Stem cell and genetic therapies for the fetus

Seminars in Fetal & Neonatal Medicine 15 (2010) 46–51

Contents lists available at ScienceDirect

Seminars in Fetal & Neonatal Medicine journal homepage: www.elsevier.com/locate/siny

Stem cell and genetic therapies for the fetus Jessica L. Roybal a, Matthew T. Santore a, b, Alan W. Flake a, b, * a b

Children’s Center for Fetal Research, Children’s Hospital of Philadelphia, PA, USA University of Pennsylvania School of Medicine, Philadelphia, PA, USA

s u m m a r y Keywords: Fetal therapy Gene therapy Hematopoietic stem cell transplantation Prenatal treatment

Advances in prenatal diagnosis have led to the prenatal management of a variety of congenital diseases. Although prenatal stem cell and gene therapy await clinical application, they offer tremendous potential for the treatment of many genetic disorders. Normal developmental events in the fetus offer unique biologic advantages for the engraftment of hematopoietic stem cells and efficient gene transfer that are not present after birth. Although barriers to hematopoietic stem cell engraftment exist, progress has been made and preclinical studies are now underway for strategies based on prenatal tolerance induction to facilitate postnatal cellular transplantation. Similarly, in-utero gene therapy shows experimental promise for a host of diseases and proof-in-principle has been demonstrated in murine models, but ethical and safety issues still need to be addressed. Here we review the current status and future potential of prenatal cellular and genetic therapy. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction In the future, the greatest impact of fetal treatment will likely be in the areas of prenatal stem cell and gene therapy. Whereas fetal surgical intervention remains limited to a few structural anomalies, prenatal stem cell and gene therapy have therapeutic potential for a large number of genetic disorders. Realization of this potential would expand fetal therapy far beyond its current focus of treating the compromised fetus. If there are biological advantages that favor prenatal over postnatal therapy, ‘fetal’ therapy may become the preferred strategy for the treatment of many anticipated pediatric and adult diseases. This review will provide our view of the rationale, current status, and potential future applications of prenatal stem cell and gene therapy.

2. In-utero stem cell therapy A stem cell can be defined as ‘a cell that can self-replicate and can give rise to more than one type of mature daughter cell’. In recent years, there have been many cell populations characterized as ‘stem cells’; however, a complete discussion of all stem cell applications in the fetus is beyond the scope of this review. It is likely that the first broad clinical application of stem cell therapy to

* Corresponding author. Address: Department of Surgery, Abramson Research Bldg, Rm. 1116B, 3615 Civic Center Blvd, Philadelphia, PA 19104-4318, USA. Tel.: þ1 215 590 3671; fax: þ1 215 590 3324. E-mail address: fl[email protected] (A.W. Flake). 1744-165X/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.siny.2009.05.005

the fetus will be in-utero hematopoietic stem cell transplantation (IUHCT). We will confine our discussion to this multipotent stem cell type as a paradigm for all prenatal stem cell therapy. 3. Rationale for in utero hematopoietic stem cell transplantation The hematopoietic stem cell (HSC) is a multipotent stem cell that maintains functional hematopoiesis by generation of all hematopoietic lineages throughout fetal and adult life.1 As such, it lends itself to treatment of a broad range of hematopoietic disorders. In addition, its differentiated progeny express the major histocompatability (MHC) antigens and participate in immune system development providing the opportunity for donor-specific tolerance induction. The rationale for IUHCT is based on unique opportunities provided by normal developmental events that may facilitate cellular engraftment.2 Its most important advantage is related to fetal immunologic tolerance. Early in gestation the immune system undergoes a process of self-education in which T-cells with high affinity to self-antigen are deleted, leaving a repertoire of T-cells reactive to foreign antigen. In theory, introduction of allogeneic cells by IUHCT with subsequent donor antigen presentation in the thymus prior to completion of this process, should lead to deletion of alloreactive T-cells and donor-specific tolerance. To achieve this, the transplant should be performed prior to the appearance of mature T-cells in the fetal thymus and peripheral circulation, which corresponds to 17 days of gestation in the mouse and 12–14 weeks of gestation in the human fetus.3 This makes possible strategies

