Preeclampsia is the leading cause of morbidity and mortality in pregnancy. Although the etiology of preeclampsia is still unclear, it is believed to involve rejection of the fetus, possibly due to an imbalance between regulatory (Treg) and effector T cells. To test this, we compared the frequencies of circulating CD4+ T cells expressing Foxp3, IFN-γ, IL-10, or IL-17 at the end of the third trimester of healthy and preeclamptic pregnancies. The size of the Treg cell compartment, defined by the frequency of CD4+CD25high, CD4+CD127lowCD25+, and CD4+Foxp3+ cells was significantly higher in normal compared with preeclamptic pregnancies. CD4+CD25high and CD4+CD127lowCD25+ populations in preeclampsia were not significantly different from those in nonpregnant controls, whereas CD4+Foxp3+ cells numbersre slightly lower in preeclampsia. The suppressive activity of ex vivo-sorted CD4+CD127lowCD25+ Treg cells was not significantly different between the three study groups. The percentage of CD4+IL-17-producing T cells decreased significantly in healthy compared with preeclamptic pregnancies and nonpregnant controls, whereas CD4+IL-10- and CD4+IFN-γ-producing cells remained unchanged. Consequently, the ratio of Foxp3+ Treg to IL-17-expressing CD4+ T cells was significantly increased in healthy but not in preeclamptic pregnancies. Thus, preeclampsia is associated with the absence of normal systemic skewing away from IL-17 production toward Foxp3+ expression. Finally, preeclamptic women had significantly higher levels of soluble endoglin, an inhibitor of TGF-β receptor signaling, which may bias toward IL-17 production. These results suggest that homeostasis between regulatory and proinflammatory CD4+ T cells might be pivotal for the semiallogeneic fetus to be tolerated within the maternal environment.

Existing recombinant adeno-associated virus (rAAV) serotypes for delivering in vivo gene therapy treatments for human liver diseases have not yielded combined high-level human hepatocyte transduction and favorable humoral neutralization properties in diverse patient groups. Yet, these combined properties are important for therapeutic efficacy. To bioengineer capsids that exhibit both unique seroreactivity profiles and functionally transduce human hepatocytes at therapeutically relevant levels, we performed multiplexed sequential directed evolution screens using diverse capsid libraries in both primary human hepatocytes in vivo and with pooled human sera from thousands of patients. AAV libraries were subjected to five rounds of in vivo selection in xenografted mice with human livers to isolate an enriched human-hepatotropic library that was then used as input for a sequential on-bead screen against pooled human immunoglobulins. Evolved variants were vectorized and validated against existing hepatotropic serotypes. Two of the evolved AAV serotypes, NP40 and NP59, exhibited dramatically improved functional human hepatocyte transduction in vivo in xenografted mice with human livers, along with favorable human seroreactivity profiles, compared with existing serotypes. These novel capsids represent enhanced vector delivery systems for future human liver gene therapy applications.

Adeno-associated virus (AAV) vectors are quickly becoming the vectors of choice for therapeutic gene delivery. To date, hundreds of natural isolates and bioengineered variants have been reported. While factors such as high production titer and low immunoreactivity are important to consider, the ability to deliver the genetic payload (physical transduction) and to drive high transgene expression (functional transduction) remains the most important feature when selecting AAV variants for clinical applications. Reporter expression assays are the most commonly used methods for determining vector fitness. However, such approaches are time consuming and become impractical when evaluating a large number of variants. Limited access to primary human tissues or challenging model systems further complicates vector testing. To address this problem, convenient highthroughput methods based on next-generation sequencing (NGS) are being developed. To this end, we built an AAV Testing Kit that allows inherent flexibility in regard to number and type of AAV variants included, and is compatible with in vitro, ex vivo, and in vivo applications. The Testing Kit presented here consists of a mix of 30 known AAVs where each variant encodes a CMV-eGFP cassette and a unique barcode in the 3¢-untranslated region of the eGFP gene, allowing NGS-barcode analysis at both the DNA and RNA/cDNA levels. To validate the AAV Testing Kit, individually packaged barcoded variants were mixed at an equal ratio and used to transduce cells/tissues of interest. DNA and RNA/cDNA were extracted and subsequently analyzed by NGS to determine the physical/functional transduction efficiencies. We were able to assess the transduction efficiencies of immortalized cells, primary cells, and induced pluripotent stem cells in vitro, as well as in vivo transduction in naïve mice and a xenograft liver model. Importantly, while our data validated previously reported transduction characteristics of individual capsids, we also identified novel previously unknown tropisms for some AAV variants.