Sherry Koontz

Transgene-free iPSCs generated from small volume peripheral blood nonmobilized CD34+ cells.

A variety of somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs), but CD34(+) hematopoietic stem cells (HSCs) present in nonmobilized peripheral blood (PB) would be a convenient target. We report a method for deriving iPSC from PB HSCs using immunobead purification and 2- to 4-day culture to enrich CD34(+) HSCs to 80% ± 9%, followed by reprogramming with loxP-flanked polycistronic (human Oct4, Klf4, Sox2, and c-Myc) STEMCCA-loxP lentivector, or with Sendai vectors. Colonies arising with STEMCCA-loxP were invariably TRA-1-60(+), yielding 5.3 ± 2.8 iPSC colonies per 20 mL PB (n = 17), where most colonies had single-copy STEMCCA-loxP easily excised by transient Cre expression. Colonies arising with Sendai were variably reprogrammed (10%-80% TRA-1-60(+)), with variable yield (6 to >500 TRA-1-60(+) iPSC colonies per 10 mL blood; n = 6). Resultant iPSC clones expressed pluripotent cell markers and generated teratomas. Genomic methylation patterns of STEMCCA-loxP-reprogrammed clones closely matched embryonic stem cells. Furthermore, we showed that iPSCs are derived from the nonmobilized CD34(+) HSCs enriched from PB rather than from any lymphocyte or monocyte contaminants because they lack somatic rearrangements typical of T or B lymphocytes and because purified CD14(+) monocytes do not yield iPSC colonies under these reprogramming conditions.

CRISPR-Cas9 gene repair of hematopoietic stem cells from patients with X-linked chronic granulomatous disease

CRISPR-mediated gene repair of hematopoietic stem cells from patients with X-linked chronic granulomatous disease resulted in functional human leukocytes in mice after transplantation. Seamless gene repair with CRISPR Targeted gene therapy has been hampered by the inability to correct mutations in stem cells that can reconstitute the immune system after transplant into patients. De Ravin et al. now report that CRISPR, a DNA editing technology, corrected blood stem cells from patients with an immunodeficiency disorder (chronic granulomatous disease) caused by mutations in NOX2. CRISPR-repaired human stem cells engrafted in mice after transplant and differentiated into leukocytes with a functional NOX2 protein for up to 5 months. The authors did not detect off-target treatment effects, suggesting that this gene repair strategy may benefit patients with chronic granulomatous disease or other blood disorders. Gene repair of CD34+ hematopoietic stem and progenitor cells (HSPCs) may avoid problems associated with gene therapy, such as vector-related mutagenesis and dysregulated transgene expression. We used CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated 9) to repair a mutation in the CYBB gene of CD34+ HSPCs from patients with the immunodeficiency disorder X-linked chronic granulomatous disease (X-CGD). Sequence-confirmed repair of >20% of HSPCs from X-CGD patients restored the function of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and superoxide radical production in myeloid cells differentiated from these progenitor cells in vitro. Transplant of gene-repaired X-CGD HSPCs into NOD (nonobese diabetic) SCID (severe combined immunodeficient) γc−/− mice resulted in efficient engraftment and production of functional mature human myeloid and lymphoid cells for up to 5 months. Whole-exome sequencing detected no indels outside of the CYBB gene after gene correction. CRISPR-mediated gene editing of HSPCs may be applicable to other CGD mutations and other monogenic disorders of the hematopoietic system.

NADPH Oxidase-2 Derived ROS Dictates Murine DC Cytokine-Mediated Cell Fate Decisions during CD4 T Helper-Cell Commitment

NADPH oxidase-2 (Nox2)/gp91phox and p47phox deficient mice are prone to hyper-inflammatory responses suggesting a paradoxical role for Nox2-derived reactive oxygen species (ROS) as anti-inflammatory mediators. The molecular basis for this mode of control remains unclear. Here we demonstrate that IFNγ/LPS matured p47phox−/−-ROS deficient mouse dendritic cells (DC) secrete more IL-12p70 than similarly treated wild type DC, and in an in vitro co-culture model IFNγ/LPS matured p47phox−/− DC bias more ovalbumin-specific CD4+ T lymphocytes toward a Th1 phenotype than wild type (WT) DC through a ROS-dependent mechanism linking IL-12p70 expression to regulation of p38-MAPK activation. The Nox2-dependent ROS production in DC negatively regulates proinflammatory IL-12 expression in DC by constraining p38-MAPK activity. Increasing endogenous H2O2 attenuates p38-MAPK activity in IFNγ/LPS stimulated WT and p47phox−/− DC, which suggests that endogenous Nox 2-derived ROS functions as a secondary messenger in the activated p38-MAPK signaling pathway during IL-12 expression. These findings indicate that ROS, generated endogenously by innate and adaptive immune cells, can function as important secondary messengers that can regulate cytokine production and immune cell cross-talk to control during the inflammatory response.

