on application to the muscle or the liver, achieving sustained FIX expression in adult haemophiliac dogs or mice after intramuscular or intravascular injection of adeno-associated virus.21 In clinical trials using adeno-associated virus in patients with haemophilia B, only short-term and low-level FIX expression has so far been observed however, which was due to a cell-mediated immune response to transduced hepatocytes.22,23 By contrast, intravenous injection into fetal haemophiliac mice of an HIV–lentivirus human FIX vector resulted in permanent partial phenotypic correction.12 Haemophilic mice expressed a mean human FIX plasma concentration of 12% of the normal human level for the duration of the study (up to 14 months). This was one of the first studies to prove the principle that fetal gene therapy could correct the phenotype and effectively cure a congenital disease. HOW MIGHT FETAL GENE THERAPY BE APPLIED? The vectors Vectors are agents that are used to carry the therapeutic gene into the cell so that it can have its effect. The development of efficient vector systems is crucial for the success of gene therapy in the adult and fetus. The ideal vector for fetal gene therapy would introduce a transcriptionally regulated gene into all organs relevant to the genetic disorder by a single safe application. Although none of the current vector systems meets all these criteria, many have characteristics that are beneficial to the fetal approach. Non-viral vectors These are an attractive option in fetal gene therapy because of their perceived better safety profile and their ability to transfer very large fragments of genetic material. Some fetal studies have shown encouraging results. Intrahepatic injection of the cationic polymer polyethylenimine (PEI) in late-gestation fetal mice enhanced gene transfer to the liver as compared with administration of naked DNA. Encouragingly, the marker gene expression was 40-fold higher marker per milligram of protein in fetuses compared with adults.24 Low-level gene transfer to the fetal sheep airway epithelium was also achieved using guanidium-cholesterol cationic liposomes delivered into the trachea in mid-gestation fetal sheep.25 Unfortunately, current non-viral systems are hindered by their low transfection efficiency and short expression time. Manipulation of non-viral vector particles can overcome some of their problems. For example, altering the chemical structure of carbon bonds within cationic liposome-DNA complexes improves their transfection efficiency and reduces their toxicity in vivo.26 Other developments include artificial chromosomes and Epstein—Barr-virus-based plasmids. DNA introduced as plasmid molecules remains episomal and will be lost with cell division, which is rapid in the fetus and could be a particular disadvantage. However,

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transient gene transfer might be useful in the management of a developmental condition in which therapy is only required for a relatively short time. For instance, short-term transgene expression via injection of a liposome that inhibited fibronectin synthesis into the ductus arteriosis of mid-trimester fetal sheep maintained a patent ductus arteriosus prior before for congenital heart defects in neonatal sheep.27 Adenovirus vectors These are useful vectors for proof-of-principle studies in fetal gene therapy because they achieve highly efficient gene transfer in a wide range of fetal tissues depending on the route of administration.28 Although they do not specifically have a tropism for the liver, these vectors strongly infect liver tissue after intravenous delivery.29 Gene expression is usually transient14 because the vector does not integrate into the host genome and is rapidly diluted by the active cellular proliferation taking place in the fetus. Although the vector is highly immunogenic in adults, fetal administration can produce extended gene expression and induction of immune tolerance to the transgene29 and – in some cases – also to the vector30, although immune responses to adenovirus are reported after fetal application31, even in early gestation.28 To reduce the immunogenicity and toxicity of the vector, all adenoviral coding sequences can be eliminated to generate so called ‘gutless vectors’.32,33 Novel hybrid vectors that take advantage of adenovirus infectivity and the permanent nature of integrative vectors such as retroviruses and lentiviruses might also prove useful in the fetus.34,35 Adeno-associated virus vectors (AAV) are considered to be less toxic and immunogenic than early-generation adenovirus vectors, although an immune response to transgenic protein has been observed after fetal intramuscular injection of AAV.36 Long-term transgene expression can be achieved after muscular, peritoneal or amniotic injection into the fetal mouse37–39 and rat.40 AAV vectors integrate into the genome only at low frequency and they are therefore likely to be diluted rapidly by the increasing tissue mass that occurs in the fetus. Integration of the wild-type virus is predominantly at a specific functionally unimportant location on human chromosome19, reducing the theoretical risk of insertional mutagenesis. However, certain recombinant vectors integrate preferentially into active genes in mice and might induce chromosomal deletions of up to 2 kb, which could have an impact on fetal development.41 Both wild-type adenovirus and AAV infection have been associated with miscarriage and perinatal morbidity and this will need further investigation before in-utero application can be considered in the human.42,43 Retroviruses and lentiviruses Retroviruses and the closely related lentiviruses can integrate permanently into the genome, thus offering the possibility of permanent gene delivery. Moloney leukaemia retrovirus (MLV) was the first vector to be applied fetally to investigate the dispersion of neuronal clones across the developing cerebral cortex of fetal mice.44 Since then, MLV has been used in a number of fetal studies of gene therapy, giving long-term expression in rats45, sheep46 and non-human primates47 after intraperitoneal and intrahepatic delivery. Retroviruses require dividing cells for gene transfer48, which suggests that they might be better suited for use in fetal rather than adult tissues where cells are rapidly dividing. Human serum can almost completely inactivate some retroviral particles49, which limits their use in vivo, although increased resistance to serum inactivation can be achieved by pseudotyping, which replaces the natural envelope of the