J.L. Roybal et al. / Seminars in Fetal & Neonatal Medicine 15 (2010) 46–51

based on donor-specific tolerance induction to maintain hematopoietic engraftment after IUHCT and to facilitate further cellular or organ transplantation in a tolerant recipient after birth.4,5 Other biological opportunities unique to the fetus are the normal sequential developmental migrations of HSCs to form hematopoietic compartments. Hematopoiesis starts at the yolk sac or aortogonado-mesonephric region, migrates to the fetal liver and finally resides in the bone marrow.6 Although the original theory was that development of new niches would facilitate engraftment of donor cells after IUHCT without the need for myeloablation, it is now recognized that the fetal hematopoietic system is highly competitive with a relative excess of circulating HSCs.7,8 However, if the regulatory signals controlling the migrations of HSCs can be understood, they may be manipulated to favor the engraftment of donor cells.9 Finally, the very small size of the fetus allows the transplantation of much larger cell doses per kilogram than can be achieved after birth. By this approach, it may be possible to partially overcome the competitive advantage of host hematopoiesis. 4. Experimental progress toward clinical IUHCT The potential for fetal tolerance to facilitate clinical transplantation has been recognized since Billingham et al.’s original description of ‘actively acquired tolerance’ in 1953.2 Additional support for the concept was provided by observations in several species of hematopoietic chimerism and associated tolerance in dizygotic twins who share placental circulation.10 Finally, mechanistic insight into tolerance for self-antigens (and by inference foreign antigen), and the central role of the thymus in this process, has been elucidated over the past two decades. Given these observations, the administration of allogeneic HSCs with appropriate timing to the preimmune fetus should theoretically result in engraftment of donor cells and consistent donor-specific tolerance. However, historically that has not been the case. Since Billingham et al.’s original report, subsequent studies by many investigators on fetal and neonatal tolerance have documented the entire spectrum of immune response from donor specific tolerance to immunization. Over the past 20 years we, and others, have focused on the barriers that exist to engraftment in the fetus. Perhaps the most important barrier is host cell competition. Following IUHCT, the fetus has a vigorous hematopoietic compartment that is absent after the myeloablative conditioning routinely used prior to postnatal bone marrow transplantation (BMT). Therefore, the success of IUHCT relies on the assumption that donor hematopoiesis can effectively compete with host hematopoiesis to achieve significant donor cell expression. There is abundant evidence that competition from the host hematopoietic compartment is a formidable barrier to successful engraftment. When donor cells have a competitive advantage, even the engraftment of a relatively limited number of cells can ultimately reconstitute the recipient. The high level of donor hematopoiesis achieved in c-kit-deficient mouse strains in which there is a proliferative defect in host HSCs is an extreme example. In this model, as few as one or two normal HSCs were shown to fully reconstitute the hematopoietic compartment after IUHCT.11 Studies of IUHCT performed in the mouse severe combined immunodeficiency (SCID) model also illustrate the importance of host cell competition.12 In this model, donor lymphoid cells have a survival and proliferative advantage. IUHCT results in complete reconstitution of the lymphoid compartment with minimal engraftment of other lineages, where progenitors maintain their competitive capacity.11 The converse is also true. Although a relationship between donor cell dose and levels of engraftment clearly exists, transplantation of even massive doses of donor cells (2  1011 cells/kg) in a congenic strain combination

47

results in average levels of chimerism of <10%. Fortunately, donorspecific tolerance does not require high levels chimerism. Our studies and those of others suggest that the threshold for consistent induction of donor-specific tolerance is between 1% and 2% in the murine model, a clinically achievable range. For many years the murine model of IUHCT was extremely difficult to engraft with only the achievement of microchimerism (detectable by polymerase chain reaction (PCR)) by ourselves and other investigators.13–15 However, mixed hematopoietic chimerism across full MHC barriers with associated donor-specific tolerance is now routinely observed. Mechanistic analysis of tolerance in chimeric mice supported a primary mechanism of deletion of donor-reactive lymphocytes, although deletion was not complete, implicating the presence of peripheral tolerance mechanisms as well.5,16 Thus IUHCT appeared to result in ‘normal’ immunologic processing of donor cells with high-level deletion of donor-reactive lymphocytes in the thymus and presumably generation of donorspecific T-regulatory cells to suppress donor-reactive cells that escape thymic deletion. The achievement of donor-specific tolerance allowed us to perform proof-in-principle studies of the promising clinical strategy of prenatal tolerance induction by IUHCT followed by postnatal non-toxic bone marrow transplantation to increase low levels of chimerism to levels that would be therapeutic for diseases such as the hemoglobinopathies. Three different non-toxic postnatal strategies were demonstrated to work: (1) preparative low-dose total body irradiation followed by T-cell-depleted BMT16; (2) postnatal donor-specific lymphocyte infusion without BMT5; and (3) low-dose busulfan as a single-agent preparative regimen, followed by T-cell-depleted BMT.4 In each study, complete or near-complete replacement of host hematopoiesis by donor cells was achieved, essentially without toxicity or graft vs host disease (GVHD). These studies form the basis for what we believe will be the first successful clinical strategy for application of IUHCT to competitive hematologic disorders. Despite the success in the murine model, there were unexplained observations that suggested an additional barrier to engraftment beyond host cell competition. First, what appeared to be consistent delivery of donor cells resulted in long-term donor chimerism in only approximately one-third of recipients. Second, engraftment differed significantly between strain combinations. By performing early tracking of donor cells and long-term assessment of donor chimerism, we were able to document that 100% of allogeneic and congenic recipients maintained high levels of engraftment up to 3 weeks after IUHCT. However, between 3 and 5 weeks 70% of allogeneic animals lost their engraftment whereas 100% of congenic animals remained chimeric. The difference in the incidence of chimerism between congenic and allogeneic donors supported the presence of an adaptive immune barrier to engraftment after IUHCT.17 We have now confirmed that there is an allospecific cellular and humoral response that is quantitatively higher in non-chimeric vs chimeric animals. This finding was incongruous with our previous demonstration of long-term chimerism in some animals and the presence of deletional tolerance. The pivotal observation that explains this contradiction and should influence all subsequent studies in the murine model is our recent observation that the immune response is in reality a maternal immune response that is transferred to the neonate via maternal breast milk.18 If pups are fostered with a surrogate mother who has not been exposed to donor antigen, the frequency of chimerism remains 100%. The mechanism of the activation of the immune response in the neonate is currently under investigation. We feel that the most important finding in these studies is not the identification of the maternal immune response as the key factor in loss of chimerism, but rather the observation that in the absence of maternal influence allogeneic engraftment and

