Moving away from one disease at a time: Screening, trial design, and regulatory implications of novel platform technologies.

Most rare diseases are caused by single-gene mutations, and as such, lend themselves to a host of new gene-targeted therapies and technologies including antisense oligonucleotides, phosphomorpholinos, small interfering RNAs, and a variety of gene delivery and gene editing systems. Early successes are encouraging, however, given the substantial number of distinct rare diseases, the ability to scale these successes will be unsustainable without new development efficiencies. Herein, we discuss the need for genomic newborn screening to match pace with the growing development of targeted therapeutics and ability to rapidly develop individualized therapies for rare variants. We offer approaches to move beyond conventional "one disease at a time" preclinical and clinical drug development and discuss planned regulatory innovations that are necessary to speed therapy delivery to individuals in need. These proposals leverage the shared properties of platform classes of therapeutics and innovative trial designs including master and platform protocols to better serve patients and accelerate drug development. Ultimately, there are risks to these novel approaches; however, we believe that close partnership and transparency between health authorities, patients, researchers, and drug developers present the path forward to overcome these challenges and deliver on the promise of gene-targeted therapies for rare diseases.

Data sharing to advance gene-targeted therapies in rare diseases.

Recent advancements in gene-targeted therapies have highlighted the critical role data sharing plays in successful translational drug development for people with rare diseases. To scale these efforts, we need to systematize these sharing principles, creating opportunities for more rapid, efficient, and scalable drug discovery/testing including long-term and transparent assessment of clinical safety and efficacy. A number of challenges will need to be addressed, including the logistical difficulties of studying rare diseases affecting individuals who may be scattered across the globe, scientific, technical, regulatory, and ethical complexities of data collection, and harmonization and integration across multiple platforms and contexts. The NCATS/NIH Gene-Targeted Therapies: Early Diagnosis and Equitable Delivery meeting series held during June 2021 included data sharing models that address these issues and framed discussions of areas that require improvement. This article describes these discussions and provides a series of considerations for future data sharing.

CRISPR-Cas9 Gene Editing for Sickle Cell Disease and ß-Thalassemia.

Transfusion-dependent ß-thalassemia (TDT) and sickle cell disease (SCD) are severe monogenic diseases with severe and potentially life-threatening manifestations. BCL11A is a transcription factor that represses γ-globin expression and fetal hemoglobin in erythroid cells. We performed electroporation of CD34+ hematopoietic stem and progenitor cells obtained from healthy donors, with CRISPR-Cas9 targeting the BCL11A erythroid-specific enhancer. Approximately 80% of the alleles at this locus were modified, with no evidence of off-target editing. After undergoing myeloablation, two patients - one with TDT and the other with SCD - received autologous CD34+ cells edited with CRISPR-Cas9 targeting the same BCL11A enhancer. More than a year later, both patients had high levels of allelic editing in bone marrow and blood, increases in fetal hemoglobin that were distributed pancellularly, transfusion independence, and (in the patient with SCD) elimination of vaso-occlusive episodes. (Funded by CRISPR Therapeutics and Vertex Pharmaceuticals; ClinicalTrials.gov numbers, NCT03655678 for CLIMB THAL-111 and NCT03745287 for CLIMB SCD-121.).

Pharmacokinetic and Drug-Drug Interaction Profiles of the Combination of Tezacaftor/Ivacaftor.