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retrovirus with an envelope from another virus.50 A particular problem with in-utero application is that amniotic fluid has a mild inhibitory effect on retrovirus infection in vitro.51 This was probably responsible for the poor gene transfer observed after intraamniotic application of retroviruses in fetal sheep52 and non-human primates.53 Lentiviruses such as those based on HIV can also infect non-dividing cells54, although gene transfer to the liver is improved by cell cycling in some lentiviruses.55 Pseudotyping improves lentivirus stability and allows vector titres to be improved by ultracentrifugation. Different viral envelopes allow gene transfer to be targeted to specific tissues. For example, intramuscular and intrahepatic injection into fetal mice of an HIV vector pseudotyped with vesicular stomatitis virus protein G (VSVG) envelope preferentially transduced the fetal liver, whereas pseudotyping with mokola or ebola envelope proteins gave the most efficient transduction of the myocytes.6 Lentivirus vectors integrate into the genome randomly and – theoretically – are therefore able to cause insertional mutagenesis (see later). Route and time of application of fetal gene therapy If fetal gene therapy is to be clinically applicable, developments in vector technology must be accompanied by improvements in minimally invasive methods of delivering vectors to the fetus. Traditionally, invasive surgical techniques such as maternal laparotomy or hysterotomy have been performed to access the fetus in small- and even large-animal models. However, in clinical practice, minimally invasive techniques such as ultrasound-guided injection, or even fetoscopy, could be used to deliver gene therapy to the fetus with less morbidity and mortality. It is likely that non-human primates will be the ultimate animal model that will be used for safety studies in the immediate preparation for a clinical trial of fetal gene therapy. However, the high maintenance costs and breeding conditions prohibit their use in the routine development of novel injection techniques. Sheep are much easier to breed and maintain and are a wellestablished animal model of human fetal physiology. Sheep have a consistent gestation period of 145 days; the development of the fetus and of the immune system is very similar to humans. Using the pregnant sheep, we have adapted ultrasound-guided injection techniques from fetal medicine practice and developed new methods to deliver gene therapy to the fetal sheep (Figure 1). Table 2 describes the different injection methods that have been tested in fetal sheep and the fetal organs that can be targeted using them. Maternal mortality in the pregnant sheep was negligible and fetal mortality was between 3 and 15%, depending on the route of injection. Over 90% of the fetal mortality was due to iatrogenic infection, usually with known fleece commensals. Invasive procedures such as tracheal injection had a complication rate of 6%, which was related to blood vessel damage within the thorax.56 Intracardiac and umbilical vein injection had an unacceptably high procedure-related fetal mortality in first-trimester fetal sheep28 and umbilical vein injection was only reliably achieved from 70 days of gestation, equivalent to 20 weeks of gestation in humans.57 More recently, ultrasound guidance has been used in non-human primates to deliver gene therapy into the amniotic cavity or for direct injection of the lung and liver parenchyma by teams in the US.47,58,59 The relevant time windows for the different application routes in humans still need to be established with respect to technical feasibility, fetal physiology and the development of the fetal immune system. In the human, fetus the immune system develops from 12 to 14 weeks of gestation, when profound increases in circulating T lymphocytes can

