The β-hemoglobinopathies, including sickle cell disease (SCD) and β-thalassemia, are caused by mutations in the β-globin gene (HBB) and affect millions of people worldwide. A curative strategy for the β-hemoglobinopathies would be ex vivo gene correction in patient-derived hematopoietic stem cells (HSCs) followed by autologous transplantation. Here we report the first CRISPR/Cas9 gene-editing platform for achieving homologous recombination (HR) at the HBB gene in HSCs by combining Cas9 ribonucleoproteins and rAAV6 HR donor delivery. Notably, we devise an enrichment paradigm to purify a population of HSPCs with >90% targeted integration. We also show efficient correction of the SCD-causing E6V mutation in patient-derived HSPCs that after differentiation into erythrocytes, express adult β-globin (HbA) mRNA, confirming intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting HSCs by HR at the HBB locus to advance the development of next generation therapies for β-hemoglobinopathies.

In T cell-deficient conditions, naive T cells undergo spontaneous "homeostatic" proliferation in response to contact with self-MHC/peptide ligands. With the aid of an in vitro system, we show here that homeostatic proliferation is also cytokine-dependent. The cytokines IL-4, IL-7, and IL-15 enhanced homeostatic proliferation of naive T cells in vitro. Of these cytokines, only IL-7 was found to be critical; thus, naive T cells underwent homeostatic proliferation in IL-4(-) and IL-15(-) hosts but proliferated minimally in IL-7(-) hosts. In addition to homeostatic proliferation, the prolonged survival of naive T cells requires IL-7. Thus, naïve T cells disappeared gradually over a 1-month period upon adoptive transfer into IL-7(-) hosts. These findings indicate that naive T cells depend on IL-7 for survival and homeostatic proliferation.

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The CRISPR-Cas9 system is a powerful tool for genome editing, which allows the precise modification of specific DNA sequences. Many efforts are underway to use the CRISPR-Cas9 system to therapeutically correct human genetic diseases [1][2][3][4][5][6] . The most widely used orthologs of Cas9 are derived from Staphylococcus aureus and Streptococcus pyogenes 5,7 . Given that these two bacterial species infect the human population at high frequencies 8,9 , we hypothesized that humans may harbor preexisting adaptive immune responses to the Cas9 orthologs derived from these bacterial species, SaCas9 (S. aureus) and SpCas9 (S. pyogenes). By probing human serum for the presence of anti-Cas9 antibodies using an enzyme-linked immunosorbent assay, we detected antibodies against both SaCas9 and SpCas9 in 78% and 58% of donors, respectively. We also found anti-SaCas9 T cells in 78% and anti-SpCas9 T cells in 67% of donors, which demonstrates a high prevalence of antigen-specific T cells against both orthologs. We confirmed that these T cells were Cas9-specific by demonstrating a Cas9-specific cytokine response following isolation, Reprints and permissions information is available at www.nature.com/reprints.

Although the mammalian immune system is generally thought to develop in a linear fashion, findings in avian and murine species argue instead for the developmentally ordered appearance (or “layering”) of unique hematopoietic stem cells (HSC) that give rise to distinct lymphocyte lineages at different stages of development. Here, we provide evidence of an analogous “layered” immune system in humans. Our results suggest that fetal and adult T cells are distinct populations that arise from different populations of HSC present at different stages of development. We also provide evidence that the fetal T cell lineage is biased towards immune tolerance. These observations offer a mechanistic explanation for the tolerogenic properties of the developing fetus and for variable degrees of immune responsiveness at birth.

The immunological mechanisms required to engender resistance have been defined in few infectious diseases of man, and the role of specific cytokines is unclear. Leprosy presents clinically as a spectrum in which resistance correlates with cell-mediated immunity to the pathogen. To assess in situ cytokine patterns, messenger RNA extracted from leprosy skin biopsy specimens was amplified by the polymerase chain reaction with 14 cytokine-specific primers. In lesions of the resistant form of the disease, messenger RNAs coding for interleukin-2 and interferon-gamma were most evident. In contrast, messenger RNAs for interleukin-4, interleukin-5, and interleukin-10 predominated in the multibacillary form. Thus, resistance and susceptibility were correlated with distinct patterns of cytokine production.

