Review

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In Utero Stem Cell and Gene Therapy: Current Status and Future Perspectives Stavros P. Loukogeorgakis1

Alan W. Flake2

1 Surgery Unit, University College London Institute of Child Health,

London, United Kingdom 2 The Children’s Hospital of Philadelphia, and The University of Pennsylvania School of Medicine - The Center for Fetal Diagnosis and Treatment, Philadelphia, Pennsylvania, United States

Address for correspondence Alan W. Flake, MD, The Centre for Fetal Research, The Children’s Hospital of Philadelphia, Abramson Research Building, 3615 Civic Centre Boulevard, Philadelphia, PA 19104, United States (e-mail: [email protected]).

Abstract

Keywords

► prenatal treatment ► fetal therapy ► stem cell transplantation ► gene therapy

Advances in prenatal diagnosis have led to the development of fetal therapies for congenital disorders. Although in utero surgical intervention has been used successfully for correction of anatomical defects that cause fetal demise or long-term disability, its clinical indications remain limited. In contrast, prenatal stem cell and gene therapy might have tremendous potential to treat multiple inherited disorders, and could dramatically expand the use of fetal intervention to a wide range of anticipated pediatric and adult diseases. Despite encouraging results from studies in animal models of disease, the clinical utility of such therapies has been restricted by poor efficacy and concerns about safety. The aim of this review is to summarize experimental progress toward clinical application of in utero stem cell transplantation and gene transfer for the treatment of congenital disease.

Introduction Significant advances in prenatal screening and diagnosis have led to the development of in utero strategies for the treatment of congenital disease. The human fetus has become an appealing target for therapy to prevent fetal mortality and neonatal organ dysfunction. Clinical applications of fetal therapy have been limited to open or minimally invasive (fetoscopic) surgical procedures targeting a small number of anatomical defects that benefit from early intervention. These include repair of myelomeningocele (open), resection of congenital lung lesions, as well as cervical and sacrococcygeal teratomas (open), laser coagulation for twin-to-twin transfusion syndrome (fetoscopic), and tracheal occlusion for diaphragmatic hernia-associated lung hypoplasia (fetoscopic).1 However, the spectrum of congenital disease is broad, and most of it remains beyond the therapeutic range of traditional fetal (surgical) interventions. Inherited conditions such as hemoglobinopathies, immunodeficiencies, and metabolic storage disorders have rela-

received May 5, 2014 accepted May 7, 2014 published online June 19, 2014

tively low prevalence individually, but they represent a large burden of disease collectively.2 Because of improvements in medical management, many patients can now survive to adulthood but require long-term care that affects their quality of life and puts considerable strain on health care resources. As a result, there is a clear need for the development of novel therapeutic strategies that might ultimately cure such disorders. Stem cell and gene therapies have shown the greatest potential for clinical use in this setting, but postnatal applications have been limited by poor efficacy and significant toxicity.3,4 A prenatal approach might alleviate such limitations by exploiting normal fetal development (immune or otherwise) to enhance therapeutic benefits and minimize the risk of harm.5 If the latter is true, in utero therapy may become the preferred treatment modality for any congenital disease that can be prenatally diagnosed and cured by stem cell transplantation and/or gene transfer. Such a paradigm shift, from prenatal treatment of a limited number of life-threatening fetal diseases to that of a wide range of genetic disorders could expand clinical applications of fetal therapy

© 2014 Georg Thieme Verlag KG Stuttgart · New York

DOI http://dx.doi.org/ 10.1055/s-0034-1382260. ISSN 0939-7248.

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Eur J Pediatr Surg 2014;24:237–245.

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dramatically. In this review, we aim to summarize the rationale, current status, and potential future application of in utero stem cell and gene therapy.

In Utero Stem Cell Therapy Recent developments in the field of regenerative medicine have intensified the search for sources of stem cells with potential for therapy. Embryonic and adult tissues can be used for the isolation of several types of stem cells, but significant limitations including complexity of isolation/culture, tumorigenicity, and (in the case of stem cells of embryonic/fetal origin) ethical concerns have hindered translation of laboratory findings to clinical practice. Although stem cells from various sources and with different proliferation/differentiation profiles might be utilized for in utero stem cell therapy (IUSCT), the most likely candidate for broad clinical application till date is the adult hematopoietic stem cell (HSC). HSC are multipotent stem cells that maintain hematopoiesis by generation of all hematopoietic linages throughout fetal and adult life.6 Currently, postnatal transplantation of HSC collected from the bone marrow and/or peripheral blood of genetically nonidentical (allogeneic) donors is used to reestablish hematopoietic function in patients with inherited hematological disorders. However, morbidity and mortality related to immune mismatch (e.g., requirement for toxic myeloablation, and graft vs. host disease) remain high and may be, for some congenital disorders, prohibitive for the majority of patients.7 With these in mind, we will confine our discussion to this stem cell type as an example for IUSCT.