210 A. L. David and D. Peebles

Figure 1. Ultrasound-guided delivery of gene therapy to the fetal sheep trachea and reporter expression in the fetal airways. (A) Ultrasonogram and (B) diagram of sheep fetus at 114 days of gestation in longitudinal section. A 20-gauge spinal needle is inserted into the fetal thorax between the third and fourth ribs, penetrates the lung parenchyma and enters the fetal trachea just proximal to the carina. After ultrasound-guided delivery of adenovirus vector containing the lacZ reporter gene, b-galactosidase expression can be seen in the small airways by X-gal staining (C, D) and by b-galactosidase immunohistochemistry (E).

be observed.60 Thus it might be necessary to deliver gene therapy before this gestational age, which will limit the routes of application that can be safely used. Experiments in the non-human primate are likely to be useful prior to clinical application. THE RISKS OF FETAL GENE THERAPY Various safety issues in relation to in-utero gene therapy need to be addressed before such therapy can be applied clinically.61,62 There is a theoretical risk that the therapeutic gene product or vector that is required later in life to correct a genetic disease could interfere with normal fetal development. This has been suggested in the case of cystic fibrosis, where in-utero infection of rats at 16–17 days gestation with a recombinant adenovirus carrying the human cystic fibrosis transmembrane receptor gene resulted in altered lung development and morphology.63 The effects of a transgenic protein on developmental processes will be difficult to predict, depending on the time of gestation and the type of protein introduced, which will require careful long-term monitoring. An established risk factor of integrating viral vectors is insertional mutagenesis. This was seen recently after gene therapy in very young children into whom haematopoietic stem cells that had been transduced ex vivo with a retroviral vector for X-linked severe combined immunodeficiency (SCID) were transplanted.64 Analysis of the lymphocytes

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Table 2. Application routes for gene delivery to different fetal organs using ultrasound-guided injection of the sheep fetus. Route of application

Gestational age of application Sheep fetus (reference) D3328 D5028 D5028 D5028 D7028,57,80

Target organ (reference)

Equivalent gestational age in human fetus (reference) From From From From From

W10 W1478,79 W14 W14 W2081

Intra-amniotic Intraperitoneal Intrahepatic Intramuscular Umbilical vein

From From From From From

Intrapleural Intracardiac

From D6082 From D10083

From W16 From W2084

Intratracheal Intragastric Cerebral ventricles

D80e11556,86,87 From D6088 D55e6557

W22e32 From W16 W15e17

Skin, fetal membranes Peritoneum, liver, diaphragm Liver Muscle Systemic delivery (predominantly liver, adrenal gland) Intercostal and diaphragm muscles Systemic delivery (predominantly liver, adrenal gland)85 Airways Stomach, small and large bowel, liver Choroid plexus, lateral ventricle and neurocortex

The equivalent gestational ages in the human fetus are based on human studies or current fetal medicine practice, or by extrapolation from experiments in the sheep fetus. The sheep gestation period is 145 days. D, days; W, weeks.

showed that the transgene had been inserted adjacent to a potential oncogene, LMO2, the product of which has been implicated in the pathogenesis of acute leukaemia.65 As well as being related to the type of vector used, the outcome of insertional mutagenesis induced by gene vectors might be influenced by the intrinsic properties of the target cell, and by extrinsic factors such as the fetal environment and disease-specific factors influencing clonal competition in vivo.66 The fetal system might be particularly sensitive to such events because integrating vectors prefer to insert their genomes into chromatin in open configuration. Recently, a very high postnatal incidence of liver tumours in prenatally treated mice was observed after application of an early form of third-generation equine infectious anaemia virus (EIAV) vectors but not when using a similar vector with an HIV backbone.67 It is not clear whether insertional mutagenesis caused this phenomenon but the observation suggests that the fetus might be particularly sensitive to adverse effects associated with this vector system. Further work is needed to address this issue and to devise strategies to determine and possibly direct integration sites. Whereas one of the aims of prenatal gene therapy is to achieve immune tolerance to the transgene and delivery system, vectors must be designed to be sufficiently different to the wild-type so that the immune system remains able to mount an effective immune response against wild-type virus infection. Germ-line transmission is another potential concern. Fetal somatic gene therapy does not aim to modify the genetic content of the germ-line but inadvertent gene transfer to the germ-line could occur and prenatal vector administration could carry a higher risk of inadvertent gene delivery to germ cells.68 In the fetus, compartmentalization of the primordial germ cells in the gonads is complete by 7 weeks of gestation