Drug-drug interaction (DDI) studies are described for tezacaftor/ivacaftor, a new cystic fibrosis transmembrane conductance regulator modulator therapy for the treatment of cystic fibrosis. Three phase I DDI studies were conducted in healthy subjects to characterize the DDI profile of tezacaftor/ivacaftor with cytochrome P450 (CYP)3A substrates, CYP3A inhibitors, and a permeability glycoprotein (P-gp) substrate. The effects of steady-state tezacaftor/ivacaftor on the pharmacokinetics (PKs) of digoxin (a P-gp substrate), midazolam, and ethinyl estradiol/norethindrone (CYP3A substrates) were evaluated. Effects of strong (itraconazole) and moderate (ciprofloxacin) CYP3A inhibitors on tezacaftor/ivacaftor PKs were also determined. Tezacaftor/ivacaftor increased digoxin area under the curve (AUC) by 30% but did not affect midazolam, ethinyl estradiol, or norethindrone exposures. Itraconazole increased the AUC of tezacaftor 4-fold and ivacaftor 15.6-fold. Ciprofloxacin had no significant effect on tezacaftor or ivacaftor exposure. Coadministration of tezacaftor/ivacaftor may increase exposure of sensitive P-gp substrates. Tezacaftor/ivacaftor is unlikely to impact exposure of drugs metabolized by CYP3A, including hormonal contraceptives. Strong CYP3A inhibitors significantly increase the exposures of tezacaftor and ivacaftor.

Tezacaftor/Ivacaftor in Subjects with Cystic Fibrosis and F508del/F508del-CFTR or F508del/G551D-CFTR.

RATIONALE: Tezacaftor (formerly VX-661) is an investigational small molecule that improves processing and trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) in vitro, and improves CFTR function alone and in combination with ivacaftor. OBJECTIVES: To evaluate the safety and efficacy of tezacaftor monotherapy and of tezacaftor/ivacaftor combination therapy in subjects with cystic fibrosis homozygous for F508del or compound heterozygous for F508del and G551D. METHODS: This was a randomized, placebo-controlled, double-blind, multicenter, phase 2 study (NCT01531673). Subjects homozygous for F508del received tezacaftor (10 to 150 mg) every day alone or in combination with ivacaftor (150 mg every 12 h) in a dose escalation phase, as well as in a dosage regimen testing phase. Subjects compound heterozygous for F508del and G551D, taking physician-prescribed ivacaftor, received tezacaftor (100 mg every day). MEASUREMENTS AND MAIN RESULTS: Primary endpoints were safety through Day 56 and change in sweat chloride from baseline through Day 28. Secondary endpoints included change in percent predicted FEV1 (ppFEV1) from baseline through Day 28 and pharmacokinetics. The incidence of adverse events was similar across treatment arms. Tezacaftor (100 mg every day)/ivacaftor (150 mg every 12 h) resulted in a 6.04 mmol/L decrease in sweat chloride and 3.75 percentage point increase in ppFEV1 in subjects homozygous for F508del, and a 7.02 mmol/L decrease in sweat chloride and 4.60 percentage point increase in ppFEV1 in subjects compound heterozygous for F508del and G551D from baseline through Day 28 (P < 0.05 for all). CONCLUSIONS: These results support continued clinical development of tezacaftor (100 mg every day) in combination with ivacaftor (150 mg every 12 h) in subjects with cystic fibrosis. Clinical trial registered with www.clinicaltrials.gov (NCT01531673).

Tezacaftor-Ivacaftor in Residual-Function Heterozygotes with Cystic Fibrosis.