Rationale The rationale for IUSCT is based on unique opportunities related to normal developmental events that may facilitate engraftment of allogeneic stem cells and avoid complications associated with postnatal transplantation.3,5 The most important of these is the development of the immune system, which allows the induction of “actively acquired” tolerance.8 Early in gestation, the immune system undergoes a process of self-education that occurs in the thymus. During this process, antigens are presented and pre–T cell clones with high affinity for self-antigen are deleted, leaving a repertoire of T cells that recognize foreign antigen but not self.9 Moreover, self-reactive T cells that escape deletion in the thymus are controlled by regulatory T cells (Treg), an important component of tolerance that prevents the development of autoimmunity.10 Thus, allogeneic donor cells (HSC or other) that are able to present antigen and are transplanted to the fetus before the completion of this process should be processed as self, resulting in complete central (deletional) and peripheral (Treg-mediated) tolerance to donor antigen.11 Donor-specific tolerance induced by this approach has the potential to create the immunological equivalent of an identical-matched donor for every fetus, allowing further cellular or organ transplantation in a tolerant recipient after birth without the need for traditional myeloablation and its associated toxicity.3 Compelling evidence for the efficacy of fetal tolerance originates from experiments of nature, in which dizygotic twins share European Journal of Pediatric Surgery

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placental circulation that allows intrauterine exchange of HSC resulting in lifelong hematopoietic chimerism and donorspecific tolerance for the sibling twin.12 In addition to the immunological advantages, there may be opportunities in normal hematopoietic development favoring prenatal transplantation of HSC. Hematopoiesis shifts from the yolk sac to the placenta and aorta-gonad-mesonephros region to the fetal liver and finally to the bone marrow.13 Manipulation of the regulatory mechanisms controlling these migrations to selectively favor donor HSC may help overcome competition from the host hematopoietic compartment and improve engraftment after IUSCT. Another key strategy in overcoming the competitive barrier is to transplant very large doses of donor HSC. At 12 to 13 weeks’ gestation, the human fetus weighs less than 35 g, allowing delivery of a relatively large cell dose on a fetal weight basis, far higher than could ever reasonably be provided in postnatal HSC transplantation.3 Finally, in some diseases (e.g., αthalassemia and glycogen storage diseases with neurological involvement) that cause fetal or perinatal death and/or endorgan damage, IUSCT has the potential to preempt manifestations of disease and to rescue fetuses with disorders otherwise incompatible with life.

Experimental Evidence Despite the potential advantages of IUSCT over postnatal transplantation, IUSCT in many normal animal models including the mouse,14 goat,15 dog,16 and nonhuman primate17 has proven much more difficult, often yielding little overall engraftment with no therapeutic relevance. These observations support the notion that, despite the logical basis for IUSCT, there are significant barriers to successful engraftment following prenatal HSC (and other stem cell) transplantation. The most formidable of these barriers is host cell competition. This is of particular importance for IUSCT, as no myeloablative conditioning (to create “space” in the hematopoietic or other stem cell niche) can be safely utilized in the fetus. The first successful animal studies of IUSCT were performed in stem cell–defective murine models. In their seminal studies, Fleischman and Mintz demonstrated that transplacental administration of even a single HSC during early gestation resulted in near complete hematopoietic replacement (multilineage chimerism) in c-kit–deficient mice.18 Subsequent studies of IUSCT in immunodeficient mouse strains (e.g., severe combined immunodeficiency; SCID) resulted in reconstitution of only the affected hematopoietic compartments (split chimerism), with minimal engraftment of other lineages where progenitors maintain their competitive capacity.19 However, in the normal fetus, stem cell and progenitor populations including fetal liver HSC and cord blood HSC have a competitive advantage over their postnatal equivalents, due to favorable cell cycling and expansion kinetics. As a result, IUSCT of large doses of donor cells (2
1011 adult bone marrow mononuclear cells/kg), made possible by the development of novel intravenous transplantation techniques, results in rather modest levels of long-term chimerism of less than 10%.20 Although, such engraftment would not be therapeutic for the majority of congenital disorders targeted

