The lack of fetal immune responses to foreign antigens, i.e., fetal immunologic tolerance, is the most compelling rationale for prenatal stem cell and gene therapy. However, the frequency of engraftment following in utero hematopoietic cell transplantation (IUHCT) in the murine model is reduced in allogeneic, compared with congenic, recipients. This observation supports the existence of an immune barrier to fetal transplantation and challenges the classic assumptions of fetal tolerance. Here, we present evidence that supports the presence of an adaptive immune response in murine recipients of IUHCT that failed to maintain engraftment. However, when IUHCT recipients were fostered by surrogate mothers, they all maintained long-term chimerism. Furthermore, we have demonstrated that the cells responsible for rejection of the graft were recipient in origin. Our observations suggest a mechanism by which IUHCT-dependent sensitization of the maternal immune system and the subsequent transmission of maternal alloantibodies to pups through breast milk induces a postnatal adaptive immune response in the recipient, which, in turn, results in the ablation of engraftment after IUHCT. Finally, we showed that non-fostered pups that maintained their chimerism had higher levels of Tregs as well as a more suppressive Treg phenotype than their non-chimeric, non-fostered siblings. This study resolves the apparent contradiction of induction of an adaptive immune response in the pre-immune fetus and confirms the potential of actively acquired tolerance to facilitate prenatal therapeutic applications.

Key Points• Optimization of IUHCT in a preclinical canine model yields stable long-term donor engraftment.• Clinically significant levels of chimerism can be achieved without conditioning, immunosuppression, or graft-versushost disease.Evidence supporting the efficacy of in utero hematopoietic cell transplantation (IUHCT) in a valid large animal model is needed prior to clinical application. The objective of this study was to establish clinically relevant levels of hematopoietic chimerism in a canine model of maternal-to-fetal IUHCT. We first assessed immune and hematopoietic ontogeny relevant to IUHCT in the canine model and identified 40 days' gestation (term 63 days) as a time point at the initiation of thymic selection, and prior to bone marrow hematopoiesis, that might be optimal for IUHCT. We next determined that intravascular administration of donor cells via intracardiac injection was far more efficient and resulted in much higher levels of donor cell engraftment than intraperitoneal injection. By applying these findings, we achieved stable long-term multilineage engraftment in 21 of 24 surviving recipients with an average level of initial chimerism of 11.7% (range 3% to 39%) without conditioning or evidence of graft-versus-host disease. Donor cell chimerism remained stable for up to 2 years and was associated with donor-specific tolerance for renal transplantation. The levels of donor cell chimerism achieved in this study would be therapeutic for many hematopoietic disorders and are supportive of a clinical trial

The ideal gene therapy for metabolical liver disorders would target hepatocytes before the onset of disease and be durable, non-toxic and non-immunogenic. Early gestational gene transfer can achieve such goals. Here, we demonstrate that prenatal gene transfer of human Atp7b reduces liver pathology and improves biochemical markers in Atp7b À/À mice, a murine model of Wilson's disease (WD). Following prenatal injection of lentivirus vector containing the human Atp7b gene under the transcriptional control of a liver-specific promoter, the full-length ATP7B was detectable in mouse livers for the entire duration of experiments (20 weeks after birth). In contrast to a marked pathology in non-injected animals, livers from age-matched treated mice consistently demonstrated normal gross and histological morphology. Hepatic copper content was decreased in the majority of treated mice, although remaining copper levels varied. Improvement of hepatic copper metabolism was further apparent from the presence of copper-bound ceruloplasmin in the sera and normalization of the mRNA levels for HMG CoAreductase. With this approach, the complete loss of copper transport function can be ameliorated, as evident from phenotypical improvement in treated Atp7b À/À mice. This study provides proof of principle for in utero gene therapy in WD and other liver-based enzyme deficiencies.

Viral vector-mediated gene transfer to the postnatal respiratory epithelium has, in general, been of low efficiency due to physical and immunological barriers, non-apical location of cellular receptors critical for viral uptake and limited transduction of resident stem/progenitor cells. These obstacles may be overcome using a prenatal strategy. In this study, HIV-1-based lentiviral vectors (LVs) pseudotyped with the envelope glycoproteins of Jaagsiekte sheep retrovirus (JSRV-LV), baculovirus GP64 (GP64-LV), Ebola Zaire-LV or vesicular stomatitis virus (VSVg-LV) and the adeno-associated virus-2/6.2 (AAV2/6.2) were compared for in utero transfer of a green fluorescent protein (GFP) reporter gene to ovine lung epithelium between days 65 and 78 of gestation. GFP expression was examined on day 85 or 136 of gestation (term is B145 days). The percentage of the respiratory epithelial cells expressing GFP in fetal sheep that received the JSRV-LV (3.18Â10 8 -6.85Â10 9 viral particles per fetus) was 24.6 ± 0.9% at 3 weeks postinjection (day 85) and 29.9±4.8% at 10 weeks postinjection (day 136). Expression was limited to the surface epithelium lining fetal airways o100 mm internal diameter. Fetal airways were amenable to VSVg-LV transduction, although the percentage of epithelial expression was low (6.6 ± 0.6%) at 1 week postinjection. GP64-LV, Ebola Zaire-LV and AAV2/6.2 failed to transduce the fetal ovine lung under these conditions. These data demonstrate that prenatal lung gene transfer with LV engineered to target apical surface receptors can provide sustained and high levels of transgene expression and support the therapeutic potential of prenatal gene transfer for the treatment of congenital lung diseases.

Children with ASD have an increased number of medical and mental health needs. Obtaining care via a medical home has been shown to decrease unmet healthcare needs. However, they are less likely to receive care through the medical home model compared with other children with special healthcare needs. Barriers identified by families include a lack of early identification, limited knowledge on educational plans, and unknown community resources. Barriers identified by providers include lack of time, training, and resources. Providing care coordination and family-centered care in a medical home model are essential for children with ASD.

In utero hematopoietic cell transplantation (IUHCT) offers the potential to achieve allogeneic engraftment and associated donor-specific tolerance without the need for toxic conditioning, as we have previously demonstrated in the murine and canine models. This strategy holds great promise in the treatment of many hematopoietic disorders, including the hemoglobinopathies. Graft-versus-host disease (GVHD) represents the greatest theoretical risk of IUHCT and has never been characterized in the context of IUHCT. We recently described a preclinical canine model of IUHCT, allowing further study of the technique and its complications. We aimed to establish a threshold T cell dose for IUHCT-induced GVHD in the haploidentical canine model and to define the GVHD phenotype. Using a range of T cell concentrations within the donor inoculum, we were able to characterize the phenotype of IUHCT-induced GVHD and establish a clear threshold for its induction between 3% and 5% graft CD3 cell content. Given the complete absence of GVHD at CD3 doses of 1% to 3% and the excellent engraftment with the lowest dose, there is a safe therapeutic index for a clinical trial of IUHCT.

Childhood apraxia of speech is a neurologic speech sound disorder in which children have difficulty constructing words and sounds due to poor motor planning and coordination of the articulators required for speech sound production. We report the case of a 3-year-old boy strongly suspected to have childhood apraxia of speech at 18 months of age who used multimodal communication to facilitate language development throughout his work with a speech language pathologist. In 18 months of an intensive structured program, he exhibited atypical rapid improvement, progressing from having no intelligible speech to achieving age-appropriate articulation. We suspect that early introduction of sign language by family proved to be a highly effective form of language development, that when coupled with intensive oro-motor and speech sound therapy, resulted in rapid resolution of symptoms.

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