Search

The potential pathogenetic mechanisms underlying the varied morphology of congenital pulmonary airway malformations (CPAMs) have not been molecularly determined, but a subset have been shown to contain clusters of mucinous cells (MCC). These clusters are believed to serve as precursors for potential invasive mucinous adenocarcinoma, and they are associated with KRAS codon 12 mutations. To assess the universality of KRAS mutations in MCCs, we sequenced exon 2 of KRAS in 61 MCCs from 18 patients, and we found a KRAS codon 12 mutation in all 61 MCCs. Furthermore, all MCCs from a single patient always had the same KRAS mutation, and the same KRAS mutation was also found in non-mucinous lesional tissue. Next generation sequencing of seven MCCs showed no other mutations or copy number variations. Sequencing of 46 additional CPAMs with MCCs revealed KRAS mutations in non-mucinous lesional tissue in all cases. RNA in situ hybridization confirmed widespread distribution of cells with mutant KRAS RNA, even extending outside of the bronchiolar type epithelium. We identified 25 additional CPAMs with overall histologic architecture similar to CPAMs with KRAS mutations but without identifiable MCCs, and we found KRAS mutations in 17 (68%). The histologic features of these KRAS mutated CPAMs included type 1 and type 3 morphology, as well as lesions with an intermediate histologic appearance, and analysis revealed a strong correlation between the specific amino acid substitution and histomorphology. These findings, together with previously published model organism data, suggests that the formation of type 1 and 3 CPAMs is driven by mosaic KRAS mutations arising in the lung epithelium early in development and places them within the growing field of mosaic RASopathies. The presence of widespread epithelial mutation explains late metastatic disease in incompletely resected patients and reinforces the recommendation for complete resection of these lesions.

Protein translational control is highly regulated step in the gene expression program during mammalian development that is critical for ensuring that the fetus develops correctly and that all of the necessary organs and tissues are formed and functional. Defects in protein expression during fetal development can lead to severe developmental abnormalities or premature death. Currently, quantitative techniques to monitor protein synthesis rates in a developing fetus (in utero) are limited. Here, we developed a novelin uterostable isotope labeling approach to quantify tissue-specific protein dynamics of the nascent proteome during mouse fetal development. Fetuses of pregnant C57BL/6J mice were injected with isotopically labeled lysine (Lys8) and arginine (Arg10) via the vitelline vein at various gestational days. After treatment, fetal organs/tissues including brain, liver, lung, and heart were harvested for sample preparation and proteomic analysis. We show that the mean incorporation rate for injected amino acids into all organs was 17.50 ± 0.6%. By analyzing the nascent proteome, unique signatures of each tissue were identified by hierarchical clustering. In addition, the quantified proteome-wide turnover rates (kobs) were calculated between 3.81E-5 and 0.424 hour-1. We observed similar protein turnover profiles for analyzed organs (e.g., liver versus brain), however, their distributions of turnover rates vary significantly. The translational kinetic profiles of developing organs displayed differentially expressed protein pathways and synthesis rates which correlated with known physiological changes during mouse development.

Congenital disorders resulting in pathological protein deficiencies are most often treated postnatally with protein or enzyme replacement therapies. However, treatment of these disorders in utero before irreversible disease onset could significantly minimize disease burden, morbidity, and mortality. One possible strategy for the prenatal treatment of congenital disorders is in utero delivery of messenger RNA (mRNA). mRNA is a nucleic acid therapeutic that has previously been investigated as a platform for protein replacement therapies and gene editing technologies. While viral vectors have been explored to induce intracellular expression of mRNA, they are limited in their clinical application due to risks associated with immunogenicity and genomic integration. As an alternative to viral vectors, safe and efficient in utero mRNA delivery can be achieved using ionizable lipid nanoparticles (LNPs). While LNPs have demonstrated potent in vivo mRNA delivery to the liver following intravenous administration, intra-amniotic delivery has the potential to deliver mRNA to cells and tissues beyond those in the liver, such as in the skin, lung, and digestive tract. However, LNP stability in fetal amniotic fluid and how this stability affects mRNA delivery has not been previously investigated. Here, we engineered a library of LNPs using orthogonal design of experiments (DOE) to evaluate how LNP structure affects their stability in amniotic fluid ex utero and whether a lead candidate identified from these stability measurements enables intra-amniotic mRNA delivery in utero. We used a combination of techniques including dynamic light scattering (DLS), transmission electron microscopy (TEM), and chromatography followed by protein content quantification to screen LNP stability in amniotic fluids. These results identified multiple lead LNP formulations that are highly stable in amniotic fluids ranging from small animals to humans, including mouse, sheep, pig, and human amniotic fluid samples. We then demonstrate that stable LNPs from the ex utero screen in mouse amniotic fluid enabled potent mRNA delivery in primary fetal lung fibroblasts and in utero following intra-amniotic injection in a murine model. This exploration of ex utero stability in amniotic fluids demonstrates a means by which to identify novel LNP formulations for prenatal treatment of congenital disorders via in utero mRNA delivery.

Gene therapy has traditionally involved the delivery of exogenous genetic material to a cell-most commonly to replace defective genes causing monogenic disorders. This allows cells to produce proteins that are otherwise absent in sufficient quantities, ideally for a therapeutic purpose. Since its inception over 40 years ago, the field of gene therapy has significantly expanded and now includes targeted gene editing strategies, including, but not limited to, clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs).