48

J.L. Roybal et al. / Seminars in Fetal & Neonatal Medicine 15 (2010) 46–51

long-term chimerism uniformly occur. This confirms the absence of an adaptive immune barrier in the preimmune fetus and validates the potential for practical application of ‘actively acquired tolerance’ to facilitate allogeneic cellular and/or organ transplantation. It raises the question of whether maternal immunization is an issue in large animal models and clinical circumstances, and whether it is a limitation to engraftment after IUHCT. It also raises the question of the importance of the innate immune system as a barrier to engraftment. Although natural killer (NK) cells have recently been implicated in loss of low-level engraftment after IUHCT,19 their effect appears to be lost in circumstances of absence of maternal influence. It is possible that the presence of maternal allospecific antibody can directly activate NK cells via the mechanism of antibody-dependent cell-mediated cytotoxicity. These are critical questions to answer prior to clinical application and ultimately will require the development of relevant large animal models. With the exception of the sheep, there has been very limited success after IUHCT in large animal models, although recently that has begun to change. Successful achievement of measurable multilineage chimerism after IUHCT with associated donor-specific tolerance for swine leukocyte antigen (SLA)-matched kidney transplants has been demonstrated in the SLA inbred pig model.20 With our success in the murine model we have recently begun translational studies in the canine model using dogs that have the canine analog of human leukocyte adhesion deficiency (canine leukocyte adhesion deficiency (CLAD)). CLAD-affected dogs have a severe immunodeficiency that results in death prior to 6 months of age, whereas the CLAD carrier is phenotypically normal. Neither the affected nor carrier dogs have a significant competitive defect in the HSC compartment or in any of the lineages. Therefore, the CLAD model should be representative of the degree of host cell competition expected for most target diseases. This is supported by prior experience in the canine model by Blakemore in which minimal levels of engraftment have been achieved after IUHCT.21 In addition, the canine model has been extensively used for BMT experiments and has been validated as a preclinical model from the perspective of GVHD.22 In our first study in the canine model, we have demonstrated that low-level chimerism can be achieved by IUHCT, and that these levels of chimerism can: (1) ameliorate or cure the clinical phenotype of CLAD; and (2) can result in associated donor specific tolerance in some animals that is adequate to facilitate postnatal enhancement of chimerism to potentially therapeutic levels using the single agent, low-dose busulfan conditioning regimen, followed by transplantation of T-celldepleted bone marrow from the same donor.23 In this study, we saw no significant toxicity and no GVHD. We are encouraged that the results of IUHCT in the canine model appear remarkably similar to our results in the murine model, suggesting that our results in the murine model can be translated to clinical application. The clinical experience thus far can be summarized as disappointing with the exception of SCID, which has been successfully treated by IUHCT in a number of centers.24–26 However, SCID is a unique disorder that provides a survival and proliferative advantage for donor T-cells, and the engraftment achieved has only been documented to reconstitute the T-cell lineage (split chimerism). Thus it can be stated that IUHCT has not been clinically successful in establishing engraftment in a hematopoietically competitive recipient. As most of the anticipated target disorders such as the hemoglobinopathies, immunodeficiencies, and the lysosomal storage diseases are competitively normal in fetal life, methods must be developed to overcome host cell competition prior to further attempts at clinical application. The strategy of prenatal tolerance induction to facilitate non-toxic postnatal BMT lowers the threshold of chimerism required for clinical application of IUHCT. Methods to selectively enhance donor cell competition