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in humans and it is unlikely, therefore, that any therapy applied after this time would result in germ-line gene transfer. The chances of germ-line transduction occurring in the mother are also low, because there is a blood – follicle barrier present in the ovary and the eggs are held in meiotic metaphase arrest until fertilization. Examination of germ cells after delivery of retroviral vectors46,69 or adenoviral vectors28 to earlygestation fetal sheep, and after intraperitoneal delivery of AAV to fetal mice39,70, has not shown any detectable transmission. Results from evaluation of maternal tissues in studies of large animals after fetal gene therapy are reassuring and suggest no germ-line gene transfer to the mother.28,47,58 Many of these issues are not confined to fetal or even adult gene therapy and concerns regarding germ-line transmission can be raised for chemotherapy and infertility treatment in particular.71 Tissue-specific vector targeting is an important and potentially safer strategy that may target vectors to specific tissues.50 Fetal gene therapy would give a third option to parents following prenatal diagnosis of inherited disease, where currently the only choices are termination of pregnancy or acceptance of an affected child. When faced with a baby with a genetic disease, many parents decide to terminate the pregnancy in a procedure that is very safe for the mother and totally effective. A prenatal gene-therapy strategy will have to be extremely safe, reliable and effective at treating the disease.72 Any fetal therapy or procedure poses risks of infection, immune reactions and the induction of preterm labour for the fetus and the potential to harm the mother. A conflict of interest might potentially arise because treating the fetus might not be in the mother’s best interest, but in UK law a fetus has no rights per se. Currently used fetal treatments, such as fetal blood transfusion for anaemia, are effective and carry a low risk for the mother, such that the risk – benefit analysis falls heavily on the side of treatment. For experimental fetal procedures, the risk – benefit analysis is uncertain and it is therefore especially important that the mother gives informed consent.73 This can be difficult because the decision to participate in a fetal gene therapy trial will occur close to the time of prenatal diagnosis of the condition. The professionals involved in counselling the parents must present the information in a non-biased way and ensure that resources are set aside for long-term surveillance of the mother and fetus after birth. The parents must also consider that fetal treatment in this pregnancy could pose risks for a future pregnancy by potentially affecting the mother’s health. For parents who would not have continued with an affected pregnancy, a partial cure of an affected child resulting in a poor quality of life would be the worst-case scenario, and we must not forget the first rule of medicine to ‘do no harm’. Testing the fetus after gene-therapy treatment to evaluate its effectiveness is an option. This presents a risk to the pregnancy but allows termination of pregnancy if no effective gene expression can be detected. Such a strategy was used in a case report of in-utero stem-cell transplanation for X-linked SCID, in which a couple requested evaluation of stem-cell engraftment following transplantation.74 Following intraperitoneal injection of fetal liver cells at 14 weeks of gestation, analysis of fetal blood at 24 and 33 weeks of gestation showed 10% and 50% chimerism confirming engraftment, and the parents continued with the pregnancy. In practice, an effective and comprehensive prenatal screening policy for the more common genetic disorders such as cystic fibrosis would need to be implemented and parents at risk of having an affected child would be seen early in the antenatal period for counselling and therapy as appropriate. Let us consider a hypothetical syndrome X – an uncommon autosomal recessive metabolic condition that results in certain brain damage in an affected child with death by school age. Parents might know they