In utero hematopoietic cell transplantation (IUHCT) is an experimental treatment for congenital hemoglobinopathies, including Sickle cell disease and thalassemias. One of the principal advantages of IUHCT is the predisposition of the developing fetus toward immunologic tolerance. This allows for engraftment across immune barriers without immunosuppression and, potentially, decreased susceptibility to graft-versus-host disease (GVHD). We demonstrate fetal resistance to GVHD following T cell-replete allogeneic hematopoietic cell transplantation compared with the neonate. We show that this resistance is associated with elevated fetal serum interleukin-10 conducive to regulatory T cells (Tregs) induction. Finally, we demonstrate that the adoptive transfer of Tregs from IUHCT recipients to neonates uniformly prevents GVHD, recapitulating the predisposition to tolerance observed after fetal allotransplantation. These findings demonstrate fetal resistance to GVHD following hematopoietic cell transplantation and elucidate Tregs as important contributors.

In utero hematopoietic cell transplantation (IUHCT) has the potential to cure congenital hematologic disorders including sickle cell disease. However, the window of opportunity for IUHCT closes with the acquisition of T-cell immunity, beginning at approximately 14 weeks gestation, posing significant technical challenges and excluding from treatment fetuses evaluated after the first trimester. Here we report that regulatory T cells can promote alloengraftment and preserve allograft tolerance after the acquisition of T-cell immunity in a mouse model of late-gestation IUHCT. We show that allografts enriched with regulatory T cells harvested from either IUHCT-tolerant or naive mice engraft at 20 days post coitum (DPC) with equal frequency to unenriched allografts transplanted at 14 DPC. Long-term, multilineage donor cell chimerism was achieved in the absence of graft-versus-host disease or mortality. Decreased alloreactivity among recipient T cells was observed consistent with donor-specific tolerance. These findings suggest that donor graft enrichment with regulatory T cells could be used to successfully perform IUHCT later in gestation.

Background Congenital heart disease (CHD) is associated with abnormal fetal brain development, a phenomenon that may be related to decreased cerebral oxygen delivery in utero. We used an artificial womb model to test the hypothesis that decreasing fetal oxygen delivery would reproduce physiologic changes identified in fetuses with CHD. Methods Experimental (hypoxemic) fetal lambs (mean gestational age, 111 ± 3 days; n = 4) and control animals (112 days; n = 5) were maintained in the artificial womb for a mean of 22 ± 6 days. Oxygen delivery was reduced to 15.6 ± 1.0 mL/kg/min in the hypoxemia animals versus 21.6 ± 2.0 mL/kg/min in the control animals. Blood chemistry analysis and sonographic evaluation were performed daily. An additional control group (n = 7) was maintained in utero and harvested for analysis at gestational age 134 ± 4 days. Results Physiologic variables were monitored continuously, and no statistical differences between the groups were identified. Fetal oxygen delivery and arterial partial pressure of oxygen were remarkably lower in the experimental group longitudinally. Increased umbilical artery and decreased middle cerebral artery resistance resulted in a lower cerebral to umbilical resistance ratio, similar to the brain sparing effect observed in human fetuses with CHD. Experimental brains were smaller than control brains in relation to the calvarium on magnetic resonance. Conclusions Sustained hypoxemia in fetal sheep leads to altered cerebrovascular resistances and loss of brain mass, similar to human fetuses with CHD. This unique model provides opportunities to investigate the pathologic process underlying CHD-associated brain dysmaturity and to evaluate potential fetal neuroprotective therapies.

Significant advances in the safety and efficacy of gene therapy have sparked a new frontier in therapeutics for genetic diseases as evidenced by the greater than 700 active gene therapy investigational new drug applications reported by the NIH and the US Food and Drug Association. Although postnatal gene therapy trials are encouraging, limitations to effective therapy including an immune barrier and initiation of treatment after disease onset can exist. Advances in prenatal diagnostics provide hope that many genetic abnormalities will be able to be diagnosed before birth. Prenatal gene therapy has the potential to take advantage of normal developmental properties of the fetus and overcome some of the current limitations to efficient postnatal gene therapy. The rationale for prenatal gene therapy includes the small fetal size, the tolerogenic fetal immune system, the presence of highly proliferative and accessible stem/progenitor cells of multiple organs, and, ultimately, the ability to treat diseases in which irreversible pathology begins prior to birth. This rationale is based on and supported by a number of published animal studies. Unique ethical considerations exist in the context of prenatal gene therapy, including the importance of rigorous evaluation of the effect of the therapy on fetal germ cells and developing organs as well as the mother. To date, animal studies have not demonstrated any significant germline or maternal effect of prenatal gene therapy. Finally, practical considerations of future clinical prenatal gene therapy will include, but not be limited to, determining the initial target disease characteristics and the importance of non-directive prenatal counseling of families carrying a fetus with a genetic diagnosis.