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by IUSCT, it allows the induction of donor-specific tolerance that could be combined with nonmyeloablative postnatal HSC transplantation to achieve clinically relevant replacement of host hematopoiesis by donor cells.21 Despite the presumptive preimmune status of the fetal recipient, accumulating evidence suggested the presence of an immune barrier to engraftment following IUSCT. Early murine studies comparing chimerism after congenic and allogeneic IUSCT failed to demonstrate a significant advantage in the congenic setting.22 However, these studies were limited by low engraftment (microchimerism; engraftment of less than 1%) in both groups, potentially obscuring any differences. Moreover, the fact that clinical IUSCT till date has only been successful in immunodeficiency disorders (e.g., X-linked SCID; XSCID),23 further suggests that the fetus might have some degree of immune competence by the time IUSCT is feasible. Indisputable evidence for the presence of an occult fetal immune barrier was provided by Peranteau et al in 2007.20 Taking advantage of the hematopoietic macrochimerism (engraftment of more than 1%) achieved by intravenous (intravitelline) IUSCT in mice, they demonstrated that although all congenic and allogenic recipients maintained similar high levels of engraftment for up to 3 weeks following prenatal transplantation of adult bone marrow mononuclear cells, more than 70% of allogenic animals lost their engraftment soon after whereas congenic animals remained chimeric.20 This finding supports the presence of an adaptive (acquired) immune barrier following IUSCT, and was somewhat inconsistent with previously observed long-term chimerism by a mechanism of deletional tolerance.24 The pivotal observation that explains this contradiction is the recent demonstration that the immune response seen in animals undergoing allogenic IUSCT is in reality a maternal immune response that results in immune system activation in the offspring via antibody transfer in breast milk.25 If pups are fostered with a surrogate mother that has not been exposed to donor antigen, chimerism is maintained in all allogenic recipients. In a separate study, using a similar murine model of IUSCT but hematopoietic cells derived from the fetal liver, maternal fetal transplacental T cell trafficking was implicated (albeit indirectly) in chimerism loss.26 These studies in murine models of IUSCT demonstrate that in the absence of maternal influence there is no adaptive immune barrier in the immunologically immature fetus and validates the potential for application of donor specific tolerance to facilitate allogenic cellular transplantation. Until the role of the maternal immune response is defined in relevant large animal models, the use of maternal cells would be a practical and safe choice in human trials of IUSCT. Following early promising results of allogenic as well as xenogenic (human HSC) IUSCT in the unusually permissive sheep model,27 there has been limited success of IUSCT in large animal models. However, recent studies in swine and canine models are much more encouraging. Lee et al demonstrated that IUSCT with bone marrow cells in swine leukocyte antigen-mismatched pigs results in stable multilineage engraftment with subsequent tolerance to donor-matched kidney transplantation.28 Moreover, our group have performed

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translational studies in a canine model of human leukocyte adhesion deficiency (canine leukocyte adhesion deficiency; CLAD) and have shown that intraperitoneal IUSCT of bone marrow-derived cells results in low-level engraftment that can ameliorate the clinical phenotype of CLAD, and induce donor-specific tolerance that is adequate in some animals to facilitate postnatal enhancement of chimerism to potentially therapeutic levels.16 More importantly, we have demonstrated that changing the route of cell administration from the intraperitoneal to the intravascular (intracardiac) and optimizing the timing of IUSCT results in markedly improved levels of engraftment, comparable to those achieved in the murine model (J. D. Vrecenak, MD, unpublished data, January 2014). Although further work is required, the similarities in results obtained in canine and murine models of IUSCT suggest that it might be possible to translate observations in mice to clinically relevant therapeutic modalities in human congenital disorders. Although HSC have been the classic cellular candidates for IUSCT, it is important to briefly highlight progress made with prenatal transplantation of other stem cell populations. Mesenchymal stem cells (MSC) are multipotent cells with a mesoderm potential, able to differentiate toward adipogenic, chondrogenic, and osteogenic lineages.29 They were first identified as a subpopulation of the bone marrow, and then quickly discovered in several sites of the human body such as the skeletal muscle, dental pulp, umbilical cord blood, adipose tissue, synovial membrane, and tendon.29 Xenogenic IUSCT with human MSC in the sheep model led to persistence of detectable donor cells in multiple tissues for over a year following transplantation.30 Intriguingly, these findings were noted when IUSCT was performed both before and after the establishment of fetal immune competence. In addition, intraperitoneal IUSCT using human fetal MSC in a murine model of Duchenne muscular dystrophy resulted in preferential long-term engraftment in muscle, although levels of chimerism were subtherapeutic.31 Finally, Guillot et al demonstrated that human fetal MSC IUSCT in a mouse model of intermediate severity type III osteogenesis imperfecta led to significantly fewer fractures as well as improved bone strength, length, and thickness.32 These studies, although limited in number, support the potential utility of MSC for IUSCT.