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were at risk either by screening or by having an affected child. For the next pregnancy, the parents would have five options: (1) take a 1: 4 chance of having an affected baby; (2) prenatal diagnosis and continuation of the pregnancy; (3) prenatal diagnosis and termination of an affected child; (4) prenatal gene therapy of an affected child; or (5) preimplantation genetic diagnosis (PGD) resulting in a pregnancy with an unaffected child. Option (4) would require diagnosis of syndrome X in the fetus before the gestational age for optimum gene-therapy treatment by non-invasive prenatal diagnosis using cellfree fetal DNA if available or by chorionic villus sampling. Pre-implantation genetic diagnosis (option 5) is often proposed as the most sensible option for parents at risk of having an affected fetus. As with in-vitro fertilization, the main limitations of PGD are the ovulation induction and invasive procedures that the woman is required to undergo, the fact that only 20–30% of couples achieve a pregnancy per cycle75 and the fact that some embryos will be disposed of, which for some individuals is of concern.76 CHALLENGES FOR THE FUTURE The application of fetal gene therapy in humans will critically depend on our ability to demonstrate its safety and efficiency in preventing or treating severe genetic disease. Improvements in vector design and safety, and in delivery techniques to the fetus, are key. A better understanding of the development of the fetal immune response to vector and gene products, as well as improved knowledge of the candidate diseases to be treated is also vital. Animal models of severe genetic diseases in the mouse and the generation of large-animal transgenic models will be useful to demonstrate proof or principle for in-utero treatment, although ultimately it is likely that some safety studies will need to be performed in non-human primates. Regulating the expression of transgenic protein will need to be explored to prevent any adverse effects by overexpression. One criticism levelled at fetal gene therapy is a belief that couples pregnant with an affected child would be unlikely to proceed with prenatal gene therapy and would instead opt for a termination. The general public remains concerned that ethical discussion about issues such as gene therapy, cloning and the Human Genome Project are falling behind the technology.77 There is almost no research in this area, and the views of the general public and patient groups need to be solicited as this technology comes closer to the clinic. Research is also needed into how adequate information on the risks and benefits of these novel techniques can best be provided for the general public. This will enable couples to have an educated involvement in the decision-making process alongside health professionals. SUMMARY Fetal gene therapy offers the potential for clinicians not only to diagnose but also to treat inherited genetic disease. Fetal application might prove better than application in the adult for the treatment or prevention of early-onset genetic disorders such as cystic fibrosis and Duchenne muscular dystrophy. Gene transfer to the developing fetus targets rapidly expanding stem cell populations that are inaccessible after birth. Integrating vector systems give permanent gene transfer. In animal models of congenital disease the functionally immature fetal immune system does not respond to the product of the introduced gene, and therefore immune tolerance can be induced.

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For the treatment to be acceptable, it must be safe for both mother and fetus, and should preferably avoid germ-line transmission. Recent developments in the understanding of genetic disease, vector design and minimally invasive delivery techniques have brought fetal gene therapy closer to clinical practice. However, more research is needed before it can be introduced as a therapy: Which vectors can provide longterm regulated gene expression preferably for the lifetime of the individual? What is the best route of administration and the optimum gestational age to target gene therapy for specific diseases? How can informed consent best be obtained from the couple who are embarking on gene therapy treatment for their fetus? Currently, fetal gene therapy remains an experimental procedure. Better understanding of the development of genetic disease in the fetus and improvements in vector design and targeting of fetal tissues should allow this technology to move into clinical practice.

Practice points  Gene transfer to the developing fetus targets rapidly expanding stem cell populations that might be inaccessible after birth.  Gene transfer to some organs such as the lung, skin and brain might be achieved more easily during fetal life that in the adult.  Delivery of lentivirus or adeno-associated virus vectors into the fetus gives long-term therapeutic gene expression at sufficiently high levels to provide phenotypic cure in some transgenic mouse animal models of disease.  In animal models of congenital disease, the functionally immature fetal immune system does not respond to the product of the introduced gene, and therefore immune tolerance can be induced.  Minimally invasive ultrasound-guided injection techniques can be used to target gene therapy to fetal organs.  Observed risks of fetal gene therapy include insertional mutagenesis, vector toxicity, fetal immune response, maternal and fetal morbidity and mortality.  Theoretical risks such as germ-line gene transfer and aberrant fetal development have not yet been seen in preclinical studies.

Research agenda  Develop vectors that can provide long-term regulated gene expression preferably for the lifetime of the individual.  Determine the best route of administration and the optimum gestational age to target gene therapy for specific diseases.  Understand how informed consent can best be obtained from couples who are embarking on gene therapy treatment for their fetus.  Characterize the fetal immune response to gene therapy and the development of immune tolerance.

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