Background and Aims The Extra-Uterine Environment for Neonatal Development (EXTEND) aims to avoid the complications of prematurity, such as NEC. Our goal was to determine if bowel development occurs normally in EXTEND-supported lambs, with specific emphasis on markers of immaturity associated with NEC. Methods We compared terminal ileum from 17 pre-term lambs supported on EXTEND for 2- 4 weeks to bowel from age-matched fetal lambs that developed in utero. We evaluated morphology, markers of epithelial integrity and maturation, enteric nervous system structure, and bowel motility. Results EXTEND-supported lamb ileum had normal villus height, crypt depth, density of mucin-containing goblet cells, and enteric neuron density. Expression patterns for I-FABP, activated caspase-3 and EGFR were normal in bowel epithelium. Transmural resistance assessed in Ussing chambers was normal. Bowel motility was also normal as assessed by ex vivo organ bath and video imaging. However, Peyer’s patch organization did not occur normally in EXTEND ileum, resulting in fewer circulating B cells in experimental animals. Conclusion EXTEND supports normal ileal epithelial and enteric nervous system maturation in pre-term lambs. The classic morphologic changes and cellular expression profiles associated with NEC are not seen. However, immune development within the EXTEND supported lamb bowel does not progress normally.

Neuroimmune cells, particularly microglia and astrocytes, play a critical role in neurodevelopment. Neurocognitive delays are common in children with congenital heart disease, but their etiology is poorly understood. Our objective was to determine whether prenatal hypoxemia, at levels common in congenital heart disease, induced neuroimmune activation to better understand the origins of neurobehavioral disorders in congenital heart disease.Eight fetal sheep at gestational age 109 ± 3 days (term ∼145 days) were cannulated onto a pumpless extracorporeal oxygenator via the umbilical vessels and supported in a fluid environment for 22 ± 2 days under normoxic (n = 4) or hypoxic (n = 4) conditions. Control fetuses (n = 7) were harvested at gestational age 133 ± 4 days. At necropsy, brains were stained with ionized calcium-binding adaptor molecule 1 and glial fibrillary acidic protein antibodies to quantify microglia and astrocytes, respectively, in gray and white matter in frontotemporal and cerebellar sections. Microglia were classified into 4 morphologic types based on cell shape. Data were analyzed with 1-way analysis of variance or Fisher exact test, as appropriate.Oxygen delivery was significantly reduced in hypoxic fetuses (15.6 ± 1.8 mL/kg/min vs 24.3 ± 2.3 mL/kg/min; P .01). Rates of apoptosis were similar in hypoxic, normoxic, and intrauterine control animals in all examined areas. There were also no differences between groups in area occupied by glial fibrillary acidic protein-labeled astrocytes or ionized calcium-binding adaptor molecule 1-labeled microglia in all examined areas. However, round microglia were significantly increased in hypoxic animals compared with normoxic animals (33% vs 6%; P .01) and control animals (33% vs 11%; P .01).Prenatal hypoxemia altered microglial morphology without significant gliosis. Additional studies characterizing these mechanisms may provide insight into the origins of neurobehavioral disabilities in children with congenital heart disease.

In utero base editing has the potential to correct disease-causing mutations before the onset of pathology. Mucopolysaccharidosis type I (MPS-IH, Hurler syndrome) is a lysosomal storage disease (LSD) affecting multiple organs, often leading to early postnatal cardiopulmonary demise. We assessed in utero adeno-associated virus serotype 9 (AAV9) delivery of an adenine base editor (ABE) targeting the Idua G→A (W392X) mutation in the MPS-IH mouse, corresponding to the common IDUA G→A (W402X) mutation in MPS-IH patients. Here we show efficient long-term W392X correction in hepatocytes and cardiomyocytes and low-level editing in the brain. In utero editing was associated with improved survival and amelioration of metabolic, musculoskeletal, and cardiac disease. This proof-of-concept study demonstrates the possibility of efficiently performing therapeutic base editing in multiple organs before birth via a clinically relevant delivery mechanism, highlighting the potential of this approach for MPS-IH and other genetic diseases., Lysosomal storage diseases like mucopolysaccharidosis type I (MPS I) cause pathology before birth and result in early morbidity and mortality. Here, the authors show that in utero base editing mediates multi-organ phenotypic and survival benefits in a mouse model recapitulating a common human MPSI mutation.

In utero transplantation (IUT) of hematopoietic stem cells (HSC) has been proposed as a strategy for the prenatal treatment of congenital hematological diseases. However, levels of long-term hematopoietic engraftment achieved in experimental IUT to date are sub-therapeutic, likely due to host fetal HSC out-competing their bone-marrow (BM) derived donor equivalents for space in the hematopoietic compartment. In the present study we demonstrate that amniotic fluid stem cells (AFSC; c-Kit+/Lin-) have hematopoietic characteristics and, thanks to their fetal origin, favourable proliferation kinetics in vitro and in vivo, which are maintained when the cells are expanded. IUT of autologous/congenic freshly-isolated or cultured AFSC resulted in stable mutli-lineage hematopoietic engraftment, far higher to that achieved with BM-HSC. Intravascular IUT of allogenic AFSC was not successful as recently reported after intraperitoneal IUT. Herein we demonstrated that this likely due to a failure of timely homing of donor cells to the host fetal thymus resulted in lack of tolerance induction and rejection. This study reveals that intravascular IUT leads to a remarkable hematopoietic engraftment of AFSC in the setting of autologous/congenic IUT, and confirms the requirement for induction of central tolerance for allogenic IUT to be successful. Autologous, gene engineered and in-vitro expanded AFSC could be used as a stem cell/gene therapy platform for the in utero treatment of inherited disorders of hematopoiesis. SIGNIFICANCE STATEMENT: Amniotic fluid stem cells can be expanded without losing their hematopoietic characteristics In utero transplantation of allogenic AFSC results in adaptive immune response and donor cell rejection, due to failure of timely homing of donor cells to the host fetal thymus and lack of central tolerance induction. Autologous/congenic amniotic fluid stem cells can be transplanted in utero and result in stable, multi-lineage, long-term hematopoietic engraftment that is significantly higher to that achieved with bone marrow-derived cells and could be therapeutic in many inherited disorders of hematopoiesis. © AlphaMed Press 2019.

