- Email: [email protected]
Future AAVenues for In Utero Gene Therapy
Cell Stem Cell
In Translation Future AAVenues for In Utero Gene Therapy Tippi C. MacKenzie1,* 1The Department of Surgery and the Center for Maternal-Fetal Precision Medicine, University of California, San Francisco, San Francisco, CA, USA *Correspondence: [email protected] https://doi.org/10.1016/j.stem.2018.08.010
Fetal gene therapy using safe and effective viral vectors no longer remains a distant prospect. Recently in Nature Medicine, Massaro et al. (2018) demonstrated that prenatal intracranial injection of a viral vector results in improved neurologic function, raising the intriguing possibility that in utero gene therapy may be approaching clinical applications. The idea of surgically correcting severe fetal malformations within the womb was born several decades ago, and surgical techniques continue to be refined. While in utero stem cell transplantation has been performed since the late 1980s, its success has been limited to a subset of genetic diseases. Since then, scientists have honed the strategies of in utero stem cell transplantation and in utero gene therapy (IUGT) in various animal models to broaden their applicability. The rationale for in utero therapy is compelling: first, the immune system of the fetus is more primed to result in tolerance to foreign antigens; thus, in utero therapy can tolerize a patient to allogeneic cells or crucial cellular proteins. Second, in utero therapy can circumvent organ-specific disease manifestations, particularly in progressive diseases such as lysosomal storage disorders. Finally, in utero therapy can more easily cross the immature blood-brain barrier, enhancing its ability to ameliorate neurologic manifestations. In a recent paper, Massaro et al. have provided additional evidence for the safety and efficacy of IUGT in a mouse model of neuronopathic Gaucher’s disease (Massaro et al., 2018). The team used an adeno-associated virus serotype 9 (AAV9) vector, which previously facilitated neuronal expression of a reporter gene, to treat transgenic mice with a fatal neurodegenerative disease that usually results in death by 2 weeks. Fetal intracranial injection of the vector improved neuronal inflammation and remarkably, overall survival of the mice. Neonatal mice that were treated with intracranial injection also had improved survival, but the brain benefits were enhanced with fetal therapy. Interestingly,
neonatal mice treated with intravenous injection also had improved neurologic manifestations, suggesting that the vector can cross the blood-brain barrier at this age. Finally, injection of an AAV9-GFP vector in two fetal macaques in midgestation resulted in widespread distribution into both brain hemispheres at birth. The authors did not specifically test whether intravenous injection into the fetus also achieved correction in the brain, but since the neonatal intravenous injection was successful, an in utero systemic injection might also mitigate both the neurologic and visceral manifestations of the disease. This manuscript is an exciting addition to the current resurgence of interest in various fetal molecular therapies. Intraamniotic injection of recombinant ectodysplasin A was recently shown to be successful in treating two patients with X-linked hypohidrotic ectodermal dysplasia, in which the missing protein is necessary only during a short period in gestation (Schneider et al., 2018). One could extrapolate that providing other missing proteins (such as intracellular enzymes lacking in lysosomal storage diseases) may have a similar salutary effect, although these would have to be administered repeatedly. There is also a rebirth of interest in in utero stem cell transplantation to treat hematopoietic disorders; transplantation of maternalderived hematopoietic stem cells is currently FDA approved for a phase 1 clinical trial for fetal alpha thalassemia major (clinicaltrials.gov number NCT02986698). IUGT, however, has not yet been attempted in human patients. In 1999, the NIH Recombinant DNA Advisory Committee (RAC) issued a position paper on prenatal gene transfer, outlining requirements for