BACKGROUND: Cystic fibrosis is an autosomal recessive disease caused by mutations in the CFTR gene that lead to progressive respiratory decline. Some mutant CFTR proteins show residual function and respond to the CFTR potentiator ivacaftor in vitro, whereas ivacaftor alone does not restore activity to Phe508del mutant CFTR. METHODS: We conducted a randomized, double-blind, placebo-controlled, phase 3, crossover trial to evaluate the efficacy and safety of ivacaftor alone or in combination with tezacaftor, a CFTR corrector, in 248 patients 12 years of age or older who had cystic fibrosis and were heterozygous for the Phe508del mutation and a CFTR mutation associated with residual CFTR function. Patients were randomly assigned to one of six sequences, each involving two 8-week intervention periods separated by an 8-week washout period. They received tezacaftor-ivacaftor, ivacaftor monotherapy, or placebo. The primary end point was the absolute change in the percentage of predicted forced expiratory volume in 1 second (FEV1) from the baseline value to the average of the week 4 and week 8 measurements in each intervention period. RESULTS: The number of analyzed intervention periods was 162 for tezacaftor-ivacaftor, 157 for ivacaftor alone, and 162 for placebo. The least-squares mean difference versus placebo with respect to the absolute change in the percentage of predicted FEV1 was 6.8 percentage points for tezacaftor-ivacaftor and 4.7 percentage points for ivacaftor alone (P<0.001 for both comparisons). Scores on the respiratory domain of the Cystic Fibrosis Questionnaire-Revised, a quality-of-life measure, also significantly favored the active-treatment groups. The incidence of adverse events was similar across intervention groups; most events were mild or moderate in severity, with no discontinuations of the trial regimen due to adverse events for tezacaftor-ivacaftor and few for ivacaftor alone (1% of patients) and placebo (<1%). CONCLUSIONS: CFTR modulator therapy with tezacaftor-ivacaftor or ivacaftor alone was efficacious in patients with cystic fibrosis who were heterozygous for the Phe508del deletion and a CFTR residual-function mutation. (Funded by Vertex Pharmaceuticals and others; EXPAND ClinicalTrials.gov number, NCT02392234 .).

Tezacaftor-Ivacaftor in Patients with Cystic Fibrosis Homozygous for Phe508del.

BACKGROUND: Combination treatment with the cystic fibrosis transmembrane conductance regulator (CFTR) modulators tezacaftor (VX-661) and ivacaftor (VX-770) was designed to target the underlying cause of disease in patients with cystic fibrosis. METHODS: In this phase 3, randomized, double-blind, multicenter, placebo-controlled, parallel-group trial, we evaluated combination therapy with tezacaftor and ivacaftor in patients 12 years of age or older who had cystic fibrosis and were homozygous for the CFTR Phe508del mutation. Patients were randomly assigned in a 1:1 ratio to receive either 100 mg of tezacaftor once daily and 150 mg of ivacaftor twice daily or matched placebo for 24 weeks. The primary end point was the absolute change in the percentage of the predicted forced expiratory volume in 1 second (FEV1) through week 24 (calculated in percentage points); relative change in the percentage of the predicted FEV1 through week 24 (calculated as a percentage) was a key secondary end point. RESULTS: Of the 510 patients who underwent randomization, 509 received tezacaftor-ivacaftor or placebo, and 475 completed 24 weeks of the trial regimen. The mean FEV1 at baseline was 60.0% of the predicted value. The effects on the absolute and relative changes in the percentage of the predicted FEV1 in favor of tezacaftor-ivacaftor over placebo were 4.0 percentage points and 6.8%, respectively (P<0.001 for both comparisons). The rate of pulmonary exacerbation was 35% lower in the tezacaftor-ivacaftor group than in the placebo group (P=0.005). The incidence of adverse events was similar in the two groups. Most adverse events were of mild severity (in 41.8% of patients overall) or moderate severity (in 40.9% overall), and serious adverse events were less frequent with tezacaftor-ivacaftor (12.4%) than with placebo (18.2%). A total of 2.9% of patients discontinued the assigned regimen owing to adverse events. Fewer patients in the tezacaftor-ivacaftor group than in the placebo group had respiratory adverse events, none of which led to discontinuation. CONCLUSIONS: The combination of tezacaftor and ivacaftor was efficacious and safe in patients 12 years of age or older who had cystic fibrosis and were homozygous for the CFTR Phe508del mutation. (Funded by Vertex Pharmaceuticals; EVOLVE ClinicalTrials.gov number, NCT02347657 .).

Continuous intravesical lidocaine treatment for interstitial cystitis/bladder pain syndrome: safety and efficacy of a new drug delivery device.