and thereby further enhance donor chimerism achieved by IUHCT have been and are being developed in animal models and need translational application in appropriate preclinical large animal models. 5. In-utero gene therapy (IUGT) Many genetic disorders may not be amenable to stem cell therapy, and for some it may be preferable to genetically correct the stem cell abnormality in situ. At the present time, there are well-documented risks associated with viral vector-mediated gene transfer that need to be addressed prior to consideration of any clinical application in the fetus. However, if safe and effective methods for fetal gene transfer can be developed, a large number of disorders would be amenable to IUGT. There is an increasing body of experimental evidence that supports the therapeutic potential of IUGT. The goal of gene therapy is to deliver genetic material to cells for therapeutic benefit. Although gene therapy has been applied to human disease for two decades, there have been many obstacles that limit its success. Many of these obstacles could be overcome by an in-utero approach. By far, the most compelling rationale for inutero gene therapy is the potential to prevent disease prior to the onset of irreversible organ damage. There are several reasons why the fetus offers a favorable environment for the transfer of genetic material. During fetal development, stem and progenitor cells exist at high frequency and are exposed within various tissue compartments. Prior to the distribution of these cells within organs or tissue compartments, a window of opportunity exists when they are accessible for gene transfer. Transgenes can be targeted to these expanding cell populations, which will be inaccessible later in life. In addition to accessibility of stem cells, immunogenic vectors and transgenes can take advantage of the immature immune system of the fetus. Immunologic tolerance not only ensures long-term, stable transduction, but should also make postnatal treatment with the same vector and transgene possible. Finally, the small size of the fetus means that extremely high vector-to-cell ratios can be achieved with a limited amount of vector. 6. Modes of gene transfer Gene delivery to cells depends on a vector, which ideally would specifically target a single organ and require only one application. Non-viral vectors, such as the gene gun, have been proposed as a safer alternative to viruses. However, the introduced DNA remains episomal and is lost with cell division, resulting in limited expression.27 Viral vectors are more efficient agents for gene delivery because they easily penetrate host cells and take advantage of the host cell machinery to replicate. They are engineered to have an attenuated viral genome so that only transgenes, not viral genes, are copied. Choice of viral vector type depends on a number of factors: immunogenicity, packaging capacity, targeted tissue, and the desired duration of expression. Adenoviral vectors have been successfully used for fetal gene transfer models. However, the vectors do not integrate into the host genome, so transgene expression in rapidly dividing fetal cells is of is limited duration. In addition, adenoviral vectors are highly immunogenic in adults and later-gestational fetuses.28 They are useful when a short duration of expression is desired. Adenoassociated virus vectors (AAVs) are less immunogenic than adenoviral vectors. The AAV serotype influences tissue specificity. Because AAVs integrate into the genome at low frequency and have a slow expression profile, peak expression may take a few weeks. Adequate duration of expression can be achieved in tissues with low cell turnover, such as skeletal muscle, liver, and the central

J.L. Roybal et al. / Seminars in Fetal & Neonatal Medicine 15 (2010) 46–51

nervous system. To attain long-term expression, a vector must integrate into the host genome. Retroviral and lentiviral vectors both integrate but lentiviruses can infect dividing and non-dividing cells, whereas retroviral vectors only infect dividing cells. Lentiviral vectors, including those derived from a replication-incompetent human immunodeficiency virus (HIV), have low immunogenicity. Lentiviruses can be pseudotyped with a unique viral envelope to improve tissue targeting and lentiviral stability.29 The downside of lentiviral integration into the host DNA is the risk of insertional mutagenesis. 7. Route and timing of prenatal gene therapy Once a vector is chosen, transduction of a targeted population of stem cells then depends on the gestational age of the fetus and the site of vector administration. Stem and progenitor cells will integrate into tissues and differentiate as the fetus grows, therefore earlier gestational gene therapy will generally result in more efficient transduction of stem cells than later gestation gene therapy. For example, stem cells that will form the skin and skin appendages can be transduced between E8 and E10 by intra-amniotic vector injection, but after that window the periderm forms and skin stem cells become inaccessible to the amniotic fluid.30 Although the technical difficulty varies, any fetal organ or embryonic cavity that can be visualized can be injected with vector. Ultrasound-guided transuterine injection is the most common method used for prenatal gene therapy in animal models. Direct administration into the lung, liver, and brain has led to localized gene expression,31–33 whereas vector injected into a body cavity such as the peritoneal or amniotic cavities can potentially transduce several different progenitor cell populations. Developmental stage at the time of injection will determine which progenitor cell populations are exposed to vector and more than one population may be transduced at a time. 8. Proof-in-principle of prenatal gene therapy The diseases best treated by prenatal gene therapy are those caused by a mutation in a single gene, those that can be diagnosed before birth, and those that cause irreversible organ damage in the fetus or early postnatal period. Using rodent models of human genetic diseases and a variety of transduction methods, prenatal gene transfer has been targeted to a range of organs. In several disease models, phenotypic rescue has been accomplished (see Table 1).32–37 Preclinical models of prenatal gene therapy are currently under investigation. Fetal sheep are ideal models because they share physiological and developmental characteristics with human

