Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design.

The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from Ideonella sakaiensis carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.

Rugged fitness landscapes minimize promiscuity in the evolution of transcriptional repressors.

How a protein's function influences the shape of its fitness landscape, smooth or rugged, is a fundamental question in evolutionary biochemistry. Smooth landscapes arise when incremental mutational steps lead to a progressive change in function, as commonly seen in enzymes and binding proteins. On the other hand, rugged landscapes are poorly understood because of the inherent unpredictability of how sequence changes affect function. Here, we experimentally characterize the entire sequence phylogeny, comprising 1,158 extant and ancestral sequences, of the DNA-binding domain (DBD) of the LacI/GalR transcriptional repressor family. Our analysis revealed an extremely rugged landscape with rapid switching of specificity, even between adjacent nodes. Further, the ruggedness arises due to the necessity of the repressor to simultaneously evolve specificity for asymmetric operators and disfavors potentially adverse regulatory crosstalk. Our study provides fundamental insight into evolutionary, molecular, and biophysical rules of genetic regulation through the lens of fitness landscapes.

Evaluation for Bleeding Disorders in Suspected Child Abuse.

Bruising or bleeding in a child can raise the concern for child abuse. Assessing whether the findings are the result of trauma and/or whether the child has a bleeding disorder is critical. Many bleeding disorders are rare, and not every child with bruising/bleeding that may raise a concern for abuse requires an evaluation for bleeding disorders. However, in some instances, bleeding disorders can present in a manner similar to child abuse. Bleeding disorders cannot be ruled out solely on the basis of patient and family history, no matter how extensive. The history and clinical evaluation can be used to determine the necessity of an evaluation for a possible bleeding disorder, and prevalence and known clinical presentations of individual bleeding disorders can be used to guide the extent of laboratory testing. This clinical report provides guidance to pediatricians and other clinicians regarding the evaluation for bleeding disorders when child abuse is suspected.

Monitoring Hemostasis During Extracorporeal Life Support.

To balance the risk of bleeding versus circuit thrombosis during extracorporeal life support (ECLS), it is important to monitor anticoagulants and hemostasis. We evaluated the prothrombin time (PT), partial thromboplastin time (PTT), activated clotting time (ACT), and antifactor Xa heparin activity (aXa) correlation with changes in coagulation factor and heparin levels using in vitro and in vivo samples. aXa correlated with heparin (r = 0.97) and antithrombin (r = 0.98) but was unaffected by other parameters. PT correlated with coagulation factors (r = 0.88) but was minimally affected by heparin or other parameters. When single parameters were changed, ACT was insensitive to <0.5 U/ml heparin, correlated with coagulation factors (r = 0.99), and was affected by factor XII and platelets. When multiple parameters changed in vitro and in vivo, ACT was not correlated with heparin or coagulation factors. PTT correlated with heparin and coagulation factors individually but had low correlation when multiple parameters changed in vitro and in vivo. In conclusion, aXa is the most specific for heparin levels, and PT is most specific for coagulation factor levels making these assays well suited to monitor anticoagulation and hemostasis for patients on ECLS. PTT is highly variable when multiple parameters are changing in vitro and in vivo, but may be useful when aXa cannot be used because of interference. ACT is too insensitive to heparin, sensitive to too many other factors, and too imprecise to be useful for monitoring hemostasis during ECLS.

A Prospective Study of the Causes of Bruises in Premobile Infants.