We tested the hypothesis that chronic fetal hypoxia, at a severity present in many types of congenital heart disease, would lead to abnormal neurodevelopment.Eight mid-gestation fetal sheep were cannulated onto a pumpless extracorporeal oxygenator via the umbilical vessels and supported in a fluid-filled environment for 22 ± 2 days under normoxic or hypoxic conditions. Total parenteral nutrition was provided. Control fetuses (n = 7) were harvested at gestational age 133 ± 4 days. At necropsy, brains were fixed for histopathology. Neurons were quantified in white matter tracts, and the thickness of the external granular layer of the cerebellum was measured to assess neuronal migration. Capillary density and myelination were quantified in white matter. Data were analyzed with unpaired Student t tests or 1-way analysis of variance, as appropriate.Oxygen delivery was reduced in hypoxic fetuses (15.6 ± 1.8 mL/kg/min vs 24.3 ± 2.3 mL/kg/min, P .01), but umbilical blood flow and caloric delivery were not different between the 2 groups. Compared with normoxic and control animals, hypoxic fetuses had reduced neuronal density and increased external granular layer thickness. Compared with normoxic and control animals, hypoxic fetuses had increased capillary density in white matter. Cortical myelin integrity score was lower in the hypoxic group compared with normoxic and control animals. There was a significant negative correlation between myelin integrity and capillary density.Chronic fetal hypoxia leads to white matter hyper-vascularity, decreased neuronal density, and impaired myelination, similar to the neuropathologic findings observed in children with congenital heart disease. These findings support the hypothesis that fetal hypoxia, even in the setting of normal caloric delivery, impairs neurodevelopment.

Asymptomatic cystic lung lesions-congenital pulmonary airway malformations (CPAMs), sequestrations, and bronchogenic cysts-are commonly diagnosed prenatally. Indications to resect are to eliminate risk of malignancy or infection. CPAMs consist of a spectrum of malformations, with type 1 historically considered the most common. Mucinous cell clusters, seen almost exclusively in type 1, are premalignant lesions at risk for progression to mucinous adenocarcinoma. We reviewed and classified 2.5 years of consecutive, prenatally diagnosed lesions as extralobar sequestration, intralobar sequestration, type 1 CPAM, type 2 CPAM/bronchial atresia, or "other" to determine the distribution of lesion types and risk of malignancy. One hundred eighty-four lesions in 174 patients showed type 1 CPAM to be least common subtype. Type 1 CPAMs had more severe presentation, infrequently had features of obstruction, and usually had cysts ≥2 cm. Fifteen of eighteen type 1 CPAMs had mucinous cell clusters (total risk, 8%), with mucous cells outside main cyst in 12/15. No pleuropulmonary blastomas were identified. Additional historic cases were reviewed to further evaluate risk of malignancy. Over 14 years, 28 infants with fetal/type 1 lesions were identified, with clusters of mucinous cells in 75% of cases. A total of 9 pleuropulmonary blastomas were diagnosed in 6 patients over 16 years. Contrary to historical studies, type 1 CPAMs are much less common than type 2, likely related to detection of asymptomatic lesions prenatally. A majority of type 1 CPAMs contain mucinous cell clusters. This data is useful in management of patients in centers that do not resect asymptomatic lesions.

The American Thoracic Society Core Curriculum updates clinicians annually in adult and pediatric pulmonary disease, medical critical care, and sleep medicine, in a 3- to 4-year recurring cycle of topics. These topics will be presented at the 2020 International Conference. Below is the pediatric pulmonary medicine core, including pediatric hypoxemic respiratory failure; modalities in noninvasive management of chronic respiratory failure in childhood; surgical and nonsurgical management of congenital lung malformations; an update on smoke inhalation lung injury; an update on vaporizers, e-cigarettes, and other electronic delivery systems; pulmonary complications of sarcoidosis; pulmonary complications of congenital heart disease; and updates on the management of congenital diaphragmatic hernia.

ispartof: MOLECULAR THERAPY vol:28 issue:11 pages:2316-2319 ispartof: location:United States status: published

Lipid nanoparticles deliver mRNA to mouse fetuses, which may ultimately enable in utero therapy to treat fetal genetic diseases., Clinical advances enable the prenatal diagnosis of genetic diseases that are candidates for gene and enzyme therapies such as messenger RNA (mRNA)–mediated protein replacement. Prenatal mRNA therapies can treat disease before the onset of irreversible pathology with high therapeutic efficacy and safety due to the small fetal size, immature immune system, and abundance of progenitor cells. However, the development of nonviral platforms for prenatal delivery is nascent. We developed a library of ionizable lipid nanoparticles (LNPs) for in utero mRNA delivery to mouse fetuses. We screened LNPs for luciferase mRNA delivery and identified formulations that accumulate within fetal livers, lungs, and intestines with higher efficiency and safety compared to benchmark delivery systems, DLin-MC3-DMA and jetPEI. We demonstrate that LNPs can deliver mRNAs to induce hepatic production of therapeutic secreted proteins. These LNPs may provide a platform for in utero mRNA delivery for protein replacement and gene editing.