49

fetuses and have large fetuses that are amenable to early gestational manipulation. In addition, the ovine immune system has been extensively studied and the ovine life span is long enough to allow for long-term studies of safety and efficacy. Sheep studies have compared different injection techniques and vector types, but only low-level expression has been reported. For example, injection of retroviral vectors into preimmune fetal sheep was shown to transduce hematopoetic cells with low-level expression observed for 5 years.38 Intra-tracheal adenoviral and lentiviral injections have led to low-level gene expression in peripheral airways.39,40 Further research with large animal models is needed to refine the technical aspects as well as the timing of potential fetal gene therapy in humans. 9. Candidate diseases Promising candidates for human application include the hemophilias, muscular dystrophy, and central nervous system disorders. Progress in hemophilia research provides one example of how prenatal gene therapy might be used. Current treatment for hemophilia is expensive and requires repeated transfusions and infusions of the missing factor. Postnatal gene therapy does not prevent manifestations of the disease and has been hampered by the host immune response.41 Prenatal gene therapy would be a rational approach to overcome these limitations and experimental data thus far have been promising. The factors that make hemophilia an optimal disease target for prenatal gene are: the disease results from the absence of a single gene; the inheritance pattern is known and prenatal diagnosis is available; the disease presents in early life and occasionally in the fetus; and low-level, unregulated protein expression is curative. In addition, targeted expression is not required because, theoretically, the deficient protein could be secreted by any cell. Hemophilias have been extensively studied in murine models with gene transfer performed at various gestational ages, via different routes of injection, and using different vector types. Schneider et al. compared intraperitoneal, intramuscular, and intravenous injections of human factor IX driven by adenoviral and AAV serotype 2 into E15.5 fetuses and found that adenoviral vectors resulted in initially higher levels of factor IX. Although transgene expression decreased over time with both vectors, adenoviral vector-injected mice maintained a therapeutic level for 6 months and no antibodies developed against either vector or transgene.42 Sabatino et al. observed low-level human factor IX expression after intramuscular injection of AAV-1 and -2 in E14 fetuses and neonatal mice. Tolerance induced to AAV-1 but not AAV-2 allowed the postnatal re-administration of the AAV-1-driven transgene and subsequent therapeutic factor IX levels.43 The most impressive

Table 1 Proof-in-principle of prenatal gene therapy in rodent models of metabolic, central nervous system, and musculoskeletal diseases. Disease

Mutation

Animal model

Vector

Cystic fibrosis

CTFR

Mouse

Adenovirus Intra-amniotic

Crigler–Najjar

UGT 1A1

Rat

Lentivirus

Intrahepatic

Pompe’s disease

GAA

Mouse

AAV-2

Leber’s congenital amarosis MPS VII (Sly syndrome)

RPE 65

Mouse

AAV-2

Intrahepatic, intraperitoneal Subretinal

b-Glucuronidase Mouse

AAV-1

Intraventricular

Duchenne’s muscular dystrophy

Dystrophin

Adenovirus Intramuscular

Mouse

Approach

Results

Authors

Reversal of the fatal intestinal cystic fibrosis phenotype, survival 250 days Decreased serum bilirubin by 45% for >1 year, but developed antibodies Transduction of the diaphragm with normal contractile function for 6 months Restoration of vision for 2.5 months, gene expression for 6 months CNS expression for 1 year

Larson et al. (1997)34 Seppen (2003)33

Rucker et al. (2004)35 Dejneka et al. (2004)36 Karolewski et al. (2006)32 Gene expression in 2–3 muscles of the injected hindlimb, Reay et al. (2008)37 muscle degeneration at 9 weeks

CFTR, cystic fibrosis transmembrane conductance regulator; UGT 1A1, UDP-glucuronyl transferase; AAV, adeno-associated virus; GAA, acid a-glucosidase; RPE 65, retinal pigment epithelium 65; MPS VII, mucopolysaccharidosis VII.