OBJECTIVE: This study had 2 objectives. First, to determine the behavior of physicians evaluating premobile infants with bruises. Second, and most importantly, to learn whether infants with unexplained bruising who had been initially evaluated by primary care and emergency department (ED) physicians are as likely to have their bruises attributed to child abuse as those children evaluated by child abuse physicians. METHODS: Primary care, ED, and child abuse pediatricians (CAPs) in King County, Washington, San Mateo, Calif, Albuquerque, NM, La Crosse, Wis, and Torrance, Calif prospectively identified and studied infants younger than 6 months with less than 6 bruises, which were judged by the evaluating clinician to be explained or unexplained after their initial clinical examination. RESULTS: Between March 1, 2010, and March 1, 2017, 63 infants with initially explained and 46 infants with initially unexplained bruises were identified. Infants with unexplained bruises had complete coagulation and abuse evaluations less frequently if they were initially identified by primary care pediatricians or ED providers than by CAPs. After imaging, laboratory, and follow-up, 54.2% (26) of the infants with initially unexplained bruises, including 2 who had been initially diagnosed with accidental injuries, were diagnosed as abused. Three (6.2%) infants had accidental bruising, 6 (12.4%) abuse mimics, 1 (2.5%) self-injury, 1 (2.5%) medical injury, and 11 (22.9%) remained of unknown causation. None had causal coagulation disorders. A total of 65.4% of the 26 abused infants had occult injuries detected by their imaging and laboratory evaluations. Six (23.1%) abused infants were not diagnosed until after they sustained subsequent injuries. Three (11.5%) were recognized abused by police investigation alone. Thirty-eight percent of the abused, bruised infants had a single bruise. Clinicians' estimates of abuse likelihood based on their initial clinical evaluation were inaccurate. Primary care, ED, and child abuse physicians identified abused infants at similar rates. CONCLUSIONS: More than half of premobile infants with initially unexplained bruises were found to be abused. Abuse was as likely for infants identified by primary care and ED providers as for those identified by CAPs. Currently, physicians often do not obtain full abuse evaluations in premobile infants with unexplained bruising. Their initial clinical judgment about abuse likelihood was inadequate. Bruised infants often have clinically occult abusive injuries or will sustain subsequent serious abuse. Bruised infants should have full abuse evaluations and referral for Protective Services and police assessments.

The American Society of Pediatric Hematology/Oncology workforce assessment: Part 1-Current state of the workforce.

The American Society of Pediatric Hematology/Oncology (ASPHO) recognized recent changes in medical practice and the potential impact on pediatric hematology-oncology (PHO) workforce. ASPHO surveyed society members and PHO Division Directors between 2010 and 2016 and studied PHO workforce data collected by the American Board of Pediatrics and the American Medical Association to characterize the current state of the PHO workforce. The analysis of this information has led to a comprehensive description of PHO physicians, professional activities, and workplace. It is important to continue to collect data to identify changes in composition and needs of the PHO workforce.

The use of antifibrinolytics in pediatric patients with hypoproliferative thrombocytopenia.

Despite the use of evidence-based platelet transfusion therapy during periods of hypoproliferative thrombocytopenia, a large proportion of pediatric hematology/oncology patients continue to suffer from clinically significant bleeding. Antifibrinolytic (AF) drugs have been shown in certain surgical and trauma settings to decrease bleeding, blood transfusion, and improve survival. We conducted a retrospective assessment of the safety of using AF drugs in pediatric patients with hypoproliferative thrombocytopenia at our center as well as the impact on bleeding occurrence and severity.

T cells from hemophilia A subjects recognize the same HLA-restricted FVIII epitope with a narrow TCR repertoire.

Factor VIII (FVIII)-neutralizing antibodies ("inhibitors") are a serious problem in hemophilia A (HA). The aim of this study was to characterize HLA-restricted T-cell responses from a severe HA subject with a persistent inhibitor and from 2 previously studied mild HA inhibitor subjects. Major histocompatibility complex II tetramers corresponding to both of the severe HA subject's HLA-DRA-DRB1 alleles were loaded with peptides spanning FVIII-A2, C1, and C2 domains. Interestingly, only 1 epitope was identified, in peptide FVIII2194-2213, and it was identical to the HLA-DRA*01-DRB1*01:01-restricted epitope recognized by the mild HA subjects. Multiple T-cell clones and polyclonal lines having different avidities for the peptide-loaded tetramer were isolated from all subjects. Only high- and medium-avidity T cells proliferated and secreted cytokines when stimulated with FVIII2194-2213 T-cell receptor ß (TCRB) gene sequencing of 15 T-cell clones from the severe HA subject revealed that all high-avidity clones expressed the same TCRB gene. High-throughput immunosequencing of high-, medium-, and low-avidity cells sorted from a severe HA polyclonal line revealed that 94% of the high-avidity cells expressed the same TCRB gene as the high-avidity clones. TCRB sequencing of clones and lines from the mild HA subjects also identified a limited TCRB gene repertoire. These results suggest a limited number of epitopes in FVIII drive inhibitor responses and that the T-cell repertoires of FVIII-responsive T cells can be quite narrow. The limited diversity of both epitopes and TCRB gene usage suggests that targeting of specific epitopes and/or T-cell clones may be a promising approach to achieve tolerance to FVIII.