Adenosine A₂A receptor agonist-mediated increase in donor-derived regulatory T cells suppresses development of graft-versus-host disease.

Graft-versus-host disease (GVHD) remains a significant complication of allogeneic transplantation. We previously reported that the adenosine A2A receptor (A2AR) specific agonist, ATL146e, decreases the incidence and severity of GVHD in a mouse transplant model. There is increasing interest in treatments that increase CD4+CD25highFoxp3+ regulatory T cells (Tregs) to suppress GVHD. Our current study found in vitro that A2AR selective agonists enhanced TGF-β–induced generation of mouse Tregs 2.3- to 3-fold. We demonstrated in vivo suppression of GVHD with specific A2AR agonists in two different murine GVHD transplant models associated with profound increases in both circulating and target tissue Tregs of donor origin. Three different A2AR agonists of differing potency, ATL146e, ATL370, and ATL1223, all significantly inhibited GVHD-associated weight loss and mortality. At the same time, Tregs shown to be of donor origin increased 5.1- to 7.4-fold in spleen, 2.7- to 4.6-fold in peripheral blood, 2.3- to 4.7-fold in colon, and 3.8- to 4.6-fold in skin. We conclude that specific activation of A2AR inhibits acute GVHD through an increase of donor-derived Tregs. Furthermore, the increased presence of Tregs in target tissues (colon and skin) of A2AR-specific agonist-treated mice is likely the mechanistic basis for the anti-inflammatory effect preventing acute GVHD.

p47phox Directs Murine Macrophage Cell Fate Decisions

Macrophage differentiation and function are pivotal for cell survival from infection and involve the processing of microenvironmental signals that determine macrophage cell fate decisions to establish appropriate inflammatory balance. NADPH oxidase 2 (Nox2)-deficient chronic granulomatous disease (CGD) mice that lack the gp91(phox) (gp91(phox-/-)) catalytic subunit show high mortality rates compared with wild-type mice when challenged by infection with Listeria monocytogenes (Lm), whereas p47(phox)-deficient (p47(phox-/-)) CGD mice show survival rates that are similar to those of wild-type mice. We demonstrate that such survival results from a skewed macrophage differentiation program in p47(phox-/-) mice that favors the production of higher levels of alternatively activated macrophages (AAMacs) compared with levels of either wild-type or gp91(phox-/-) mice. Furthermore, the adoptive transfer of AAMacs from p47(phox-/-) mice can rescue gp91(phox-/-) mice during primary Lm infection. Key features of the protective function provided by p47(phox-/-) AAMacs against Lm infection are enhanced production of IL-1α and killing of Lm. Molecular analysis of this process indicates that p47(phox-/-) macrophages are hyperresponsive to IL-4 and show higher Stat6 phosphorylation levels and signaling coupled to downstream activation of AAMac transcripts in response to IL-4 stimulation. Notably, restoring p47(phox) protein expression levels reverts the p47(phox)-dependent AAMac phenotype. Our results indicate that p47(phox) is a previously unrecognized regulator for IL-4 signaling pathways that are important for macrophage cell fate choice.

Molecular Analysis of Neutrophil Differentiation from Human Induced Pluripotent Stem Cells Delineates the Kinetics of Key Regulators of Hematopoiesis.