Progress toward Clinical Translation The early success of IUSCT in the sheep model was followed by several attempts worldwide to perform IUSCT for various human congenital diseases of hematopoiesis. Till date, approximately 50 cases of IUSCT using hematopoietic cells have been reported, targeting a wide variety of inherited disorders that include XSCID,23,33 chronic granulomatous disease,34,35 α- and β-thalassemia,36,37 and metabolic storage diseases.38 The clinical experience has been rather discouraging with the exception of XSCID, in which more than 10 cases of successful IUSCT have been reported.23,33,39 However, XSCID is a unique disorder that provides survival and proliferation advantages for donor T cells, and (predictably) engraftment post-IUSCT has been achieved only in the T cell compartment as is the European Journal of Pediatric Surgery

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case with postnatal treatment.40,41 Thus, it can be stated that IUSCT has not been clinically successful in hematopoietically competitive recipients. As most of the target disorders for IUSCT are competitively normal, methods should be developed to overcome host cell competition before further attempts at clinical application are performed. The strategy of prenatal tolerance induction to allow nonmyeloablative/nontoxic postnatal transplantation lowers the threshold of chimerism required for clinical application of IUSCT (a threshold of 1–2% is generally considered to be sufficient for donor specific tolerance21). This “two-step” approach is likely to be utilized in the setting of disorders requiring high levels of engraftment for clinical effect (e.g., clinical amelioration of various hemoglobinopathies would necessitate chimerism levels of around 20%).42,43 If adequate engraftment can be achieved to consistently induce tolerance without graft versus host disease in a preclinical model (such as the canine model), then clinical trials of IUSCT for prenatally diagnosed genetic disorders should be initiated. A compelling target disorder for the first human clinical trial would be sickle cell disease, which is relatively common, and despite the fact that it is curable by postnatal bone marrow transplantation, is rarely treated due to the low frequency of available HLA-matched sibling donors and the adverse effects of postnatal transplantation.7

manifestations before birth. However, in most circumstances (as in the case of IUSCT) the rationale for prenatal gene therapy relates to opportunities for gene transfer presented by normal events during fetal development. In adult life, stem cells are low-frequency populations, which are difficult to access because of tissue distribution and anatomical barriers. In contrast, stem and progenitor cell populations exist at high-relative frequencies in the fetus, and (depending on gestational age) may be accessible for gene transfer.47 The latter provides a unique opportunity for targeted gene delivery to all stem cells of specific tissue compartments. Moreover, the immature immune system of the fetus might allow tolerance to be developed for immunogenic transgenes and/ or viral products that would be rejected postnatally by the intact immune system.5,46 The lack of a fetal immune response against both the vector and transgene would make long-term transduction possible and allow postnatal treatment with the same vector/transgene combination. Finally, IUGT enables the delivery of much higher vector-to-cell ratios because of the relatively small size of the fetus.5,46 The latter should result in significantly improved transduction efficiencies (compared with postnatal gene transfer), and would limit prohibitive costs associated with large-scale vector production.

Gene Transfer Modalities

In Utero Gene Therapy Gene therapy is defined as the transfer of target genes to the cells of an individual to achieve therapeutic benefit. Several gene transfer methods have been developed that can be broadly divided into viral and nonviral. Nonviral gene delivery modalities (e.g., electroporation,44 gene “gun”45) are generally considered to be safer alternatives to viral vectors, but in vivo utilization is restricted by their limited efficiency and practicality. In contrast, gene transfer by viral vectors is a relatively efficient and versatile approach. The mainstay of vector-mediated gene delivery is to harness viral infection and replication pathways but avoid expression of innate viral genes that cause cellular toxicity. This is traditionally achieved by deleting some of the coding regions from the viral genome (leaving intact sequences essential for vector function), and inserting expression cassettes containing genes of interest. Gene therapy is an appealing therapeutic modality for the treatment of inherited disorders that are not amenable to stem cell therapy, and in which there is a single, wellcharacterized genetic defect. Although gene therapy has been applied (postnatally) to human disease for over two decades, there have been several obstacles limiting success. These include poor accessibility of target stem cell populations, and the development of an immune response against the transgenic (therapeutic) protein and/or the vector itself.5,46 Many of these issues could be theoretically overcome by an in utero gene therapy (IUGT) approach.