Eltrombopag for the treatment of children with persistent and chronic immune thrombocytopenia (PETIT): a randomised, multicentre, placebo-controlled study.

BACKGROUND: The oral thrombopoietin receptor agonist eltrombopag is approved for treatment of adults with chronic immune thrombocytopenia. In the PETIT trial, we aimed to investigate the efficacy and safety of eltrombopag in children with persistent or chronic immune thrombocytopenia. METHODS: PETIT was a three-part, randomised, multicentre, placebo-controlled study done at 22 centres in the USA, UK, Canada, Spain, France, and the Netherlands. Patients aged 1-17 years with immune thrombocytopenia lasting for 6 months or longer and platelets less than 30 × 10(9) per L who had received at least one previous treatment were enrolled. We enrolled patients into three cohorts consisting of patients aged 12-17, 6-11, and 1-5 years. We established patients' starting doses with an open-label, dose-finding phase with five patients in each cohort. During the dose-finding phase, patients aged 6-17 years started eltrombopag at 25 mg once per day (12·5 mg for those weighing <27 kg) and patients aged 1-5 years received 0·7 mg/kg per day to a maximum of 2 mg/kg unless otherwise approved. We permitted dose adjustments on the basis of platelet response up to a maximum dosage of 75 mg per day. Additional patients were then recruited and randomly assigned (2:1) to receive either eltrombopag or placebo tablets (or oral suspension formulation if aged 1-5 years) once per day for 7 weeks at the previously established doses. Starting doses for the double-blind phase were 37·5 mg/day for patients aged 12-17 years; 50 mg/day for patients weighing 27 kg or more (25 mg for east Asian patients) and 25 mg/day for patients weighing less than 27 kg (12·5 mg once per day for east Asian patients) for patients aged 6-11 years; and 1·5 mg/kg once per day (0·8 mg/kg once per day for east Asian patients) for patients aged 1-5 years. Randomisation was done by the GlaxoSmithKline Registration/Medication Ordering System and both patients and study personnel were masked to treatment assignments. Patients who completed treatment were then enrolled into an open-label phase and all patients could receive up to 24 weeks of eltrombopag. The primary outcome was the proportion of patients achieving a platelet count of 50 × 10(9) per L or more at least once from weeks 1-6 (days 8 to 43) of the randomised phase of the study in the absence of rescue therapy. We assessed efficacy in the intent-to-treat population, which consisted of all patients assigned to treatment, and we assessed safety in all patients who received at least one dose of study treatment. This trial is registered with ClinicalTrials.gov, number NCT00908037. FINDINGS: Between Oct 2, 2009, and June 22, 2011, we recruited 15 patients, with five patients in each age cohort, into the open-label dose-finding phase who did not progress into the double-blind phase. From March 17, 2010, to Jan 15, 2013, we randomly assigned 67 patients to treatment, with 45 patients assigned to receive eltrombopag (16 children aged 12-17 years, 19 aged 6-11 years, and ten aged 1-5 years) and 22 to receive placebo (eight children aged 12-17 years, nine aged 6-11 years, and five aged 1-5 years). However, two patients assigned to receive eltrombopag did not receive the study drug and one was lost to follow-up, and one patient assigned to receive placebo was given eltrombopag. From weeks 1 to 6, 28 (62%) patients who received eltrombopag, compared with seven (32%) who received placebo, achieved the primary endpoint of platelet count 50 × 10(9) per L or more at least once without rescue (odds ratio 4·31, 95% CI 1·39-13·34, p=0·011). The most common adverse events with eltrombopag were headache (13 [30%] patients receiving eltrombopag vs nine [43%] patients receiving placebo), upper respiratory tract infection (11 [25%] patients vs two [10%] patients), and diarrhoea (seven [16%] patients vs one [5%] patient). Grade 3 or 4 adverse events occurred in five (11%) patients receiving eltrombopag and four (19%) patients receiving placebo, and serious adverse events (four [9%] patients receiving eltrombopag and two (10%) patients receiving placebo) were similarly infrequent in both groups. No thrombotic events or malignancies occurred. Increased alanine aminotransferase concentrations caused two (3%) of 65 patients to discontinue eltrombopag in the open-label phase. INTERPRETATION: Our results showed that eltrombopag could be used to increase platelet counts and reduce clinically significant bleeding in children with persistent or chronic immune thrombocytopenia. Prevalence of increased liver laboratory values was similar to that seen in adults. FUNDING: GlaxoSmithKline.