320 Cell Stem Cell 23, September 6, 2018 ª 2018 Elsevier Inc.
preclinical work in relevant animal models (U.S. National Institutes of Health Recombinant DNA Advisory Committee, 2000). Since then, there has been a large body of research in mouse, sheep, and nonhuman primate models focused on the safety and efficacy of IUGT (AlmeidaPorada et al., 2016). A critical benefit of in utero exposure to a missing protein is tolerance, and numerous reports confirm induction of tolerance to exogenous proteins, such as clotting factors, after IUGT (Sabatino et al., 2007; Waddington et al., 2004). Thus, it may be time to revisit some of the requirements raised by the RAC nearly 20 years ago. There are several prerequisites for envisioning a clinical scenario amenable for IUGT. Prenatal diagnosis of the disease should be accurate, with a good understanding of genotype/phenotype correlations that affect clinical prognosis. While some patients will be screened because of a known family history, others may be diagnosed because of abnormalities on a screening ultrasound (for example, hydrops in a patient with a lysosomal storage disease). With the growing use and diminishing costs of prenatal sequencing, this prerequisite is rapidly becoming easier to achieve. Similarly, the technical aspects of fetal gene therapy should be feasible, particularly for the most relevant technique of systemic injections (fetal umbilical vein access for in utero transfusions). Given the extensive experience with clinical fetal surgery, even more sophisticated interventions for fetal intracardiac or intracerebral delivery should not be limiting in experienced hands. The most important criteria for considering IUGT should be an appropriate developmental rationale for not waiting until the child is born, such as concerns
Cell Stem Cell
In Translation for immune reactions to the transgene encoded protein, or concerns for progressive neurological or cardiac disease. Recent successes in postnatal gene therapy trials highlight some potential diseases that could benefit from earlier correction. For example, intracerebral injection of an AAV 2/5 vector encoding the a-N-acetylglucosaminidase (NAGLU) gene has shown encouraging results in children with Sanfilippo B syndrome (Tardieu et al., 2017). Gene replacement therapy using an AAV9 vector carrying the SMN gene in children with spinal muscular atrophy type 1 showed the most improvement in the youngest patients (Mendell et al., 2017). Finally, the safety and efficacy profiles of several AAV vectors for Hemophilia A (Rangarajan et al., 2017) and Hemophilia B (George et al., 2017) are quite encouraging for future in utero applications of this strategy. Maternal safety is a critical consideration in any fetal therapy and IUGT is no exception, particularly with respect to possible exposure to the viral vectors infused into the fetus. Exposure to a viral vector may result in maternal immune responses to the capsid protein (it is likely that early trials would exclude mothers with pre-existing antibodies, which could cross the placenta) or to the recombinant protein (although unlikely, since the mother should already be producing— and therefore tolerant to—the protein missing in the fetus). For the fetus, integration of the viral vector, even AAV vec-
tors, has been reported in animal models in IUGT, although germline integration has not been found. Some of these limitations may be solved in the future by emerging technologies that allow more specific cell targeting and gene editing to minimize the risk of off-target events. Finally, an important ethical implication is partial treatment of a fatal disease: in this situation, fetal therapy may result in survival of a baby with a severely morbid and painful postnatal course. Fetal therapy hinges on the concept of non-directive counseling, in which the options of no therapy and experimental therapy— with all possible risks and benefits—are explained without the physician’s personal bias. As these and other issues continue to be addressed, rigorous preclinical studies and multidisciplinary discussions will continue to push the frontiers of fetal therapy. REFERENCES Almeida-Porada, G., Atala, A., and Porada, C.D. (2016). In utero stem cell transplantation and gene therapy: rationale, history, and recent advances toward clinical application. Mol. Ther. Methods Clin. Dev. 5, 16020. George, L.A., Sullivan, S.K., Giermasz, A., Rasko, J.E.J., Samelson-Jones, B.J., Ducore, J., Cuker, A., Sullivan, L.M., Majumdar, S., Teitel, J., et al. (2017). Hemophilia B Gene Therapy with a HighSpecific-Activity Factor IX Variant. N. Engl. J. Med. 377, 2215–2227. Massaro, G., Mattar, C.N.Z., Wong, A.M.S., Sirka, E., Buckley, S.M.K., Herbert, B.R., Karlsson, S., Perocheau, D.P., Burke, D., Heales, S., et al. (2018). Fetal gene therapy for neurodegenerative disease of infants. Nat. Med., in press. Published