Rationale The most compelling rationale for IUGT is to prevent disease onset in circumstances where the disease has devastating European Journal of Pediatric Surgery

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Several types of viral vectors have been developed, and the choice for prenatal gene transfer applications is based on factors such as desired duration of transgene expression, immunogenicity, packaging capacity, and tissue tropism. Adenoviral vectors (AV) have been successfully used for IUGT, but they do not integrate into the host genome, have broad tropism, and are highly immunogenic. This has led to short, nontargeted expression in various types of rapidly dividing fetal cells, and significant inflammatory responses when administered after completion of immune education of the fetus.48–50 Adeno-associated virus vectors (AAV) are single-stranded DNA viruses that are gaining in application because of their safety and low immunogenicity.4,51 Tissue specificity can be influenced by the AAV serotype, but peak expression might take a few weeks (due to low integration frequency and slow expression profile). AAV vectors can be used to achieve adequate (but not permanent) transgene expression in tissues with low cell turnover including muscle, hepatocytes and neurons.4,51 Permanent expression of transgene following IUGT necessitates the use of integrating viral vectors. Retroviral vectors were the first such vectors to be used, but they have been largely superseded by lentiviral vectors (LV) that are highly efficient in transducing both dividing and quiescent cell populations with low immunogenicity.52,53 LV can be pseudotyped with unique viral envelopes to improve vector stability and tropism.54 In addition to vector type, route of vector administration as well as the gestational age of the fetus at the time of IUGT are key determinants of the distribution and efficiency of transduction, and are integrally related to normal developmental events. The distribution of transduction following intra-amniotic vector administration during early gestation

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(transduction of ectoderm/neuroectoderm tissues, including the skin and neural tissues) is dramatically different to that achieved with intravascular administration at the same gestational age (hematopoietic, endothelial, cardiac, hepatic, and osteogenic transduction).47 Moreover, localized transgene expression in specific tissues such as the lung,55 liver,50 and brain56 has been achieved using ultrasound-guided vector administration. As far as the timing of IUGT is concerned, it is generally accepted that gene transfer earlier in gestation will result in more efficient transduction, as accessibility of stem cell populations is reduced over time. For instance, murine skin stem cells can be easily transduced between embryonic days 8 and 10 (E8–E10; term E21) by intra-amniotic injection, but formation of the periderm makes them inaccessible thereafter.57 Similarly, murine neural tissues can only be transduced via the amniotic cavity before the closure of the neural tube at E9.58 Finally, late intra-amniotic IUGT (E16– E17) has been used to transduce pulmonary epithelial cells in mice; the timing of administration corresponds to the onset of fetal breathing movements.59

Experimental Evidence The National Institutes of Health Recombinant DNA Advisory Committee considered that a candidate disease for prenatal gene therapy should pose serious morbidity and mortality risks to the fetus and not have effective postnatal treatment.60 Till date, IUGT has been limited to proof of principle studies in animal models of such diseases with varying degrees of success. In this section, we will summarize experimental evidence on a few target genetic disorders to highlight the therapeutic potential of IUGT. One such group of disorders are the hemophilias, which are attractive targets for IUGT as they are relatively common, may manifest clinically early in life, and can be cured by low percentage of normal protein activity secreted by any cell type (avoiding issues with vector tropism). Initial murine studies used nonintegrating AV and AAV vectors administered by various routes, and achieved therapeutic but transient levels of factor VIII and factor IX expression.61,62 A more recent study demonstrated tolerance induction following intramuscular IUGT with AAV. Despite the fact that only low-level human factor IX expression were initially observed, the development of tolerance to the vector and transgene allowed readministration postnatally, and subsequent therapeutic factor IX levels.63 Waddington et al were the first to perform IUGT with an integrating LV encoding human factor IX; in this study, intravenous administration of the vector in immunocompetent hemophilic mice resulted in stable transgene expression with phenotypic improvement and no evidence of an immune response.64 More recently, stable expression of human factor IX at therapeutic levels was demonstrated in sheep65 and nonhuman primates66 following IUGT with AAV vectors. Duchenne and other forms of muscular dystrophy are also appealing target disorders for IUGT. In order for therapy to be achieved in these disorders, the striated muscles of the limb and chest wall as well as the cardiac muscle would need to be targeted. In adult clinical trials, dystrophin gene transfer was