Limited treatment options exist for patients who suffer from a painful bladder condition known as interstitial cystitis/bladder pain syndrome (IC/BPS). Whether given systemically (orally) or by short-duration (1 to 2 hours) exposure via intravesical instillation, therapeutic agents have exhibited poor efficacy because their concentrations in the bladder are low. A previous attempt to develop a drug delivery device for use in the bladder was unsuccessful, likely as a result of poor tolerability. A continuous lidocaine-releasing intravesical system (LiRIS) was designed to be retained in the bladder and release therapeutic amounts of the drug into urine over a period of 2 weeks. The device was tested in healthy volunteers and IC/BPS patients and was found to be well tolerated in both subject groups because of its small size and freedom of movement within the bladder. The 16 women with IC/BPS who were enrolled in the study met the National Institute of Diabetes and Digestive and Kidney Diseases criteria for bladder hemorrhages or Hunner's lesions. Subjects received either LiRIS 200 mg or LiRIS 650 mg for 2 weeks. Safety, efficacy, cystoscopic appearance of the bladder, and limited pharmacokinetic data were collected. Both doses were well tolerated, and clinically meaningful reductions were seen in pain, urgency, voiding frequency, and disease questionnaires. Cystoscopic examinations showed improvement on day 14 (day of removal) compared with day 1, including resolution of Hunner's lesions in five of six subjects with baseline lesions. Global response assessment showed an overall responder rate of 64% at day 14 and a sustained overall responder rate of 64% 2 weeks later. Extended follow-up suggests that the reduction in pain was maintained for several months after the device was removed.

Effect of CC chemokine receptor 2 CCR2 blockade on serum C-reactive protein in individuals at atherosclerotic risk and with a single nucleotide polymorphism of the monocyte chemoattractant protein-1 promoter region.

CC chemokine receptor 2 (CCR2), expressed on the surface of circulating monocytes, and its ligand monocyte chemoattractant protein-1 (MCP-1; also known as CC-chemokine ligand 2) are present in atherosclerotic plaques and may have important roles in endothelial monocyte recruitment and activation. MLN1202 is a highly specific humanized monoclonal antibody that interacts with CCR2 and inhibits MCP-1 binding. The aim of this randomized, double-blind, placebo-controlled study was to measure reductions in circulating levels of high-sensitivity C-reactive protein, an established biomarker of inflammation associated with coronary artery disease, on MLN1202 treatment in patients at risk for atherosclerotic cardiovascular disease (≥2 risk factors for atherosclerotic cardiovascular disease and circulating high-sensitivity C-reactive protein >3 mg/L). Additionally, patients were genotyped for the 2518 A→G polymorphism in the promoter of the MCP-1 gene to investigate the correlation between this polymorphism and reduced C-reactive protein levels with MLN1202 treatment. Patients who received MLN1202 exhibited significant decreases in high-sensitivity C-reactive protein levels, beginning at 4 weeks and continuing through 12 weeks after dosing. Patients with A/G or G/G genotypes in the MCP-1 promoter had significantly greater reductions in high-sensitivity C-reactive protein levels than patients with the wild-type A/A genotype. In conclusion, MLN1202 treatment was well tolerated in this patient population and resulted in significant reductions in high-sensitivity C-reactive protein levels.

Phase 1B, randomized, double-blind, dose-escalation trial of CPG 10101 in patients with chronic hepatitis C virus.