In vitro generation of mature neutrophils from human induced pluripotent stem cells (iPSCs) requires hematopoietic progenitor development followed by myeloid differentiation. The purpose of our studies was to extensively characterize this process, focusing on the critical window of development between hemogenic endothelium, hematopoietic stem/progenitor cells (HSPCs), and myeloid commitment, to identify associated regulators and markers that might enable the stem cell field to improve the efficiency and efficacy of iPSC hematopoiesis. We utilized a four‐stage differentiation protocol involving: embryoid body (EB) formation (stage‐1); EB culture with hematopoietic cytokines (stage‐2); HSPC expansion (stage‐3); and neutrophil maturation (stage‐4). CD34+CD45− putative hemogenic endothelial cells were observed in stage‐3 cultures, and expressed VEGFR‐2/Flk‐1/KDR and VE‐cadherin endothelial markers, GATA‐2, AML1/RUNX1, and SCL/TAL1 transcription factors, and endothelial/HSPC‐associated microRNAs miR‐24, miR‐125a‐3p, miR‐126/126*, and miR‐155. Upon further culture, CD34+CD45− cells generated CD34+CD45+ HSPCs that produced hematopoietic CFUs. Mid‐stage‐3 CD34+CD45+ HSPCs exhibited increased expression of GATA‐2, AML1/RUNX1, SCL/TAL1, C/EBPα, and PU.1 transcription factors, but exhibited decreased expression of HSPC‐associated microRNAs, and failed to engraft in immune‐deficient mice. Mid‐stage‐3 CD34−CD45+ cells maintained PU.1 expression and exhibited increased expression of hematopoiesis‐associated miR‐142‐3p/5p and a trend towards increased miR‐223 expression, indicating myeloid commitment. By late Stage‐4, increased CD15, CD16b, and C/EBPɛ expression were observed, with 25%‐65% of cells exhibiting morphology and functions of mature neutrophils. These studies demonstrate that hematopoiesis and neutrophil differentiation from human iPSCs recapitulates many features of embryonic hematopoiesis and neutrophil production in marrow, but reveals unexpected molecular signatures that may serve as a guide for enhancing iPSC hematopoiesis. Stem Cells 2016;34:1513–1526

Role of p47phox in Antigen-Presenting Cell-Mediated Regulation of Humoral Immunity in Mice

Microbial-induced inflammation is important for eliciting humoral immunity. Genetic defects of NADPH oxidase 2-based proteins interrupt phagocyte superoxide generation and are the basis for the human immunodeficiency chronic granulomatous disease (CGD). Hyperinflammation is also a significant clinical manifestation of CGD. Herein, we evaluated humoral immunity in the phagocyte oxidase p47(phox)-deficient model of CGD and found that UV-inactivated Streptococcus pneumoniae and Listeria monocytogenes (Lm) elicited higher specific antibody (Ab) titers in p47(phox-/-) mice than wild-type (WT) mice. Both organisms elicited robust and distinct antigen-presenting cell maturation phenotypes, including IL-12 hypersecretion, and higher major histocompatibility complex II and costimulatory protein expression in Lm-stimulated p47(phox-/-) dendritic cells (DCs) relative to WT DCs. Furthermore, p47(phox-/-) DCs pulsed with Lm and adoptively transferred into naïve WT mice elicited Ab titers, whereas Lm-pulsed WT DCs did not elicit these titers. The observed robust p47(phox-/-) mouse humoral response was recapitulated with live Lm and sustained in vivo in p47(phox-/-) mice. Notably, anti-serum samples from p47(phox-/-) mice that survived secondary Lm infection were protective in WT and p47(phox-/-) mice that were rechallenged with secondary lethal Lm infection. These findings demonstrate a novel benefit of NADPH oxidase 2 deficiency (ie, dependent inflammation in antigen-presenting cell-mediated humoral immunity) and that anti-Lm Ab can be protective in an immunodeficient CGD host.

Gene-edited pseudogene resurrection corrects p47phox-deficient chronic granulomatous disease

Pseudogenes are duplicated genes with mutations rendering them nonfunctional. For single-gene disorders with homologous pseudogenes, the pseudogene might be a target for genetic correction. Autosomal-recessive p47phox-deficient chronic granulomatous disease (p47-CGD) is a life-threatening immune deficiency caused by mutations in NCF1, a gene with 2 pseudogenes, NCF1B and NCF1C. The most common NCF1 mutation, a GT deletion (ΔGT) at the start of exon 2 (>90% of alleles), is constitutive to NCF1B and NCF1C. NCF1 ΔGT results in premature termination, undetectable protein expression, and defective production of antimicrobial superoxide in neutrophils. We examined strategies for p47-CGD gene correction using engineered zinc-finger nucleases targeting the exon 2 ΔGT in induced pluripotent stem cells or CD34+ hematopoietic stem cells derived from p47-CGD patients. Correction of ΔGT in NCF1 pseudogenes restores oxidase function in p47-CGD, providing the first demonstration that targeted restoration of pseudogene function can correct a monogenic disorder.

