Mouse epiblast stem cells (EpiSCs) can be derived from a wide range of developmental stages. To characterize and compare EpiSCs with different origins, we derived a series of EpiSC lines from pregastrula stage to late-bud-stage mouse embryos. We found that the transcriptomes of these cells are hierarchically distinct from those of the embryonic stem cells, induced pluripotent stem cells (iPSCs), and epiblast/ectoderm. The EpiSCs display globally similar gene expression profiles irrespective of the original developmental stage of the source tissue. They are developmentally similar to the ectoderm of the late-gastrula-stage embryo and behave like anterior primitive streak cells when differentiated in vitro and in vivo. The EpiSC lines that we derived can also be categorized based on a correlation between gene expression signature and predisposition to differentiate into particular germ-layer derivatives. Our findings therefore highlight distinct identifying characteristics of EpiSCs and provide a foundation for further examination of EpiSC properties and potential.
Vectors based on adeno-associated virus type 2 (AAV2) are powerful tools for gene transfer and genome editing applications. The level of interest in this system has recently surged in response to reports of therapeutic efficacy in human clinical trials, most notably for those in patients with hemophilia B (ref. 3). Understandably, a recent report drawing an association between AAV2 integration events and human hepatocellular carcinoma (HCC) has generated controversy about the causal or incidental nature of this association and the implications for AAV vector safety. Here we describe and functionally characterize a previously unknown liver-specific enhancer-promoter element in the wild-type AAV2 genome that is found between the stop codon of the cap gene, which encodes proteins that form the capsid, and the right-hand inverted terminal repeat. This 124-nt sequence is within the 163-nt common insertion region of the AAV genome, which has been implicated in the dysregulation of known HCC driver genes and thus offers added insight into the possible link between AAV integration events and the multifactorial pathogenesis of HCC.
Recent clinical successes in gene therapy applications have intensified interest in using adeno-associated viruses (AAVs) as vectors for therapeutic gene delivery. Although prototypical AAV2 shows robust in vitro transduction of human hepatocyte–derived cell lines, it has not translated into an effective vector for liver-directed gene therapy in vivo. This is consistent with observations made in Fah−/−/Rag2−/−/Il2rg−/− (FRG) mice with humanized livers, showing that AAV2 functions poorly in this xenograft model. Here, we derived naturally hepatotropic AAV capsid sequences from primary human liver samples. We demonstrated that capsid mutations, likely acquired as an unintentional consequence of tissue culture propagation, attenuated the intrinsic human hepatic tropism of natural AAV2 and related human liver AAV isolates. These mutations resulted in amino acid changes that increased binding to heparan sulfate proteoglycan (HSPG), which has been regarded as the primary cellular receptor mediating AAV2 infection of human hepatocytes. Propagation of natural AAV variants in vitro showed tissue culture adaptation with resulting loss of tropism for human hepatocytes. In vivo readaptation of the prototypical AAV2 in FRG mice with a humanized liver resulted in restoration of the intrinsic hepatic tropism of AAV2 through decreased binding to HSPG. Our results challenge the notion that high affinity for HSPG is essential for AAV2 entry into human hepatocytes and suggest that natural AAV capsids of human liver origin are likely to be more effective for liver-targeted gene therapy applications than culture-adapted AAV2.
Viral vectors based on adeno-associated virus (AAV) are showing exciting promise in gene therapy trials targeting the adult liver. A major challenge in extending this promise to the pediatric liver is the loss of episomal vector genomes that accompanies hepatocellular proliferation during liver growth. Hence maintenance of sufficient transgene expression will be critical for success in infants and children. We therefore set out to explore the therapeutic efficacy and durability of liver-targeted gene transfer in the challenging context of a neonatal lethal urea cycle defect, using the argininosuccinate synthetase deficient mouse. Lethal neonatal hyperammonemia was prevented by prenatal and early postnatal vector delivery; however, hyperammonemia subsequently recurred limiting survival to no more than 33 days despite vector readministration. Antivector antibodies acquired in milk from vector-exposed dams were subsequently shown to be blocking vector readministration, and were avoided by crossfostering vector-treated pups to vector-naive dams. In the absence of passively acquired antivector antibodies, vector redelivery proved efficacious with mice surviving to adulthood without recurrence of significant hyperammonemia. These data demonstrate the potential of AAV vectors in the developing liver, showing that vector readministration can be used to counter growth-associated loss of transgene expression provided the challenge of antivector humoral immunity is addressed.
Adeno-associated viral vector-mediated (AAV-mediated) expression of allogeneic major histocompatibility complex class I (MHC class I) in recipient liver induces donor-specific tolerance in mouse skin transplant models in which a class I allele (H-2Kb or H-2Kd) is mismatched between donor and recipient. Tolerance can be induced in mice primed by prior rejection of a donor-strain skin graft, as well as in naive recipients. Allogeneic MHC class I may be recognized by recipient T cells as an intact molecule (direct recognition) or may be processed and presented as an allogeneic peptide in the context of self-MHC (indirect recognition). The relative contributions of direct and indirect allorecognition to tolerance induction in this setting are unknown. Using hepatocyte-specific AAV vectors encoding WT allogeneic MHC class I molecules, or class I molecules containing a point mutation (D227K) that impedes direct recognition of intact allogeneic MHC class I by CD8+ T cells without hampering the presentation of processed peptides derived from allogeneic MHC class I, we show here that tolerance induction depends upon recognition of intact MHC class I. Indirect recognition alone yielded a modest prolongation of subsequent skin graft survival, attributable to the generation of CD4+ Tregs, but it was not sufficient to induce tolerance.
Summary
The mechanisms by which tumour cells escape recognition by the immune system or subvert antitumour effector responses remain poorly understood. In the course of investigating the potential of costimulatory signals in anticancer immunotherapy strategies, we have observed that HeLa cells (a human cervical carcinoma cell line) cocultured with peripheral blood lymphocytes (PBL) acquire the capacity to inhibit PBL proliferation in response to interleukin‐2 (IL‐2). This immuno‐inhibitory phenotype was further shown to result from induction of the tryptophan‐catabolizing enzyme, indoleamine 2,3‐dioxygenase (IDO), by interferon‐γ (IFN‐γ) secreted from cocultured allo‐reactive PBL. This enzyme has recently been shown to be a critically important modulator of immunological responses, most notably through the capacity to protect allogeneic concepti from alloreactive maternal lymphocytes. While the cytostatic consequences of IDO activity in tumour cells has received attention, the data presented in this report support the hypothesis that IDO activity may also act to impair antitumour immune responses.
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