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hampered by low efficacy due to the development of both cellular and humoral immunity to the transgenic protein.67 This could be avoided by IUGT that may also target myogenic progenitor and satellite cells capable of differentiating into and regenerating muscle fibers.68 The fetal approach was supported by early studies in a murine model of Duchenne muscular dystrophy, in which IUGT-induced expression of dystrophin during fetal life corrected the phenotype, but postnatal gene transfer did not.69 In a subsequent study by Gregory et al, more efficient gene transfer to all necessary muscle groups was seen after delivery of LV vectors to fetal mice using multiple administration routes70; intravascular delivery targeted the heart, intraperitoneal injection reached the diaphragm and intercostal muscles, and direct injection transduced skeletal muscle. Initial experience with muscle transduction following IUGT in sheep has been less encouraging. Although AV vector gene delivery to the hind limb musculature resulted in highly efficient gene transfer,50 targeting of the heart, diaphragm, and intercostal muscles using other administration routes was not successful.50,71 Perhaps the most compelling rationale for IUGT applies to the lysosomal storage diseases. These are characterized by deposition of substrate in the central nervous system (CNS) and associated permanent neurological damage before birth. Postnatal stem cell therapy has been attempted in these disorders with disappointing results because of the inability of donor cells to cross the blood–brain barrier.72 Although it was initially thought that systemic prenatal administration of vector could cross the immature blood–brain barrier leading to efficient CNS transduction, this has not proven to be the case. The use of localized administration strategies has been much more promising; Karolewski and Wolfe demonstrated that intraventricular injection of AAV vector in fetal mucopolysaccharidosis mice resulted in efficient transduction of the brain and spinal cord with associated long-term reversal of pathology.73 We have recently achieved impressive transduction of murine neural progenitors and CNS parenchyma using intra-amniotic administration of LV vectors during early gestation.47,58 This level of gene transfer would provide correction of multiple CNS disorders, but it would require very early prenatal diagnosis that is not feasible at present. Cystic fibrosis (CF) was one of the first diseases to be studied for a prenatal gene therapy approach. The main therapeutic target for CF is the ciliated pulmonary epithelial cell of upper airways expressing the CF transmembrane conductance regulator (CFTR) protein. Strategies for pulmonary IUGT include intratracheal (with or without concomitant tracheal occlusion), direct intraparenchymal, and intraamniotic administration.46 The latter approach is the least invasive, most effective, and widely used till date. Initial reports were promising with a study in CFTR knockout mice demonstrating cure of the disease following intraamniotic IUGT with AV vector.74 Unfortunately, subsequent studies have not replicated this success.75,76 This could be attributed in part to the rather short window for intraamniotic gene transfer to the pulmonary epithelium, which is limited immediately after the onset of fetal breathing movements during late gestation.59 In our experience, European Journal of Pediatric Surgery

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administration of AV or AAV vectors in the amniotic cavity during this window (around E16 in mice) results in extensive gene transfer to the pulmonary epithelium.77 Unfortunately, integrating vectors demonstrate much less efficient transduction, and expression thus far with all nonintegrating vectors has been transient.46 Although such expression would not be curative in the case of CF, it could be utilized to ameliorate pulmonary hypoplasia associated with congenital diaphragmatic hernia. IUGT might allow induction of short-term expression of growth factors at critical stages of pulmonary development, which might be useful against the significant neonatal morbidity and mortality associated with this serious condition.78 In this setting, intratracheal IUGT could be used in conjunction with tracheal occlusion, which is currently used clinically to promote lung growth.79

Obstacles to Clinical Translation Despite significant progress made in small and large animal models of congenital disease, there are currently no clinical studies of IUGT. This is due to the fact that there are several safety concerns that must be addressed before prenatal gene transfer can be applied to humans. Insertional mutagenesis is a major concern associated with integrating viral vectors, and is relevant to both pre- and postnatal gene transfer. New mutations have been observed in the postnatal setting, with four cases of T cell leukemia following gene therapy for XSCID.80 Till date, only one study has investigated the oncogenic potential of IUGT. In this report, high incidence of postnatal hepatic cancers was demonstrated in mice after IUGT with equine infectious anemia virus LV vector but not when using a similar vector with an HIV backbone.81 Although it remains unclear whether insertional mutagenesis led to tumor formation, these results suggest that the fetus might be particularly sensitive to certain vectors. Germ line transmission following IUGT with integrating vectors is another significant safety concern as well as a bioethical issue. Targeted gene therapy that occurs after the compartmentalization of primordial germ cells should not affect the germ line. Studies by Porada et al demonstrated low-level transduction of germ cells following intraperitoneal IUGT in sheep,82 and also determined that the degree of gene transfer to germ cells is dependent on the gestational age.83 Given the likelihood that low-level germ cell transduction after systemic administration of integrating vector cannot be excluded, the frequency of transduction that is acceptable in the context of treatment of a severe genetic disorder needs to be considered. Finally, the effects of prenatal gene transfer on fetal organ development need to be investigated carefully for all IUGT modalities. Strategies involving expression of growth factors or regulatory molecules have significant potential to alter normal organ development. This is highlighted in a recent study by Gonzaga et al, who demonstrated that delivery of AV vector containing the fibroblast growth factor 10 into the fetal rat parenchyma, resulted in the formation of cystic abnormalities similar to human congenital cystic adenomatoid malformations.84 In a separate study, overexpression of transforming growth factor β in the lung of the fetal macaque resulted in pulmonary hypoplasia and fibrosis.85 European Journal of Pediatric Surgery