Background This study sought to evaluate neurodevelopmental outcome in survivors of high-risk congenital lung lesions (CLLs) who underwent prenatal intervention or postnatal surgery within the first month of life. Methods Forty-five high-risk CLL survivors underwent assessment using the Bayley Scales of Infant Development, 3rd Edition between July 2004 and December 2016. Scores were grouped as average, at-risk, and delayed based on SD intervals. Correlations between outcome and risk factors were analyzed by Fisher's exact test or two-sided t test as appropriate, with significant p values Results Open prenatal intervention was required in 13 (28.9%) children (fetal surgical resection, n = 4 , ex utero intrapartum treatment, n = 9), whereas 32 (71.1%) children had respiratory distress postnatally and required resection within the first month of life. Mean age at follow-up was 19.3 ± 10.3 months. Mean composite scores were within the expected average range. A total of 62.2% scored within the average range for all domains. At-risk scores were found in 26.7% of children in at least one domain, and 11.1% had delays in at least one domain. Neurodevelopmental outcome was similar between treatment groups. Prolonged ventilator support and neonatal intensive care unit stay, need for supplemental oxygen at day of life 30, gastroesophageal reflux disease, and delayed enteral feeding were associated with neurologic delays (all p Conclusions Neurodevelopmental scores for high-risk CLL survivors in infancy and toddlerhood are age appropriate. Neither fetal intervention nor the need for postnatal resection within the first month of life increases the risk of delays. Surrogate markers of a complicated neonatal course are predictive of adverse outcome.

KEY POINTS Bronchopulmonary dysplasia is a disease of extreme prematurity that occurs when the immature lung is exposed to gas ventilation. We designed a novel 'artificial womb' system for supporting extreme premature lambs (called EXTEND) that obviates gas ventilation by providing oxygen via a pumpless arteriovenous circuit with the lamb submerged in sterile artificial amniotic fluid. In the present study, we compare different arteriovenous cannulation strategies on EXTEND, including carotid artery/jugular vein (CA/JV), carotid artery/umbilical vein (CA/UV) and umbilical artery/umbilical vein (UA/UV). Compared to CA/JV and CA/UV cannulation, UA/UV cannulation provided significantly higher, physiological blood flows to the oxygenator, minimized flow interruptions and supported significantly longer circuit runs (up to 4 weeks). Physiological circuit blood flow in UA/UV lambs made possible normal levels of oxygen delivery, which is a critical step toward the clinical application of artificial womb technology. ABSTRACT EXTEND (EXTra-uterine Environment for Neonatal Development) is a novel system that promotes physiological development by maintaining the premature lamb in a sterile fluid environment and providing gas exchange via a pumpless arteriovenous oxygenator circuit. During the development of EXTEND, different cannulation strategies evolved with the aim of improving circuit flow. The present study examines how different cannulation strategies affect EXTEND circuit haemodynamics in extreme premature lambs. Seventeen premature lambs were cannulated at gestational ages 105-117 days (term 145-150 days) and supported on EXTEND for up to 4 weeks. Experimental groups were distinguished by cannulation strategy: carotid artery outflow and jugular vein inflow (CA/JV; n = 4), carotid artery outflow and umbilical vein inflow (CA/UV; n = 5) and double umbilical artery outflow and umbilical vein inflow (UA/UV; n = 8). Circuit flows and pressures were measured continuously. As we transitioned from CA/JV to CA/UV to UA/UV cannulation, mean duration of circuit run and weight-adjusted circuit flows increased (P

Purpose The prenatal natural history of intralobar and extralobar bronchopulmonary sequestrations (BPSs), including lesion growth patterns and need for prenatal intervention, have not been fully characterized. We review our series of BPSs to determine their natural history and outcomes in the context of the need for prenatal intervention. Methods A retrospective review of the pre/postnatal course of 103 fetuses with an intralobar (n = 44) or extralobar BPS (n = 59) managed at a single institution between 2008 and 2015 was performed. Outcomes included prenatal lesion growth trajectory, presence of hydrops, need for prenatal intervention, survival, and postnatal surgical management. Results Most extralobar (71%) and intralobar BPSs (94%) decreased in size or became isoechoic from initial to final evaluation. Peak lesion size occurred at 26–28 weeks gestation. Eight fetuses developed hydrothorax, four of which (all extralobar BPSs) also developed hydrops. All four hydropic fetuses received maternal betamethasone, and three hydropic fetuses underwent thoracentesis and/or thoracoamniotic shunt placement with subsequent hydrops resolution. All fetuses survived. Forty-one intralobar (93%) and 35 extralobar BPSs (59%) were resected after birth. Conclusions BPSs tend to decrease in size after 26–28 weeks gestation and rarely require fetal intervention. Lesions resulting in hydrothorax ± hydrops can be effectively managed with maternal steroids and/or drainage of the hydrothorax. Level of evidence IV

Objectives: Whereas left-sided congenital diaphragmatic hernias (L-CDH) have been extensively studied and their prognostic parameters delineated, right-sided hernias (R-CDH) have not. Published results remain inconclusive. The aim of this study is to evaluate if proven prognostic indicators of postnatal survival in the fetus with L-CDH apply to the fetus with R-CDH. Methods: Retrospective single-center study of R-CDH fetuses with available prenatal studies assessed for fetal lung volume by means of ultrasound-measured observed versus expected (O/E) lung area to head circumference (LHR) and magnetic resonance-calculated O/E total lung volume (TLV) in a 12-year time period. Percentage of herniated liver volume and postnatal use of extracorporeal membrane oxygenation (ECMO) were also evaluated. Results: In a cohort of 24 patients, O/E LHR, O/E TLV, percentage of herniated liver, and postnatal use of ECMO are not prognostic indicators of survival in the fetus with R-CDH. Cut-off values of O/E LHR of ≤45 or O/E TLV ≤25, known to select a population of severe cases for the L-CDH fetus, do not appear to extrapolate to the R-CDH fetus, as survival in both R-CDH groups is 60%. Conclusion: The findings in this study suggest that L- and R-CDH appear to behave differently, and that factors that make L-CDH fatal (low O/E TLV and O/E LHR, high-volume herniated liver) may not apply to the fetus with R-CDH.