Hemolytic disease of the fetus and newborn: managing the mother, fetus, and newborn.

Hemolytic disease of the fetus and newborn (HDFN) affects 3/100 000 to 80/100 000 patients per year. It is due to maternal blood group antibodies that cause fetal red cell destruction and in some cases, marrow suppression. This process leads to fetal anemia, and in severe cases can progress to edema, ascites, heart failure, and death. Infants affected with HDFN can have hyperbilirubinemia in the acute phase and hyporegenerative anemia for weeks to months after birth. The diagnosis and management of pregnant women with HDFN is based on laboratory and radiographic monitoring. Fetuses with marked anemia may require intervention with intrauterine transfusion. HDFN due to RhD can be prevented by RhIg administration. Prevention for other causal blood group specificities is less studied.

Congenital sideroblastic anemia due to mutations in the mitochondrial HSP70 homologue HSPA9.

The congenital sideroblastic anemias (CSAs) are relatively uncommon diseases characterized by defects in mitochondrial heme synthesis, iron-sulfur (Fe-S) cluster biogenesis, or protein synthesis. Here we demonstrate that mutations in HSPA9, a mitochondrial HSP70 homolog located in the chromosome 5q deletion syndrome 5q33 critical deletion interval and involved in mitochondrial Fe-S biogenesis, result in CSA inherited as an autosomal recessive trait. In a fraction of patients with just 1 severe loss-of-function allele, expression of the clinical phenotype is associated with a common coding single nucleotide polymorphism in trans that correlates with reduced messenger RNA expression and results in a pseudodominant pattern of inheritance.

Factor VIII gene variants and inhibitor risk in African American hemophilia A patients.

African American hemophilia A (HA) patients experience a higher incidence of neutralizing anti-factor VIII (FVIII) antibodies ("inhibitors") vis-à-vis white patients. Nonsynonymous single-nucleotide polymorphisms (ns-SNPs) in the F8 gene encoding FVIII-H484, FVIII-E1241, and FVIII-V2238 are more prevalent in African Americans. This study tested the hypothesis that immune responses to these sites provoke inhibitors. Blood samples were obtained from 174 African American and 198 white HA subjects and their F8 gene sequences determined. Major histocompatibility complex class II binding and T-cell recognition of polymorphic sequences were evaluated using quantitative binding assays and HLA-DRB1 tetramers. Peptides corresponding to 4 common ns-SNPs showed limited binding to 11 HLA-DRB1 proteins. CD4 T cells from 22 subjects treated with FVIII products having sequences at residues FVIII-484, 1241, and 2238 differing from those of putative proteins encoded by their F8 genes did not show high-avidity tetramer binding, whereas positive-control staining of tetanus-specific CD4 T cells was routinely successful. African Americans with an intron-22 inversion mutation showed a 2-3 times-higher inhibitor incidence than whites with the same mutation (odds ratio = 2.3 [1.1-5.0, P = .04]), but this did not correlate with any of the ns-SNPs. We conclude that immune responses to "sequence-mismatched" FVIII products are unlikely to contribute appreciably to the inhibitor incidence in African Americans.