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Combined In Utero Stem Cell–Gene Therapy using Autologous Stem Cells A clinically translatable method of delivering gene therapy to the fetus would be a combined autologous stem cell–gene transfer approach.86 This would involve harvesting of stem cells from the fetus, in vitro gene therapy to correct the genetic defect, and transplantation of these cells back to the donor fetus. Stem cells can be obtained from various fetal sources including the blood, liver, and gestational tissues (amniotic fluid and placenta). Blood and fetal liver sampling in early gestation are associated with significant miscarriage risks,87,88 and as a result it is unlikely that these stem cell sources will have clinical utility. However, fetal stem cells can be obtained easily and safely from samples collected at amniocentesis and/ or chorionic villus sampling.89 Following their first description by De Coppi et al,90 amniotic fluid stem cells (AFSC) have been the focus of intensive investigation for both pre- and postnatal applications. AFSC are appealing for therapy as they are broadly multipotent (can differentiate in cells from all three embryonic germ layers, including all hematopoietic lineages), can be easily transduced without altering their cellular characteristics, have no tumorigenic potential, and their isolation is not associated with any ethical issues.90–92 Their use in the prenatal setting could address many of the limitations of both IUSCT and IUGT. AFSC are of fetal origin and as a result should be able to compete better against host cells compared with adult stem cells. They are nonimmunogenic to the fetus at any gestational age, and are also unlikely to result in maternal immunization because of the tolerogenic properties of the placenta. Moreover, the possibility of performing in vitro gene transfer would allow cells to be checked for insertional mutagenesis before transplantation, and would obviate the risk of germ line transmission of transgenes. In a recent proof of principle study, Shaw et al showed that in utero transplantation of autologous (isolated using ultrasound-guided amniocentesis), expanded, and transduced AFSC resulted in widespread tissue engraftment in the ovine fetus.93 We are currently investigating the hematopoietic potential of AFSC following intravenous transplantation in immunocompetent fetal mice, and have obtained stable, multilineage engraftment at near-therapeutic levels using relatively small donor cell numbers (S. P. Loukogeorgakis, MD, PhD, unpublished data, April 2014). Whether in utero transplantation of AFSC would be therapeutic in models of hematological and other congenital disorders remains to be determined.

Conclusions and Future Perspectives In utero stem cell and gene therapies offer clear advantages over postnatal application. However, in order for these treatments to become acceptable clinically they need to be shown to be safe (for both the mother and fetus), as well as effective at providing a lifetime cure to the treated fetus. Although great progress has been made over the past 30 years, significant challenges remain. In the case of IUSCT, future research efforts should be focused on defining and overcoming the competitive and

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3 Vrecenak JD, Flake AW. In utero hematopoietic cell transplantation

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Conflict of Interest None. 22

References 1 Vrecenak JD, Flake AW. Fetal surgical intervention: progress and

perspectives. Pediatr Surg Int 2013;29(5):407–417 2 McCandless SE, Brunger JW, Cassidy SB. The burden of genetic disease on inpatient care in a children’s hospital. Am J Hum Genet 2004;74(1):121–127