Why some patients with seizures are successfully treated with antiepileptic drugs (AEDs) and others prove medically intractable is not known. Inadequate intraparenchymal drug concentration is a possible mechanism of resistance to AEDs. The multiple drug resistance gene (MDR1) encodes P-glycoprotein, an energy-dependent efflux pump that exports planar hydrophobic molecules from the cell. If P-glycoprotein is expressed in brain of some patients with intractable epilepsy and AEDs are exported by P-glycoprotein, lower intraparenchymal drug concentrations could contribute to lack of drug response in such patients. Eleven of 19 brain specimens removed from patients during operation for intractable epilepsy had MDR1 mRNA levels > 10 times greater than those in normal brain, as determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method. Immunohistochemistry for P-glycoprotein from 14 of the patients showed increased staining in capillary endothelium in samples from epileptic patients as compared with staining in normal brain samples. In epileptic brain specimens with high MDR1 mRNA levels, expression of P-glycoprotein in astrocytes also was identified. Last, steady-state intracellular phenytoin (PHT) concentrations in MDR1 expressing neuroectodermal cells was one fourth that in MDR1-negative cells. MDR1 expression is increased in brain of some patients with medically intractable epilepsy, suggesting that the patients' lack of response to medication may be caused by inadequate accumulation of AED in brain.

Hematopoietic stem cell transplantation (HSCT) is followed by profound immunodeficiency. Thymic function is necessary for de novo generation of T cells after HSCT. Circulating CD45RA ؉ naive T-cell levels are predictive of antigen-specific T-cell responses in the absence of graftversus-host disease (GVHD). These T cells may not represent recent thymic emigrants, since naive T cells may maintain this phenotype if not antigen-activated. To accurately measure thymic output after HSCT and determine the factors that influence thymic function, T-cell receptor excision circles (TRECs) were examined in CD4 ؉ and CD8 ؉ cells from a crosssection of patients following HSCT. TREC levels rose weeks after HSCT and could be detected in patients 6 years after HSCT. TREC levels correlated with the frequency of phenotypically naive T cells, indicating that such cells were not expanded progeny of naive T cells present in the donor graft. Chronic GVHD was the most important factor that predicted low TREC levels even years after HSCT. Patients with a history of resolved GVHD had decreased numbers of TREC, compared with those with no GVHD. Because few adults had no history of GVHD, it was not possible to determine whether age alone inversely correlated with TREC levels. Recipients of cord blood grafts had no evidence of decreased TREC induced by immunosuppressive prophylaxis drugs. Compared with unrelated donor grafts, recipients of matched sibling grafts had higher TREC levels. Collectively, these data suggest that thymopoiesis is inhibited by GVHD. Larger studies will be needed to determine the independent contributions of age and preparative regimen to posttransplant thymopoietic capacity. IntroductionFollowing hematopoietic stem cell transplant (HSCT), there is a prolonged period of profound immune deficiency, which includes defects in thymopoiesis. 1 This immune deficiency contributes to the high incidence of opportunistic infection, which continues for years after HSCT. 2,3 The etiology of the immune defect is multifactorial. Thymopoietic defects resulting in decreased ability to generate new T cells after HSCT are important since complete immune reconstitution ultimately depends on the generation of new T cells from hematopoietic stem cell (HSCs), just as long-term myeloid and erythroid reconstitution depends on HSC engraftment. Transfer of committed progenitors or mature donor-derived T cells may permit short-term immune function. Analyses of patients after HSCT have demonstrated that the presence of immune function at 1 year or later was correlated with the number of CD4 ϩ CD45RA ϩ naive T cells, suggesting that immune function at later time points is dependent on the ability to generate new T cells. 4,5 The factors that inhibit thymic function may include age, graft-versus-host disease (GVHD), and direct thymic damage from chemoradiotherapy. In multiple studies of normal individuals, recipients of high-dose chemotherapy, HSCT recipients, and patients infected with human immunodeficiency virus, age has been inversely correl...