UNLABELLED: CPG 10101, a synthetic oligodeoxynucleotide (ODN), is a toll-like receptor 9 (TLR9) agonist with antiviral and immunomodulatory properties that could potentially influence chronic infection with HCV. In this multicenter Phase 1b trial, 60 HCV-positive patients (50 genotype 1 HCV) were randomized and received either placebo or CPG 10101 at 0.25, 1, 4, 10, or 20 mg subcutaneously (SC) twice weekly for 4 weeks or at 0.5 or 0.75 mg/kg SC once weekly for 4 weeks. Dose-dependent cytokine induction was observed after administration of CPG 10101. At 24 hours after administering the highest dose of 0.75 mg/kg CPG 10101, interferon (IFN)-gamma-inducible protein 10 (IP-10) had a mean increase over baseline levels (+/-SD) of 15,057 (+/-9769) pg/ml (P < 0.01, compared to placebo); IFN-alpha had a 106 (+/-63.3) pg/ml increase (P < 0.01); and 2'5'-oligoadenylate synthetase (OAS) had a 163 (+/-120.6) pmol/dl increase (P < 0.01). Decreases in HCV RNA also were dose-dependent, with the greatest group geometric mean maximum reduction of 1.69 +/- 0.618 log(10) (P < 0.05) observed in the 0.75 mg/kg dose group. Decreases >/=1 log(10) were seen in 22 of 40 patients who received >/=1 mg CPG 10101, with 3 patients exceeding a 2.5-log(10) reduction. CPG 10101 was well tolerated, and adverse events were consistent with CPG 10101's mechanism of action. CONCLUSION: In this Phase 1 study, CPG 10101 was associated with dose-dependent increases in markers of immune activation and decreases in HCV RNA levels. The data support further clinical studies of CPG 10101 for treating chronic HCV infection.

Safety, pharmacokinetics and immune effects in normal volunteers of CPG 10101 (ACTILON), an investigational synthetic toll-like receptor 9 agonist.

UNLABELLED: CPG 10101 (ACTILON) is a novel potent and selective unmethylated cytidine-phosphate-guanosine (CpG)-containing oligodeoxynucleotide agonist of Toll-like receptor 9 (TLR9) being developed for the treatment of chronic infections such as HCV. OBJECTIVES AND METHODS: In this randomized, double-blind, placebo-controlled Phase I study in 48 normal volunteers, we investigated the safety, pharmacokinetic parameters and immune effects of subcutaneous administration of CPG 10101. Five sequential escalating doses from 0.25 to 20 mg were administered twice, 14 days apart. In addition, a 4 mg dose was administered twice weekly for four weeks. RESULTS: A maximum tolerated dose was not reached and the adverse event profile was consistent with the known immunostimulatory effects of TLR9 agonists, mostly consisting of injection site reactions or flu-like symptoms that were generally mild in intensity. CPG 10101 induced interferons, cytokines and chemokines in a pattern consistent with the biology of TLR9. The most sensitive marker was IP-10/CXCL10, whose induction was detected in some subjects even at the 0.25 mg dose. Some cytokines showed transient circulating levels, while the levels of others such as the antiviral cytokine 2',5'-oligoadenylate synthetase were sustained for several days. CONCLUSION: This study warrants further investigation of CPG 10101 for the treatment of chronic infections such as HCV.

Metabolic response of mice to a postnatal ablation of CCAAT/enhancer-binding protein alpha.

Although CCAAT/enhancer-binding protein alpha (C/EBPalpha) is essential for initiating or sustaining several metabolic processes during the perinatal period, the consequences of total ablation of C/EBPalpha during postnatal development have not been investigated. We have created a conditional knock-out model in which the administration of poly(I:C) caused a virtually total deletion of c/ebpalpha (C/EBPalpha(Delta/-) mice) in the liver, spleen, white and brown adipose tissues, pancreas, lung, and kidney of the mice. C/EBPalpha itself was completely ablated in the liver by day 4 after the injection of poly(I:C). There was no noticeable change in phenotype during the first 15 days after the injection. The mice maintained a normal level of fasting blood glucose and responded to the diabetogenic action of streptozotocin. From day 16 onward, the mice developed hypophagia, exhibited severe weight loss, lost triglyceride in white but not brown adipose tissue, became hypoglycemic and hypoinsulinemic, depleted their hepatic glycogen, and developed fatty liver. They also exhibited lowered plasma levels of free fatty acid, triglyceride, and cholesterol, as well as marked changes in hepatic mRNA for C/EBPdelta, peroxisome proliferator-activated receptor alpha, sterol regulatory element-binding protein 1, hydroxymethylglutaryl-coenzyme A reductase, and apolipoproteins. Although basal levels of hepatic mRNA for the cytosolic isoform of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase were reduced, transcription of the genes for these enzymes was inducible by dibutyryl cyclic AMP in C/EBPalpha(Delta/-) mice. The animals died about 1 month after the injection of poly(I:C). These findings demonstrate that C/EBPalpha is essential for the survival of animals during postnatal life and that its ablation leads to distinct biphasic change in metabolic processes.