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—recent progress and the potential for clinical application. Cytotherapy 2013;15(5):525–535 Mehta V, Abi Nader K, Waddington S, David AL. Organ targeted prenatal gene therapy—how far are we? Prenat Diagn 2011;31(7): 720–734 Pearson EG, Flake AW. Stem cell and genetic therapies for the fetus. Semin Pediatr Surg 2013;22(1):56–61 Weissman IL, Shizuru JA. The origins of the identification and isolation of hematopoietic stem cells, and their capability to induce donor-specific transplantation tolerance and treat autoimmune diseases. Blood 2008;112(9):3543–3553 Michlitsch JG, Walters MC. Recent advances in bone marrow transplantation in hemoglobinopathies. Curr Mol Med 2008; 8(7):675–689 Billingham RE, Brent L, Medawar PB. Actively acquired tolerance of foreign cells. Nature 1953;172(4379):603–606 Takahama Y. Journey through the thymus: stromal guides for T-cell development and selection. Nat Rev Immunol 2006;6(2):127–135 Josefowicz SZ, Lu LF, Rudensky AY. Regulatory T cells: mechanisms of differentiation and function. Annu Rev Immunol 2012; 30:531–564 Nijagal A, Derderian C, Le T, et al. Direct and indirect antigen presentation lead to deletion of donor-specific T cells after in utero hematopoietic cell transplantation in mice. Blood 2013;121(22): 4595–4602 van Dijk BA, Boomsma DI, de Man AJ. Blood group chimerism in human multiple births is not rare. Am J Med Genet 1996;61(3): 264–268 Tavian M, Péault B. Embryonic development of the human hematopoietic system. Int J Dev Biol 2005;49(2-3):243–250 Kim HB, Shaaban AF, Yang EY, Liechty KW, Flake AW. Microchimerism and tolerance after in utero bone marrow transplantation in mice. J Surg Res 1998;77(1):1–5 Lovell KL, Kraemer SA, Leipprandt JR, et al. In utero hematopoietic stem cell transplantation: a caprine model for prenatal therapy in inherited metabolic diseases. Fetal Diagn Ther 2001;16(1):13–17 Peranteau WH, Heaton TE, Gu YC, et al. Haploidentical in utero hematopoietic cell transplantation improves phenotype and can induce tolerance for postnatal same-donor transplants in the canine leukocyte adhesion deficiency model. Biol Blood Marrow Transplant 2009;15(3):293–305 Harrison MR, Slotnick RN, Crombleholme TM, Golbus MS, Tarantal AF, Zanjani ED. In-utero transplantation of fetal liver haemopoietic stem cells in monkeys. Lancet 1989;2(8677):1425–1427 Fleischman RA, Mintz B. Prevention of genetic anemias in mice by microinjection of normal hematopoietic stem cells into the fetal placenta. Proc Natl Acad Sci U S A 1979;76(11):5736–5740 Blazar BR, Taylor PA, Vallera DA. In utero transfer of adult bone marrow cells into recipients with severe combined immunodeficiency disorder yields lymphoid progeny with T- and B-cell functional capabilities. Blood 1995;86(11):4353–4366 Peranteau WH, Endo M, Adibe OO, Flake AW. Evidence for an immune barrier after in utero hematopoietic-cell transplantation. Blood 2007;109(3):1331–1333 Ashizuka S, Peranteau WH, Hayashi S, Flake AW. Busulfan-conditioned bone marrow transplantation results in high-level allogeneic chimerism in mice made tolerant by in utero hematopoietic cell transplantation. Exp Hematol 2006;34(3):359–368 Carrier E, Lee TH, Busch MP, Cowan MJ. Induction of tolerance in nondefective mice after in utero transplantation of major histocompatibility complex-mismatched fetal hematopoietic stem cells. Blood 1995;86(12):4681–4690 Flake AW, Roncarolo MG, Puck JM, et al. Treatment of X-linked severe combined immunodeficiency by in utero transplantation of paternal bone marrow. N Engl J Med 1996;335(24):1806–1810 Kim HB, Shaaban AF, Milner R, Fichter C, Flake AW. In utero bone marrow transplantation induces donor-specific tolerance by a European Journal of Pediatric Surgery

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immune barriers to engraftment in translational animal models and humans. Despite the fact that the “two-step” scheme consisting of induction of donor-specific tolerance followed by nonmyeloablative postnatal transplantation is nearing clinical application, a “single-step” IUSCT approach leading to therapeutic levels of engraftment would be preferable. Strategies that might allow us to achieve this goal include targeted fetal myeloablation (to create “space” in hematopoietic or other stem cell niches), transplantation of large numbers of cells (to provide an overwhelming excess of donor HSC or other stem cells), pretreatment of donor cells with compounds that enhance their proliferation and/or niche-homing abilities, and selective use of donor cells that do not induce an immune response in the mother (e.g., maternal HSC and autologous AFSC). Although some of these have been difficult to accomplish thus far, we are hopeful that, with better understanding of stem cell behavior and fetomaternal physiology, as well as advances in novel scientific fields (e.g., nanotechnology), such strategies can be developed in the near future. IUGT, although still in the experimental stage, has even greater potential to prevent the onset of a broad range of inherited disorders. Proof of principle studies of IUGT in animal models of congenital disease have shown significant therapeutic benefits, but clinical translation necessitates the alleviation of safety concerns including the risk of insertional mutagenesis (leading to tumor formation), the effect on organ development (resulting in treatmentrelated fetal anomalies), and the inadvertent transmission of transgenes to the germ line. While greater tissue specificity and safety can be achieved by the use of tissuespecific promoters or regulated transgene expression, safer gene transfer modalities will be needed to develop to address these issues. One such method might be the “hybrid” stem cell–gene therapy approach using ex vivo transduction and transplantation of autologous fetal cells isolated from gestational tissues. Finally, it is equally important to devise strategies that will minimize risks associated with fetal intervention, including fetal demise, infection, and preterm labor. This is likely to require the development of novel instruments and minimally invasive/image-guided delivery platforms that could be used for both IUSCT and IUGT. Regardless of current concerns, there is great potential for prenatal stem cell and gene therapy, and solving the remaining hurdles will change the way we perceive and treat congenital disease.

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In Utero Stem Cell and Gene Therapy

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