50

J.L. Roybal et al. / Seminars in Fetal & Neonatal Medicine 15 (2010) 46–51

results of prenatal gene transfer in hemophiliac mice occurred with a lentiviral vector. Waddington et al. demonstrated therapeutic levels of factor IX expression (9–16%) for 14 months, improved coagulation, and no immune response against the protein following intravenous administration of a lentiviral-driven transgene into E15 fetuses.44 These studies have shown that prenatal gene therapy in mice can result in low-level transgene expression that not only has therapeutic significance, but also in some cases induces tolerance allowing postnatal administration of the same vector and transgene for enhancement of expression. 10. Risks of prenatal gene therapy The potential safety concerns of prenatal gene therapy include those associated with fetal intervention and those due to gene transfer. As with any fetal intervention, fetal loss, infection, and preterm labor are possible. In reality, a minimally invasive approach using a fine needle under ultrasound guidance has minimal procedure related morbidity. As previously discussed, some gene transferrelated risks depend on the type of vector. The host immune response to vector or transgene, insertional mutagenesis caused by integrating vectors, and the risk of a replication-incompetent HIV vectors becoming replication-competent may all be concerns depending upon the gestational age of the recipient and the vector construct used. Common to all vectors used for prenatal gene transfer are the concerns of germ-line transmission, disruption of normal organ development, and transplacental spread of transgenes to the mother. Lentiviral vectors result in efficient transduction because they integrate into the host genome, but the DNA insertion site may have deleterious consequences. New mutations have been observed after postnatal gene therapy with integrating vectors. Four cases of T-cell leukemia were diagnosed 31–68 months after retroviralmediated gene therapy for X-linked SCID.45 Only one prenatal study has demonstrated the same phenomenon. A high incidence of liver tumors was observed in mice that received prenatal injection with an early form of third-generation equine infectious anemia virus vectors with self-inactivating configuration. The insertion sites were not identified, but no tumors were observed when a similar vector with an HIV backbone was used.46 Further studies of prenatally treated animals are needed to fully assess the risk of insertional mutagenesis. Although prenatal gene transfer has great potential for restoring normal function, manipulation of the fetus may alter normal organ development. Both the site of injection and the toxicity of the vector itself need to be evaluated. Sheep subjected to in-utero intrapulmonary and intracardiac vector injection showed no adverse effects on the postnatal heart and lung development.47 However, we have found that fibroblast growth factor 10 expression in the developing rat lung leads to cystic adenomatoid malformations illustrating how forced expression of a specific transgene can lead to malformation.48 Prenatal gene therapy is directed toward somatic cells, but inadvertent gene transfer to the germ line is a major concern for both safety and ethical reasons. Targeted gene therapy that occurs after the compartmentalization of primordial germ cells should not affect the germ line.49 Gene transfer to the germ line has been investigated by several groups. Porada et al. evaluated sheep that received intraperitoneal retroviral-mediated prenatal gene transfer. Despite negative breeding studies, PCR on the purified sperm from injected rams and immunohistochemistry of sectioned testes showed low-level transduction of germ cells.50 More recently, the same group reported that gestational age affects germ cell transduction. Given the likelihood that low-level transduction of germ cells after systemic administration of integrating vector to the fetus cannot be completely excluded, the frequency of germ-line

transduction that is acceptable in the context of treatment of a severe genetic disorder needs to be considered. 11. Future challenges Although great progress has been made, there are many remaining challenges for prenatal cellular and gene therapy. Challenges for IUHCT are primarily related to overcoming the competitive barriers to engraftment in the fetus, and better defining the innate and adaptive immune limitations to engraftment in large animals and humans. While the strategy of prenatal tolerance induction for facilitation of postnatal BMT is nearing clinical application, a single-step treatment consisting of IUHCT with achievement of therapeutic levels of engraftment would be ideal. In our opinion, it is unlikely that high levels of engraftment can be achieved in the fetus without the development of a highly specific, non-toxic method for fetal myeloablation. Fetal gene therapy has even greater potential to prevent the onset of inherited genetic diseases, but it is still in the early experimental stage. Proof-in-principle for fetal gene therapy for many disorders has already been demonstrated in rodent and large animal models. Safety concerns involving the risk of insertional mutagenesis, the effect on organ development and the importance of low-level germ cell transmission need to be extensively investigated in appropriate preclinical animal models prior to application in humans. The ethics of fetal gene therapy and its potential to alter the human genome also need to be considered. While greater tissue specificity and safety can likely be accomplished by the use of tissue-specific promoters, or regulated transgene expression, safer gene transfer technologies will need to be developed to alleviate these concerns. Conflict of interest statement None declared. Funding sources None. References 1. 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;112:3543–53. 2. Billingham RE, Brent L, Medawar PB. Actively acquired tolerance of foreign cells. Nature 1953;172:603–6. 3. Takahama Y. Journey through the thymus: stromal guides for T-cell development and selection. Nat Rev Immunol 2006;6:127–35. 4. 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;34:359–68. 5. Hayashi S, Peranteau WH, Shaaban AF, Flake AW. Complete allogeneic hematopoietic chimerism achieved by a combined strategy of in utero hematopoietic stem cell transplantation and postnatal donor lymphocyte infusion. Blood 2002;100:804–12. 6. Christensen JL, Wright DE, Wagers AJ, Weissman IL. Circulation and chemotaxis of fetal hematopoietic stem cells. PLoS Biol 2004;2:E75. 7. Harrison DE, Zhong RK, Jordan CT, Lemischka IR, Astle CM. Relative to adult marrow, fetal liver repopulates nearly five times more effectively long-term than short-term. Exp Hematol 1997;25:293–7. 8. Shaaban AF, Kim HB, Milner R, Flake AW. A kinetic model for the homing and migration of prenatally transplanted marrow. Blood 1999;94:3251–7. 9. Peranteau WH, Endo M, Adibe OO, Merchant A, Zoltick PW, Flake AW. CD26 inhibition enhances allogeneic donor-cell homing and engraftment after in utero hematopoietic-cell transplantation. Blood 2006;108:4268–74. 10. Anderson D, Billingham RE, Lampkin G, et al. The use of skin grafting to distinguish between monozygotic and dizygotic twins in cattle. Heredity 1951;5:379. 11. 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 USA 1979;76:5736–40.