Human neutrophil collagenase expression is C/EBP-dependent during myeloid development.

OBJECTIVE: Human neutrophil collagenase (HNC) is one of several secondary granule proteins (SGP) expressed late in the myeloid maturation pathway. SGPs are encoded by unlinked and functionally diverse genes that are hypothesized to be coordinately regulated at the transcriptional level and demonstrate uniform dysregulation in leukemic cells. In support of the hypothesis that tissue and stage-specific expression of SGP genes is regulated by shared factor(s), we sought to identify factors responsible for positive regulation of the SGP genes. METHODS: Using 5' deletion analysis, we identified a minimal HNC promoter located within the first 193 bp upstream of the transcription start site. Three CCAAT enhancer binding protein (C/EBP) sites were identified within this region and their functional importance was confirmed by mutational analysis, gel retardation, and oligonucleotide pulldown assays. Using chromatin immunoprecipitation (ChIP), we demonstrated that C/EBPalpha binds to the SGP gene promoters lactoferrin and HNC in nonexpressing cells. Upon induction of maturation, C/EBPalpha binds to these promoters and this binding correlates with the expression of both SGP genes. CONCLUSION: We conclude that in the later stages of myeloid development, SGP genes are coordinately upregulated, and that members of the C/EBP family of transcription factors, in particular C/EBPalpha and C/EBPepsilon, play specific and unique roles in upregulating their expression.

Human platelets exhibit chemotaxis using functional N-formyl peptide receptors.

OBJECTIVE: Activated platelets participate in inflammatory and microbicidal processes by upregulation of surface selectins, shedding of CD40 ligand, and release of platelet microbicidal proteins and microparticles. Given their myeloid lineage, we hypothesized that platelets express functional N-formyl peptide receptors and respond to the bacterially derived chemotactic peptide N-formyl peptide with gradient-driven chemotaxis. METHODS AND RESULTS: Here we show specific binding of N-formyl peptides to the surface of activated platelets. Platelet expression and function of the formyl peptide receptor, FPR, was verified by RT-PCR of the differentiated megakaryocyte MEG-01 cell line, immunoblotting of platelet proteins, and calcium mobilization in platelets with formyl peptide binding. Furthermore, we demonstrate gradient-driven chemotaxis of platelets by video microscopy and transwell migration toward formyl peptides. We also show that endogenous formyl peptides, released by eukaryotic mitochondria from necrotic cells, induce chemotaxis using formyl peptide receptors expressed by thrombin-activated platelets. Conversely, supernatants from cells undergoing apoptotic cell death do not induce platelet chemotaxis. Platelet chemotaxis to formyl peptides was blocked with FPR-specific antibody as well as by pertussis toxin inhibition of the formyl peptide G-coupled receptor. CONCLUSION: These data establish a new role for platelets in host defense and suggest reexamination of their active function in microbicidal and other host defense activities.

Inhibition of human neutrophil IL-8 production by hydrogen peroxide and dysregulation in chronic granulomatous disease.