online July 16, 2018. https://doi.org/10.1038/ s41591-018-0106-7. Mendell, J.R., Al-Zaidy, S., Shell, R., Arnold, W.D., Rodino-Klapac, L.R., Prior, T.W., Lowes, L., Alfano, L., Berry, K., Church, K., et al. (2017). Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy. N. Engl. J. Med. 377, 1713–1722. Rangarajan, S., Walsh, L., Lester, W., Perry, D., Madan, B., Laffan, M., Yu, H., Vettermann, C., Pierce, G.F., Wong, W.Y., and Pasi, K.J. (2017). AAV5-Factor VIII Gene Transfer in Severe Hemophilia A. N. Engl. J. Med. 377, 2519–2530. Sabatino, D.E., Mackenzie, T.C., Peranteau, W., Edmonson, S., Campagnoli, C., Liu, Y.L., Flake, A.W., and High, K.A. (2007). 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. 15, 1677–1685. Schneider, H., Faschingbauer, F., SchuepbachMallepell, S., Ko¨rber, I., Wohlfart, S., Dick, A., Wahlbuhl, M., Kowalczyk-Quintas, C., Vigolo, M., Kirby, N., et al. (2018). Prenatal Correction of X-Linked Hypohidrotic Ectodermal Dysplasia. N. Engl. J. Med. 378, 1604–1610. Tardieu, M., Ze´rah, M., Gougeon, M.L., Ausseil, J., de Bournonville, S., Husson, B., Zafeiriou, D., Parenti, G., Bourget, P., Poirier, B., et al. (2017). Intracerebral gene therapy in children with mucopolysaccharidosis type IIIB syndrome: an uncontrolled phase 1/2 clinical trial. Lancet Neurol. 16, 712–720. U.S. National Institutes of Health Recombinant DNA Advisory Committee (2000). Prenatal gene transfer: scientific, medical, and ethical issues: a report of the Recombinant DNA Advisory Committee. Hum. Gene Ther. 11, 1211–1229. Waddington, S.N., Nivsarkar, M.S., Mistry, A.R., Buckley, S.M., Kemball-Cook, G., Mosley, K.L., Mitrophanous, K., Radcliffe, P., Holder, M.V., Brittan, M., et al. (2004). Permanent phenotypic correction of hemophilia B in immunocompetent mice by prenatal gene therapy. Blood 104, 2714– 2721.
Cell Stem Cell 23, September 6, 2018 321
In Translation Future AAVenues for In Utero Gene Therapy Tippi C. MacKenzie1,* 1The Department of Surgery and the Center for Maternal-Fetal Precision Medicine, University of California, San Francisco, San Francisco, CA, USA *Correspondence: [email protected] https://doi.org/10.1016/j.stem.2018.08.010
Fetal gene therapy using safe and effective viral vectors no longer remains a distant prospect. Recently in Nature Medicine, Massaro et al. (2018) demonstrated that prenatal intracranial injection of a viral vector results in improved neurologic function, raising the intriguing possibility that in utero gene therapy may be approaching clinical applications. The idea of surgically correcting severe fetal malformations within the womb was born several decades ago, and surgical techniques continue to be refined. While in utero stem cell transplantation has been performed since the late 1980s, its success has been limited to a subset of genetic diseases. Since then, scientists have honed the strategies of in utero stem cell transplantation and in utero gene therapy (IUGT) in various animal models to broaden their applicability. The rationale for in utero therapy is compelling: first, the immune system of the fetus is more primed to result in tolerance to foreign antigens; thus, in utero therapy can tolerize a patient to allogeneic cells or crucial cellular proteins. Second, in utero therapy can circumvent organ-specific disease manifestations, particularly in progressive diseases such as lysosomal storage disorders. Finally, in utero therapy can more easily cross the immature blood-brain barrier, enhancing its ability to ameliorate neurologic manifestations. In a recent paper, Massaro et al. have provided additional evidence for the safety and efficacy of IUGT in a mouse model of neuronopathic Gaucher’s disease (Massaro et al., 2018). The team used an adeno-associated virus serotype 9 (AAV9) vector, which previously facilitated neuronal expression of a reporter gene, to treat transgenic mice with a fatal neurodegenerative disease that usually results in death by 2 weeks. Fetal intracranial injection of the vector improved neuronal inflammation and remarkably, overall survival of the mice. Neonatal mice that were treated with intracranial injection also had improved survival, but the brain benefits were enhanced with fetal therapy. Interestingly,