Antithrombin concentrates use in children on extracorporeal membrane oxygenation: a retrospective cohort study.

OBJECTIVE: To investigate whether receipt of any antithrombin concentrate improves laboratory and clinical outcomes in children undergoing extracorporeal membrane oxygenation for respiratory failure during their hospitalization compared with those who did not receive antithrombin. DESIGN: Retrospective cohort study. SETTING: Single, tertiary-care pediatric hospital. PATIENTS: Sixty-four neonatal and pediatric patients who underwent extracorporeal membrane oxygenation for respiratory failure between January 2007 and September 2011. INTERVENTION: Exposure to any antithrombin concentrate during their extracorporeal membrane oxygenation course compared with similar children who never received antithrombin concentrate. MEASUREMENTS AND MAIN RESULTS: Thirty patients received at least one dose of antithrombin during their extracorporeal membrane oxygenation course and 34 patients did not receive any. The median age at admission was less than 1-month old. Age, duration of extracorporeal membrane oxygenation, or first antithrombin level did not differ significantly between the two cohorts. The mean plasma antithrombin level in those who never received antithrombin was 42.2% compared with 66% in those who received it. However, few levels reached the targeted antithrombin level of 120% and those who did fell back to deficient levels within an average of 6.8 hours. For those who received antithrombin concentrate, heparin infusion rates decreased by an average of 10.2 U/kg/hr for at least 12 hours following administration. No statistical differences were noted in the number of extracorporeal membrane oxygenation circuit changes, in vivo clots or hemorrhages, transfusion requirements, hospital or ICU length of stay, or in-hospital mortality. CONCLUSIONS: Intermittent, on-demand dosing of antithrombin concentrate in pediatric patients on extracorporeal membrane oxygenation for respiratory failure increased antithrombin levels, but not typically to the targeted level. Patients who received antithrombin concentrate also had decreased heparin requirements for at least 12 hours after dosing. However, no differences were noted in the measured clinical endpoints. A prospective, randomized study of this intervention may require different dosing strategies; such a study is warranted given the unproven efficacy of this costly product.

Inherited disorders of platelet function.

Inherited platelet function disorders are of variable severity and unknown frequency and may be difficult to diagnose. Nevertheless, they are increasingly recognized as an important cause of bleeding in pediatrics, particularly in adolescent girls with menorrhagia, where they may be more common than von Willebrand disease. This article reviews the presentation of these disorders, summarizes the most common types of platelet function disorders, discusses the challenges in diagnostic testing, and details treatment and supportive care options.

Fanconi anemia-like presentation in an infant with constitutional deletion of 21q including the RUNX1 gene.

We describe a newborn female with a de novo interstitial deletion of chromosome 21q21.1-22.12 including the RUNX1 gene who had developmental delay, multiple congenital anomalies, tetralogy of Fallot, anemia, and chronic thromobocytopenia requiring frequent platelet transfusions from birth. Because of her physical and hematologic abnormalities, she was tested for Fanconi anemia (FA). Lymphocytes and fibroblasts from this patient demonstrated increased chromosome breakage with exposure to the clastogen mitomycin C, but not, in contrast to most FA patients, to diepoxybutane. Further testing by Western analysis and complementation testing did not show a defect in the function of known Fanconi proteins. Her constitutional deletion was later found to span 13.2 Mb by chromosome microarray analysis, encompassing the RUNX1 gene that has been implicated in thrombocytopenia and predisposition to acute myelogenous leukemia (AML) when in the haploinsufficient state. We compare her phenotype to other individuals with similar 21q deletions and thrombocytopenia, as well as those with FA. We suggest that deletion of RUNX1 or another critical gene within the deleted region may result in chromosomal instability similar to that seen in FA.