Recombinant Pregnancy-Specific Glycoprotein 1 Has a Protective Role in a Murine Model of Acute Graft-versus-Host Disease

Acute graft-versus-host disease (aGVHD) is an immune-mediated reaction that can occur after hematopoietic stem cell transplantation in which donor T cells recognize the host antigens as foreign, destroying host tissues. Establishment of a tolerogenic immune environment while preserving the immune response to infectious agents is required for successful bone marrow transplantation. Pregnancy-specific glycoprotein 1 (PSG1), which is secreted by the human placenta into the maternal circulation throughout pregnancy, likely plays a role in maintaining immunotolerance to prevent rejection of the fetus by the maternal immune system. We have previously shown that PSG1 activates the latent form of transforming growth factor β1 (TGF-β), a cytokine essential for the differentiation of tolerance-inducing CD4+FoxP3+ regulatory T cells (Tregs). Consistent with this observation, treatment of naïve murine T cells with PSG1 resulted in a significant increase in FoxP3+ cells that was blocked by a TGF-β receptor I inhibitor. We also show here that PSG1 can increase the availability of active TGF-β in vivo. As the role of CD4+FoxP3+ cells in the prevention of aGVHD is well established, we tested whether PSG1 has beneficial effects in a murine aGHVD transplantation model. PSG1-treated mice had reduced numbers of tissue-infiltrating inflammatory CD3+ T cells and had increased expression of FoxP3 in T cells compared with vehicle-treated mice. In addition, administration of PSG1 significantly inhibited aGVHD-associated weight loss and mortality. On the other hand, administration of PSG1 was less effective in managing aGVHD in the presence of an alloimmune reaction against a malignancy in a graft-versus-leukemia experimental model. Combined, this data strongly suggests that PSG1 could be a promising treatment option for patients with aGVHD following bone marrow transplantation for a nonmalignant condition, such as an autoimmune disorder or a genetic immunodeficiency.

41. Therapeutic Level CRISPR-Oligomer-Mediated Correction of X-CGD Patient Hematopoietic Stem Cells Using Non-Viral, cGMP Compliant, Scalable, and Closed System

Gene therapy using integrating viral vectors in hematopoietic stem cells (HSC) has shown clinical benefit in genetic diseases. However, there remain safety concerns associated with random integration and the lack of regulation of gene expression. Efficient and site-specific correction of mutation(s) in HSC using non-viral methods may improve safety and regulation of gene expression. Chronic granulomatous disease (CGD) due to defective phagocyte NADPH oxidase complex and lack of bactericidal superoxide and other reactive oxidative species (ROS) is characterized by severe infections and hyperinflammation. Although the X-linked form of CGD with gp91phox deficiency results from mutations that span the CYBB gene, we identified a ‘hotspot’ mutation at Exon 7 c. 676C>T, causing a premature stop codon in 17 out of 285 patients with X-linked CGD at the NIH. Here we report the result of the efficient correction of the hotspot CYBB mutation using highly efficient CRISPR (Cas9 and sgRNA) system with an oligomer as donor repair template, using MaxCytes commercially/clinically validated cGMP/regulatory compliant and closed platform technology. Plasmids encoding Cas9 and gRNA were purchased from the Genomic Engineering Center at Washington University (St. Louis, MO). The mRNA encoding Cas9 and gRNA were in vitro transcribed at MaxCyte using mMESSAGE mMACHINE® T7 Ultra kit, (Ambion, Austin, TX). We screened and selected best gRNA from four gRNA candidates for correction and then optimized transfection conditions with EBV-transformed B cell line (B-LCL) derived from an adult patient (P1) with the hotspot CYBB mutation. Transfected B-LCL exhibit 80±6% viability, minimal detectable toxicity as determined by cell proliferation rate referenced to control cells, and efficient site-specific gene correction with 20-50% WT gp91 expression. These developed protocols were used to treat G-CSF and pleraxifor mobilized CD34+ HSC from P1. Following optimization, in vitro treated HSC from P1 achieved 20-30% WT gp91 expression, with >50% viability, and minimal loss of cell proliferation capacity compared with control cells. CD34+ HSCs undergo myeloid differentiation in DMEM supplemented with G-CSF prior to functional evaluation using flow cytometric dihydrorhodamine (DHR) assay, demonstrating ~20% ROS+ cells in treated samples compared to ~80% in normal controls. P1s HSC treated the same way were transplanted into immunodeficient mice, and analyzed 8 weeks later. Bone marrow from mice transplanted with P1 treated cells showed CD45+ human cell engraftment rates at 50-80%, and of the forward/side scatter-gated granulocytes, 11-26% express gp91phox relative to 68% in normal control. Peripheral blood from mice demonstrated 11-23% human CD45+ cells, of which 9-21% expressed gp91phox, compared to 79% in normal controls. Deep sequencing of human CD45+ cells sorted from mouse bone marrow confirmed high rates (up to 21%) of genetic correction from the ‘T’ mutation to the wildtype ‘C’. Since female carriers of X-CGD with ~10-15% normal functioning neutrophils appear to have normal resistance to infections, this level of correction at 10-20% in human CD45+ cells from transplanted mice suggest CRISPR/oligo approach a feasible therapeutic option for treatment of CGD patients with the Ex7, c. 676C>T mutation.