The innate immune response to bacterial infections includes neutrophil chemotaxis and activation, but regulation of inflammation is less well understood. Formyl peptides, byproducts of bacterial metabolism as well as mitochondrial protein biosynthesis, induce neutrophil chemotaxis, the generation of reactive oxygen intermediates (ROI), and the production of the neutrophil chemoattractant, IL-8. Patients with chronic granulomatous disease (CGD) exhibit deficient generation of ROI and hydrogen peroxide and susceptibility to bacterial and fungal pathogens, with associated dysregulated inflammation and widespread granuloma formation. We show in this study that in CGD cells, fMLF induces a 2- to 4-fold increase in IL-8 production and a sustained IL-8 mRNA response compared with normal neutrophils. Moreover, normal neutrophils treated with catalase (H(2)O(2) scavenger) or diphenyleneiodonium chloride (NADPH oxidase inhibitor) exhibit IL-8 responses comparable to those of CGD neutrophils. Addition of hydrogen peroxide or an H(2)O(2)-generating system suppresses the sustained IL-8 mRNA and increased protein production observed in CGD neutrophils. These results indicate that effectors downstream of the activation of NADPH oxidase negatively regulate IL-8 mRNA in normal neutrophils, and their absence in CGD cells results in prolonged IL-8 mRNA elevation and enhanced IL-8 levels. ROI may play a critical role in regulating inflammation through this mechanism.

Enhancement of hematopoietic stem cell repopulating capacity and self-renewal in the absence of the transcription factor C/EBP alpha.

The transcription factor C/EBP alpha is required for granulopoiesis and frequently disrupted in human acute myeloid leukemia (AML). Here, we show disruption of C/EBP alpha blocks the transition from the common myeloid to the granulocyte/monocyte progenitor but is not required beyond this stage for terminal granulocyte maturation. C/EBP alpha-deficient hematopoietic stem cells (HSCs) have increased expression of Bmi-1 and enhanced competitive repopulating activity. Bone marrow in adult C/EBP alpha-deficient mice was filled with myeloblasts, similar to human AML, supporting the notion that disruption of C/EBP alpha cooperates with other events in the development of leukemia. Therefore, C/EBP alpha is not only essential for granulocyte development but, in addition, is a regulator of hematopoietic stem cell activity.

Platelets deliver costimulatory signals to antigen-presenting cells: a potential bridge between injury and immune activation.

The danger model of immunity and tolerance holds that antigen-presenting cells (APCs), activated by stress, injury, or necrosis, but not by physiological (apoptotic) cell death, initiate adaptive immune responses. APC activation is fundamentally associated with binding of CD40 to its ligand CD154. Platelets express CD154 upon activation and are thus potential primal danger signals linking the homeostatic response to trauma to activation of the acquired immune system. Previously, we showed that platelets can undergo gradient-driven migration, or chemotaxis, toward supernatants from cells injured by repeated freeze/thaws, UV light, or ischemia/reperfusion. Herein, we demonstrate that platelet-derived CD154 induces immature dendritic cell maturation with upregulation of costimulatory molecules and IL-12p40 production. Overall, these results provide a mechanism for platelet activation of APC facilitating the induction of adaptive immunity in environments of cell injury.

Novel combinatorial interactions of GATA-1, PU.1, and C/EBPepsilon isoforms regulate transcription of the gene encoding eosinophil granule major basic protein.