In utero hematopoietic cell transplantation (IUHCT) is a novel nonmyeloablative approach that results in donor-specific tolerance and mixed allogeneic chimerism. Clinical application is limited by low levels of donor cell engraftment. Competition from endogenous hematopoietic stem cells (HSCs) for limited "space" in fetal hematopoietic organs remains a significant barrier to successful IUHCT. AMD3100, a CXCR4 inhibitor, and firategrast, an α4β1 and α4β7 integrin inhibitor (α4β1/7), have been shown to disrupt HSC retention in the postnatal hematopoietic niche. We hypothesized that maternal administration of AMD3100 and/or firategrast prior to IUHCT would mobilize endogenous HSCs from the fetal liver (FL) and result in preferential FL homing of donor HSCs and enhanced long-term engraftment following IUHCT in an allogeneic mouse model. We demonstrate that (1) both agents cross the placenta with rapidly detectable fetal serum concentrations following maternal administration; (2) firategrast treatment alone or with AMD3100 mobilizes endogenous HSCs from the FL and results in increased FL homing of donor HSCs following IUHCT; and (3) enhanced donor HSC homing following firategrast treatment translates into increased long-term multilineage donor cell engraftment. This approach highlights the potential of mobilization strategies to overcome barriers to successful engraftment and increase the clinical promise of IUHCT.

Nevus comedonicus syndrome (NCS) is a rare epidermal nevus syndrome characterized by ocular, skeletal, and central nervous system anomalies. We present a 23-month-old boy with a history of a congenital pulmonary airway malformation (CPAM) of the lung and a congenital cataract who developed progressive linear and curvilinear plaques of dilated follicular openings with keratin plugs (comedones) on parts of his scalp, face, and body consistent with nevus comedonicus. MRI of the brain demonstrated an aneurysm of the right internal carotid artery. Genetic testing identified NEK9 c.1755_1757del (p.Thr586del) at mean allele frequency of 28% in the nevus comedonicus. This same mutation was present in the CPAM tissue. This is the first case of a CPAM in a patient with an epidermal nevus syndrome. This case expands the phenotype of nevus comedonicus syndrome to include CPAM and vascular anomalies.

Monogenic lung diseases that are caused by mutations in surfactant genes of the pulmonary epithelium are marked by perinatal lethal respiratory failure or chronic diffuse parenchymal lung disease with few therapeutic options. Using a CRISPR fluorescent reporter system, we demonstrate that precisely timed in utero intra-amniotic delivery of CRISPR-Cas9 gene editing reagents during fetal development results in targeted and specific gene editing in fetal lungs. Pulmonary epithelial cells are predominantly targeted in this approach, with alveolar type 1, alveolar type 2, and airway secretory cells exhibiting high and persistent gene editing. We then used this in utero technique to evaluate a therapeutic approach to reduce the severity of the lethal interstitial lung disease observed in a mouse model of the human SFTPC(I73T) mutation. Embryonic expression of SftpC(I73T) alleles is characterized by severe diffuse parenchymal lung damage and rapid demise of mutant mice at birth. After in utero CRISPR-Cas9-mediated inactivation of the mutant SftpC(I73T) gene, fetuses and postnatal mice showed improved lung morphology and increased survival. These proof-of-concept studies demonstrate that in utero gene editing is a promising approach for treatment and rescue of monogenic lung diseases that are lethal at birth.

Host cell competition is a major barrier to engraftment after in utero hematopoietic cell transplantation (IUHCT). Here we describe a cell-engineering strategy using glycogen synthase kinase-3 (GSK3) inhibitor-loaded nanoparticles conjugated to the surface of donor hematopoietic cells to enhance their proliferation kinetics and ability to compete against their fetal host equivalents. With this approach, we achieved remarkable levels of stable, long-term hematopoietic engraftment for up to 24 weeks post-IUHCT. We also show that the salutary effects of the nanoparticle-released GSK3 inhibitor are specific to donor progenitor/stem cells and achieved by a pseudoautocrine mechanism. These results establish that IUHCT of hematopoietic cells decorated with GSK3 inhibitor-loaded nanoparticles can produce therapeutic levels of long-term engraftment and could therefore allow single-step prenatal treatment of congenital hematological disorders.