J.L. Roybal et al. / Seminars in Fetal & Neonatal Medicine 15 (2010) 46–51 12. Waldschmidt TJ, Panoskaltsis-Mortari A, McElmurry RT, Tygrett LT, Taylor PA, Blazar BR. Abnormal T cell-dependent B-cell responses in SCID mice receiving allogeneic bone marrow in utero. Severe combined immune deficiency. Blood 2002;100:4557–64. 13. Blazar BR, Taylor PA, Vallera DA. Adult bone marrow-derived pluripotent hematopoietic stem cells are engraftable when transferred in utero into moderately anemic fetal recipients. Blood 1995;85:833–41. 14. Carrier E, Gilpin E, Lee TH, Busch MP, Zanetti M. Microchimerism does not induce tolerance after in utero transplantation and may lead to the development of alloreactivity. J Lab Clin Med 2000;136:224–35. 15. 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;77:1–5. 16. Peranteau WH, Hayashi S, Hsieh M, Shaaban AF, Flake AW. High-level allogeneic chimerism achieved by prenatal tolerance induction and postnatal nonmyeloablative bone marrow transplantation. Blood 2002;100:2225–34. 17. Peranteau WH, Endo M, Adibe OO, Flake AW. Evidence for an immune barrier after in utero hematopoietic-cell transplantation. Blood 2007;109:1331–3. 18. Merianos D, Heaton T, Flake AW. In utero hematopoietic stem cell transplantation: progress toward clinical application. Biol Blood Marrow Transplant 2008;14:729–40. 19. Durkin ET, Jones KA, Rajesh D, Shaaban AF. Early chimerism threshold predicts sustained engraftment and NK-cell tolerance in prenatal allogeneic chimeras. Blood 2008;112:5245–53. 20. Lee PW, Cina RA, Randolph MA, et al. Stable multilineage chimerism across full MHC barriers without graft-versus-host disease following in utero bone marrow transplantation in pigs. Exp Hematol 2005;33:371–9. 21. Blakemore K, Hattenburg C, Stetten G, et al. In utero hematopoietic stem cell transplantation with haploidentical donor adult bone marrow in a canine model. Am J Obstet Gynecol 2004;190:960–73. 22. Storb R, Deeg HJ, Raff R, et al. Prevention of graft-versus-host disease. Studies in a canine model. Ann NY Acad Sci 1995;770:149–64. 23. 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;15:293–305. 24. 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;335:1806–10. 25. Touraine JL, Raudrant D, Laplace S. Transplantation of hemopoietic cells from the fetal liver to treat patients with congenital diseases postnatally or prenatally. Transplant Proc 1997;29:712–3. 26. Westgren M, Ringden O, Bartmann P, et al. Prenatal T-cell reconstitution after in utero transplantation with fetal liver cells in a patient with X-linked severe combined immunodeficiency. Am J Obstet Gynecol 2002;187:475–82. 27. Yoshizawa J, Li XK, Fujino M, et al. Successful in utero gene transfer using a gene gun in midgestational mouse fetuses. J Pediatr Surg 2004;39:81–4. 28. David A, Cook T, Waddington S, et al. Ultrasound-guided percutaneous delivery of adenoviral vectors encoding the beta-galactosidase and human factor IX genes to early gestation fetal sheep in utero. Hum Gene Ther 2003;14:353–64. 29. Cronin J, Zhang XY, Reiser J. Altering the tropism of lentiviral vectors through pseudotyping. Curr Gene Ther 2005;5:387–98. 30. Endo M, Zoltick PW, Peranteau WH, et al. Efficient in vivo targeting of epidermal stem cells by early gestational intraamniotic injection of lentiviral vector driven by the keratin 5 promoter. Mol Ther 2008;16:131–7.