GATA-1 and the ets factor PU.1 have been reported to functionally antagonize one another in the regulation of erythroid versus myeloid gene transcription and development. The CCAAT enhancer binding protein epsilon (C/EBPepsilon) is expressed as multiple isoforms and has been shown to be essential to myeloid (granulocyte) terminal differentiation. We have defined a novel synergistic, as opposed to antagonistic, combinatorial interaction between GATA-1 and PU.1, and a unique repressor role for certain C/EBPepsilon isoforms in the transcriptional regulation of a model eosinophil granulocyte gene, the major basic protein (MBP). The eosinophil-specific P2 promoter of the MBP gene contains GATA-1, C/EBP, and PU.1 consensus sites that bind these factors in nuclear extracts of the eosinophil myelocyte cell line, AML14.3D10. The promoter is transactivated by GATA-1 alone but is synergistically transactivated by low levels of PU.1 in the context of optimal levels of GATA-1. The C/EBPepsilon(27) isoform strongly represses GATA-1 activity and completely blocks GATA-1/PU.1 synergy. In vitro mutational analyses of the MBP-P2 promoter showed that both the GATA-1/PU.1 synergy, and repressor activity of C/EBPepsilon(27) are mediated via protein-protein interactions through the C/EBP and/or GATA-binding sites but not the PU.1 sites. Co-immunoprecipitations using lysates of AML14.3D10 eosinophils show that both C/EBPepsilon(32/30) and epsilon(27) physically interact in vivo with PU.1 and GATA-1, demonstrating functional interactions among these factors in eosinophil progenitors. Our findings identify novel combinatorial protein-protein interactions for GATA-1, PU.1, and C/EBPepsilon isoforms in eosinophil gene transcription that include GATA-1/PU.1 synergy and repressor activity for C/EBPepsilon(27).

C/EBPalpha is required for proteolytic cleavage of cyclin A by calpain 3 in myeloid precursor cells.

In this report, we present novel findings that implicate CCAAT/enhancer-binding protein (C/EBPalpha) in regulating the expression and activity of calpain 3 in vivo and data showing a new physiological substrate for calpain 3, cyclin A. Our results demonstrate that cleavage of cyclin A by calpain 3 occurs in mouse and human myeloid precursor cells. Calpain 3 cleaves cyclin A in vitro and in vivo, resulting in the production of a truncated product that lacks the N-terminal destruction box required for its degradation at the end of mitosis. The cleaved form of cyclin A retains the cyclin-dependent kinase (cdk) binding domain and forms active complexes with cdk2. Calpain 3-mediated cleavage of cyclin A is lacking in C/EBPalpha-/- mice, which are not able to produce mature granulocytes. Our data support a model in which calpain 3-mediated cleavage of cyclin A in dividing myeloid progenitor cells is important for the onset of differentiation. Deficits in this pathway in C/EBPalpha-/- mice might contribute to the failure of these mice to produce mature granulocytes. These data reveal a new pathway involving tightly controlled post-translational processing of cyclin A during differentiation of granulocytes.

Inherited Neutrophil Disorders: Molecular Basis and New Therapies.

Recent advances in our understanding of the molecular basis of inherited neutrophil disorders and complementary studies in transgenic mouse models have provided new insights into the normal mechanisms regulating myelopoiesis and the functional responses of mature neutrophils. Neutrophil specific granule deficiency is a rare disorder of neutrophil function characterized by a lack of neutrophil secondary granule proteins and associated with recurrent bacterial infections. The CCAAT/enhancer binding protein (C/EBP) epsilon, a leucine zipper transcription factor expressed primarily in myeloid cells, and C/EBPepsilon-deficient mice generated by gene targeting lack specific granules and have impaired host defense are discussed by Dr. Lekstrom-Himes in Section I. The similarity between these phenotypes led to the identification of a loss of function mutation in the C/EBPepsilon gene in a subset of patients with specific granule deficiency. Dr. Dale reviews the clinical features and management of congenital neutropenia and cyclic hematopoiesis in Section II. Inherited mutations in the neutrophil elastase gene have recently been identified in both disorders. Specific mutations identified in cyclic and congenital neutropenia are described along with possible mechanisms for regulation of hematopoiesis by neutrophil elastase. In Section III, Dr. Dinauer reviews the molecular genetics of chronic granulomatous disease and studies in knockout mouse models. This work has revealed important features of the regulation of the respiratory burst oxidase and its role in host defense and inflammation. Results from preclinical studies and phase 1 clinical trials for gene therapy for CGD are summarized, in addition to alternative approaches using allogeneic bone marrow transplantation with nonmyeloablative conditioning.