neonatal mice treated with intravenous injection also had improved neurologic manifestations, suggesting that the vector can cross the blood-brain barrier at this age. Finally, injection of an AAV9-GFP vector in two fetal macaques in midgestation resulted in widespread distribution into both brain hemispheres at birth. The authors did not specifically test whether intravenous injection into the fetus also achieved correction in the brain, but since the neonatal intravenous injection was successful, an in utero systemic injection might also mitigate both the neurologic and visceral manifestations of the disease. This manuscript is an exciting addition to the current resurgence of interest in various fetal molecular therapies. Intraamniotic injection of recombinant ectodysplasin A was recently shown to be successful in treating two patients with X-linked hypohidrotic ectodermal dysplasia, in which the missing protein is necessary only during a short period in gestation (Schneider et al., 2018). One could extrapolate that providing other missing proteins (such as intracellular enzymes lacking in lysosomal storage diseases) may have a similar salutary effect, although these would have to be administered repeatedly. There is also a rebirth of interest in in utero stem cell transplantation to treat hematopoietic disorders; transplantation of maternalderived hematopoietic stem cells is currently FDA approved for a phase 1 clinical trial for fetal alpha thalassemia major (clinicaltrials.gov number NCT02986698). IUGT, however, has not yet been attempted in human patients. In 1999, the NIH Recombinant DNA Advisory Committee (RAC) issued a position paper on prenatal gene transfer, outlining requirements for
320 Cell Stem Cell 23, September 6, 2018 ª 2018 Elsevier Inc.
preclinical work in relevant animal models (U.S. National Institutes of Health Recombinant DNA Advisory Committee, 2000). Since then, there has been a large body of research in mouse, sheep, and nonhuman primate models focused on the safety and efficacy of IUGT (AlmeidaPorada et al., 2016). A critical benefit of in utero exposure to a missing protein is tolerance, and numerous reports confirm induction of tolerance to exogenous proteins, such as clotting factors, after IUGT (Sabatino et al., 2007; Waddington et al., 2004). Thus, it may be time to revisit some of the requirements raised by the RAC nearly 20 years ago. There are several prerequisites for envisioning a clinical scenario amenable for IUGT. Prenatal diagnosis of the disease should be accurate, with a good understanding of genotype/phenotype correlations that affect clinical prognosis. While some patients will be screened because of a known family history, others may be diagnosed because of abnormalities on a screening ultrasound (for example, hydrops in a patient with a lysosomal storage disease). With the growing use and diminishing costs of prenatal sequencing, this prerequisite is rapidly becoming easier to achieve. Similarly, the technical aspects of fetal gene therapy should be feasible, particularly for the most relevant technique of systemic injections (fetal umbilical vein access for in utero transfusions). Given the extensive experience with clinical fetal surgery, even more sophisticated interventions for fetal intracardiac or intracerebral delivery should not be limiting in experienced hands. The most important criteria for considering IUGT should be an appropriate developmental rationale for not waiting until the child is born, such as concerns
Cell Stem Cell