51

31. Henriques-Coelho T, Gonzaga S, Endo M, et al. Targeted gene transfer to fetal rat lung interstitium by ultrasound-guided intrapulmonary injection. Mol Ther 2007;15:340–7. 32. Karolewski BA, Wolfe JH. Genetic correction of the fetal brain increases the lifespan of mice with the severe multisystemic disease mucopolysaccharidosis type VII. Mol Ther 2006;14:14–24. 33. Seppen J, van der Rijt R, Looije N, van Til NP, Lamers WH, Oude Elferink RP. Long-term correction of bilirubin UDPglucuronyltransferase deficiency in rats by in utero lentiviral gene transfer. Mol Ther 2003;8:593–9. 34. Larson JE, Morrow SL, Happel L, Sharp JF, Cohen JC. Reversal of cystic fibrosis phenotype in mice by gene therapy in utero. Lancet 1997;349:619–20. 35. Rucker M, Fraites Jr TJ, Porvasnik SL, et al. Rescue of enzyme deficiency in embryonic diaphragm in a mouse model of metabolic myopathy: Pompe disease. Development 2004;131:3007–19. 36. Dejneka NS, Surace EM, Aleman TS, et al. In utero gene therapy rescues vision in a murine model of congenital blindness. Mol Ther 2004;9:182–8. 37. Reay DP, Bilbao R, Koppanati BM, et al. Full-length dystrophin gene transfer to the mdx mouse in utero. Gene Ther 2008;15:531–6. 38. Porada CD, Park P, Almeida-Porada G, Zanjani ED. The sheep model of in utero gene therapy. Fetal Diagn Ther 2004;19:23–30. 39. Peebles D, Gregory LG, David A, et al. Widespread and efficient marker gene expression in the airway epithelia of fetal sheep after minimally invasive tracheal application of recombinant adenovirus in utero. Gene Ther 2004;11:70–8. 40. Yu ZY, McKay K, van Asperen P, et al. Lentivirus vector-mediated gene transfer to the developing bronchiolar airway epithelium in the fetal lamb. J Gene Med 2007;9:429–39. 41. Zaiss AK, Muruve DA. Immunity to adeno-associated virus vectors in animals and humans: a continued challenge. Gene Ther 2008;15:808–16. 42. Schneider H, Muhle C, Douar AM, et al. Sustained delivery of therapeutic concentrations of human clotting factor IX – a comparison of adenoviral and AAV vectors administered in utero. J Gene Med 2002;4:46–53. 43. Sabatino DE, Mackenzie TC, Peranteau W, et al. Persistent expression of hF.IX After tolerance induction by in utero or neonatal administration of AAV-1-F.IX in hemophilia B mice. Mol Ther 2007;15:1677–85. 44. Waddington SN, Nivsarkar MS, Mistry AR, et al. Permanent phenotypic correction of hemophilia B in immunocompetent mice by prenatal gene therapy. Blood 2004;104:2714–21. 45. Hacein-Bey-Abina S, Garrigue A, Wang GP, et al. Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J Clin Invest 2008;118:3132–42. 46. Themis M, Waddington SN, Schmidt M, et al. Oncogenesis following delivery of a nonprimate lentiviral gene therapy vector to fetal and neonatal mice. Mol Ther 2005;12:763–71. 47. Tarantal AF, McDonald RJ, Jimenez DF, et al. Intrapulmonary and intramyocardial gene transfer in rhesus monkeys (Macaca mulatta): safety and efficiency of HIV-1-derived lentiviral vectors for fetal gene delivery. Mol Ther 2005;12:87–98. 48. Gonzaga S, Henriques-Coelho T, Davey M, et al. Cystic adenomatoid malformations are induced by localized FGF10 overexpression in fetal rat lung. Am J Respir Cell Mol Biol 2008;39:346–55. 49. David AL, Peebles D. Gene therapy for the fetus: is there a future? Best Pract Res Clin Obstet Gynaecol 2008;22:203–18. 50. Porada CD, Park PJ, Tellez J, et al. Male germ-line cells are at risk following direct-injection retroviral-mediated gene transfer in utero. Mol Ther 2005;12:754–62.