In utero hypoxia is a major cause of neonatal morbidity and mortality and predisposes to adult cardiovascular disease. No therapies exist to correct fetal hypoxia. In a new ex utero fetal support system, we tested the hypothesis that hypoxemic support of the fetus impairs myocardial development, whereas normoxic support allows normal myocardial development. Preterm fetal lambs were connected via umbilical vessels to a low-resistance oxygenator and placed in a sterile-fluid environment. Control normoxic fetuses received normal fetal oxygenation, and hypoxemic fetuses received subphysiologic oxygenation. Fetuses with normal in utero development served as normal controls. Hypoxemic fetuses exhibited decreased maximum cardiac output in both ventricles, diastolic function, myocyte and myocyte nuclear size, and increased myocardial capillary density versus control normoxic fetuses. There were no differences between control normoxic fetuses in the fetal support system and normal in utero controls. Chronic fetal hypoxemia resulted in significant abnormalities in myocyte architecture and myocardial capillary density as well as systolic and diastolic cardiac function, whereas control fetuses showed no differences. This ex utero fetal support system has potential to become a significant research tool and novel therapy to correct fetal hypoxia.

Pulmonary hypoplasia and hypertension is a leading cause of morbidity and mortality in congenital diaphragmatic hernia (CDH). The etiologic insult occurs early in gestation highlighting the potential of prenatal interventions. We evaluated prenatal pharmacologic therapies in the nitrofen CDH model. Olive oil or nitrofen were administered alone or with dexamethasone (DM), sildenafil, or DM+sildenafil to pregnant rats. Newborn pups were assessed for lung function, structure and pulmonary artery (PA) flow and resistance. Prenatal DM treatment of CDH pups increased alveolar volume density (Vva), decreased interalveloar septal thickness, increased tidal volumes and improved ventilation without improving oxygenation or PA resistance. Sildenafil decreased PA resistance and improved oxygenation without improving ventilation or resulting in significant histologic changes. DM+sildenafil decreased PA resistance, improved oxygenation and ventilation while increasing Vva and decreasing interalveolar septal and pulmonary arteriole medial wall thickness. Lung and body weights were decreased in pups treated with DM and/or sildenafil. Prenatal DM or sildenafil treatment increased pulmonary compliance and decreased pulmonary vascular resistance respectively, and was associated with improved neonatal gas exchange but had a detrimental effect on lung and fetal growth. This study highlights the potential of individual and combined prenatal pharmacologic therapies for CDH management.

In utero hematopoietic cell transplantation (IUHCT) has the potential to treat a number of congenital hematologic disorders. Clinical application is limited by low levels of donor engraftment. Techniques that optimize donor cell delivery to the fetal liver (FL), the hematopoietic organ at the time of IUHCT, have the potential to enhance engraftment and the clinical success of IUHCT. We compared the 3 clinically applicable routes of injection (intravenous [i.v.], intraperitoneal [i.p.], and intrahepatic [i.h.]) and assessed short- and long-term donor cell engraftment and fetal survival in the murine model of IUHCT. We hypothesized that the i.v. route would promote direct donor cell homing to the FL, resulting in increased engraftment and allowing for larger injectate volumes without increased fetal mortality. We demonstrate that the i.v. route results in (1) rapid diffuse donor cell population of the FL compared with delayed diffuse engraftment after the i.p. and i.h. routes; (2) higher FL and spleen engraftment at early prenatal time points; (3) enhanced stable long-term peripheral blood donor cell engraftment; and (4) improved survival at higher injectate volumes, allowing for higher donor cell doses and increased long-term engraftment. These findings support the use of an i.v. route for clinical protocols of IUHCT.

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

In utero gene editing has the potential to prenatally treat genetic diseases that result in significant morbidity and mortality before or shortly after birth. We assessed the viral vector-mediated delivery of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated 9 (CRISPR-Cas9) or base editor 3 (BE3) in utero, seeking therapeutic modification of Pcsk9 or Hpd in wild-type mice or the murine model of hereditary tyrosinemia type 1 (HT1), respectively. We observed long-term postnatal persistence of edited cells in both models, with reduction of plasma PCSK9 and cholesterol levels following in utero Pcsk9 targeting and rescue of the lethal phenotype of HT1 following in utero Hpd targeting. The results of this proof-of-concept work demonstrate the possibility to efficiently perform gene editing before birth, pointing to a potential new therapeutic approach for select congenital genetic disorders.

In utero hematopoietic cell transplantation (IUHCT) is a nonmyeloablative nonimmunosuppressive alternative to postnatal hematopoietic stem cell transplantation for the treatment of congenital hemoglobinopathies. Anti-HLA donor-specific Abs (DSA) are associated with a high incidence of graft rejection following postnatal hematopoietic stem cell transplantation. We determine if DSA present in the mother can similarly cause graft rejection in the fetus following IUHCT. Ten million C57BL/6 (B6, H2kb) bone marrow cells were transplanted in utero into gestational day 14 BALB/c (H2kd) fetuses. The pregnant BALB/c dams carrying these fetuses either had been previously sensitized to B6 Ag or were injected on gestational days 13–15 with serum from B6-sensitized BALB/c females. Maternal–fetal Ab transmission, Ab opsonization of donor cells, chimerism, and frequency of macrochimeric engraftment (chimerism >1%) were assessed by flow cytometry. Maternal IgG was transmitted to the fetus and rapidly opsonized donor cells following IUHCT. Donor cell rejection was observed as early as 4 h after IUHCT in B6-sensitized dams and 24 h after IUHCT in dams injected with B6-sensitized serum. Efficient opsonization was strongly correlated with decreased chimerism. No IUHCT recipients born to B6-sensitized dams or dams injected with B6-sensitized serum demonstrated macrochimeric engraftment at birth compared with 100% of IUHCT recipients born to naive dams or dams injected with naive serum (p < 0.001). In summary, maternal donor–specific IgG causes rapid, complete graft rejection in the fetus following IUHCT. When a third-party donor must be used for clinical IUHCT, the maternal serum should be screened for DSA to optimize the chance for successful engraftment.