In Translation for immune reactions to the transgene encoded protein, or concerns for progressive neurological or cardiac disease. Recent successes in postnatal gene therapy trials highlight some potential diseases that could benefit from earlier correction. For example, intracerebral injection of an AAV 2/5 vector encoding the a-N-acetylglucosaminidase (NAGLU) gene has shown encouraging results in children with Sanfilippo B syndrome (Tardieu et al., 2017). Gene replacement therapy using an AAV9 vector carrying the SMN gene in children with spinal muscular atrophy type 1 showed the most improvement in the youngest patients (Mendell et al., 2017). Finally, the safety and efficacy profiles of several AAV vectors for Hemophilia A (Rangarajan et al., 2017) and Hemophilia B (George et al., 2017) are quite encouraging for future in utero applications of this strategy. Maternal safety is a critical consideration in any fetal therapy and IUGT is no exception, particularly with respect to possible exposure to the viral vectors infused into the fetus. Exposure to a viral vector may result in maternal immune responses to the capsid protein (it is likely that early trials would exclude mothers with pre-existing antibodies, which could cross the placenta) or to the recombinant protein (although unlikely, since the mother should already be producing— and therefore tolerant to—the protein missing in the fetus). For the fetus, integration of the viral vector, even AAV vec-
tors, has been reported in animal models in IUGT, although germline integration has not been found. Some of these limitations may be solved in the future by emerging technologies that allow more specific cell targeting and gene editing to minimize the risk of off-target events. Finally, an important ethical implication is partial treatment of a fatal disease: in this situation, fetal therapy may result in survival of a baby with a severely morbid and painful postnatal course. Fetal therapy hinges on the concept of non-directive counseling, in which the options of no therapy and experimental therapy— with all possible risks and benefits—are explained without the physician’s personal bias. As these and other issues continue to be addressed, rigorous preclinical studies and multidisciplinary discussions will continue to push the frontiers of fetal therapy. REFERENCES Almeida-Porada, G., Atala, A., and Porada, C.D. (2016). In utero stem cell transplantation and gene therapy: rationale, history, and recent advances toward clinical application. Mol. Ther. Methods Clin. Dev. 5, 16020. George, L.A., Sullivan, S.K., Giermasz, A., Rasko, J.E.J., Samelson-Jones, B.J., Ducore, J., Cuker, A., Sullivan, L.M., Majumdar, S., Teitel, J., et al. (2017). Hemophilia B Gene Therapy with a HighSpecific-Activity Factor IX Variant. N. Engl. J. Med. 377, 2215–2227. Massaro, G., Mattar, C.N.Z., Wong, A.M.S., Sirka, E., Buckley, S.M.K., Herbert, B.R., Karlsson, S., Perocheau, D.P., Burke, D., Heales, S., et al. (2018). Fetal gene therapy for neurodegenerative disease of infants. Nat. Med., in press. Published
online July 16, 2018. https://doi.org/10.1038/ s41591-018-0106-7. Mendell, J.R., Al-Zaidy, S., Shell, R., Arnold, W.D., Rodino-Klapac, L.R., Prior, T.W., Lowes, L., Alfano, L., Berry, K., Church, K., et al. (2017). Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy. N. Engl. J. Med. 377, 1713–1722. Rangarajan, S., Walsh, L., Lester, W., Perry, D., Madan, B., Laffan, M., Yu, H., Vettermann, C., Pierce, G.F., Wong, W.Y., and Pasi, K.J. (2017). AAV5-Factor VIII Gene Transfer in Severe Hemophilia A. N. Engl. J. Med. 377, 2519–2530. Sabatino, D.E., Mackenzie, T.C., Peranteau, W., Edmonson, S., Campagnoli, C., Liu, Y.L., Flake, A.W., and High, K.A. (2007). 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. 15, 1677–1685. Schneider, H., Faschingbauer, F., SchuepbachMallepell, S., Ko¨rber, I., Wohlfart, S., Dick, A., Wahlbuhl, M., Kowalczyk-Quintas, C., Vigolo, M., Kirby, N., et al. (2018). Prenatal Correction of X-Linked Hypohidrotic Ectodermal Dysplasia. N. Engl. J. Med. 378, 1604–1610. Tardieu, M., Ze´rah, M., Gougeon, M.L., Ausseil, J., de Bournonville, S., Husson, B., Zafeiriou, D., Parenti, G., Bourget, P., Poirier, B., et al. (2017). Intracerebral gene therapy in children with mucopolysaccharidosis type IIIB syndrome: an uncontrolled phase 1/2 clinical trial. Lancet Neurol. 16, 712–720. U.S. National Institutes of Health Recombinant DNA Advisory Committee (2000). Prenatal gene transfer: scientific, medical, and ethical issues: a report of the Recombinant DNA Advisory Committee. Hum. Gene Ther. 11, 1211–1229. Waddington, S.N., Nivsarkar, M.S., Mistry, A.R., Buckley, S.M., Kemball-Cook, G., Mosley, K.L., Mitrophanous, K., Radcliffe, P., Holder, M.V., Brittan, M., et al. (2004). Permanent phenotypic correction of hemophilia B in immunocompetent mice by prenatal gene therapy. Blood 104, 2714– 2721.
Cell Stem Cell 23, September 6, 2018 321