Rationale: Disruption of normal pulmonary development is a leading cause of morbidity and mortality in infants. Congenital lung malformations are a unique model to study the molecular pathogenesis of isolated structural birth defects, as they are often surgically resected. Objectives: To provide insight into the molecular pathogenesis of congenital lung malformations through analysis of cell-type and gene expression changes in these lesions. Methods: Clinical data, and lung tissue for DNA, RNA, and histology, were obtained from 58 infants undergoing surgical resection of a congenital lung lesion. Transcriptome-wide gene expression analysis was performed on paired affected and unaffected samples from a subset of infants (n = 14). A three-dimensional organoid culture model was used to assess isolated congenital lung malformation epithelium (n = 3). Measurements and Main Results: Congenital lung lesions express higher levels of airway epithelial related genes, and dysregulated expression of genes related to the Ras and PI3K–AKT–mTOR (phosphatidylinositol 3-kinase–AKT–mammalian target of rapamycin) signaling pathways. Immunofluorescence confirmed differentiated airway epithelial cell types throughout all major subtypes of congenital lung lesions, and three-dimensional cell culture demonstrated a cell-autonomous defect in the epithelium of these lesions. Conclusions: This study provides the first comprehensive analysis of the congenital lung malformation transcriptome and suggests that disruptions in Ras or PI3K–AKT–mTOR signaling may contribute to the pathology through an epithelial cell-autonomous defect.

Sickle cell disease (SCD) and thalassemias (Thal) are common congenital disorders, which can be diagnosed early in gestation and result in significant morbidity and mortality. Hematopoietic stem cell transplantation, the only curative therapy for SCD and Thal, is limited by the absence of matched donors and treatment-related toxicities. In utero hematopoietic stem cell transplantation (IUHCT) is a novel nonmyeloablative transplant approach that takes advantage of the immunologic immaturity and normal developmental properties of the fetus to achieve mixed allogeneic chimerism and donor-specific tolerance (DST). We hypothesized that a combined strategy of IUHCT to induce DST, followed by postnatal nonmyeloablative same donor "booster" bone marrow (BM) transplants in murine models of SCD and Thal would result in high levels of allogeneic engraftment and donor hemoglobin (Hb) expression with subsequent phenotypic correction of SCD and Thal. Our results show that: (1) IUHCT is associated with DST and low levels of allogeneic engraftment in the murine SCD and Thal models; (2) low-level chimerism following IUHCT can be enhanced to high-level chimerism and near complete Hb replacement with normal donor Hb with this postnatal "boosting" strategy; and (3) high-level chimerism following IUHCT and postnatal "boosting" results in phenotypic correction in the murine Thal and SCD models. This study supports the potential of IUHCT, combined with a postnatal nonmyelablative "boosting" strategy, to cure Thal and SCD without the toxic conditioning currently required for postnatal transplant regimens while expanding the eligible transplant patient population due to the lack of a restricted donor pool.

Nature Communications 8: Article number: 15112 (2017); Published 25 April 2017; Updated 23 May 2017 A patent based on the work reported in this Article was inadvertently omitted from the Competing interests section of this article. The Competing interests statement should read: E.M., A.F. and M.D. are co-authors on a patent entitled ‘Extracorporeal life support system and methods of use thereof’ (Patent no.

To evaluate whether infants with congenital diaphragmatic hernia (CDH) can be safely resuscitated with a reduced starting fraction of inspired oxygen (FiO2) of 0.5. A retrospective cohort study comparing 68 patients resuscitated with starting FiO2 0.5 to 45 historical controls resuscitated with starting FiO2 1.0. Reduced starting FiO2 had no adverse effect upon survival, duration of intubation, need for ECMO, duration of ECMO, or time to surgery. Furthermore, it produced no increase in complications, adverse neurological events, or neurodevelopmental delay. The need to subsequently increase FiO2 to 1.0 was associated with female sex, lower gestational age, liver up, lower lung volume–head circumference ratio, decreased survival, a higher incidence of ECMO, longer time to surgery, periventricular leukomalacia, and lower neurodevelopmental motor scores. Starting FiO2 0.5 may be safe for the resuscitation of CDH infants. The need to increase FiO2 to 1.0 during resuscitation is associated with worse outcomes.

In the developed world, extreme prematurity is the leading cause of neonatal mortality and morbidity due to a combination of organ immaturity and iatrogenic injury. Until now, efforts to extend gestation using extracorporeal systems have achieved limited success. Here we report the development of a system that incorporates a pumpless oxygenator circuit connected to the fetus of a lamb via an umbilical cord interface that is maintained within a closed ‘amniotic fluid' circuit that closely reproduces the environment of the womb. We show that fetal lambs that are developmentally equivalent to the extreme premature human infant can be physiologically supported in this extra-uterine device for up to 4 weeks. Lambs on support maintain stable haemodynamics, have normal blood gas and oxygenation parameters and maintain patency of the fetal circulation. With appropriate nutritional support, lambs on the system demonstrate normal somatic growth, lung maturation and brain growth and myelination., The ability to support the development of a premature fetus in the form of an extracorporeal system has had limited success. Here, the authors show that an extra-uterine device that mimics the intra-uterine environment can provide physiologic support for the extreme premature lamb fetus for four weeks.