Successful unrelated donor cord blood transplantation for Glanzmann's thrombasthenia.

GT, a rare disorder of platelet function, can lead to life-threatening bleeding, particularly following the development of antiplatelet antibodies. Curative therapy includes HCT but previous reports are limited predominantly to matched siblings and have excluded CBT. Delayed or non-engraftment of platelets because of antiplatelet antibodies might be particularly concerning after CBT for GT. Here, we report two successful unrelated cord blood transplants for GT. Recurrent life-threatening bleeding was the primary indication for HCT, with one patient developing antiplatelet antibodies pre-HCT. Bleeding risks associated with delivery of the conditioning regimen and the toxicity that follows should be carefully considered, including tunneled central venous line catheter placement, inclusion of B cell-specific therapy to potentially decrease antiplatelet antibody production, and targeted busulfan dosing. This is the first report of successful unrelated cord blood HCT for GT and indicates that modifications to supportive care can improve the safety of this potentially curative therapy for patients with severe, life-threatening disease manifestations.

Allogeneic hematopoietic cell transplantation after conditioning with 131I-anti-CD45 antibody plus fludarabine and low-dose total body irradiation for elderly patients with advanced acute myeloid leukemia or high-risk myelodysplastic syndrome.

We conducted a study to estimate the maximum tolerated dose (MTD) of (131)I-anti-CD45 antibody (Ab; BC8) that can be combined with a standard reduced-intensity conditioning regimen before allogeneic hematopoietic cell transplantation. Fifty-eight patients older than 50 years with advanced acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS) were treated with (131)I-BC8 Ab and fludarabine plus 2 Gy total body irradiation. Eighty-six percent of patients had AML or MDS with greater than 5% marrow blasts at the time of transplantation. Treatment produced a complete remission in all patients, and all had 100% donor-derived CD3(+) and CD33(+) cells in the blood by day 28 after the transplantation. The MTD of (131)I-BC8 Ab delivered to liver was estimated to be 24 Gy. Seven patients (12%) died of nonrelapse causes by day 100. The estimated probability of recurrent malignancy at 1 year is 40%, and the 1-year survival estimate is 41%. These results show that CD45-targeted radiotherapy can be safely combined with a reduced-intensity conditioning regimen to yield encouraging overall survival for older, high-risk patients with AML or MDS. This study was registered at www.clinicaltrials.gov as #NCT00008177.

Pretargeted radioimmunotherapy using anti-CD45 monoclonal antibodies to deliver radiation to murine hematolymphoid tissues and human myeloid leukemia.

Radioimmunotherapy (RIT) for treatment of hematologic malignancies frequently fails because of disease recurrence. We therefore conducted pretargeted (P)RIT studies to augment the efficacy in mice of therapy using a pretargeted anti-human (h)CD45 antibody (Ab)-streptavidin (SA) conjugate followed by a biotinylated clearing agent and radiolabeled 1,4,7,10-tetraazacylodode cane N,N',N",N'''-tetraacetic (DOTA)-biotin. Tumor-to-blood ratios at 24 hours were 20:1 using pretargeted anti-hCD45 RIT and <1:1 with conventional RIT. In vivo imaging studies confirmed that the PRIT approach provided high-contrast tumor images with minimal blood-pool activity, whereas directly labeled anti-hCD45 Ab produced distinct tumor images but the blood pool retained a large amount of labeled Ab for a prolonged time. Therapy experiments showed that (90)Y-DOTA-biotin significantly prolonged survival of mice treated with pretargeted anti-hCD45 Ab-SA compared with mice treated with conventional RIT using (90)Y-labeled anti-hCD45 Ab at 200 muCi. Because human CD45 antigens are confined to xenograft tumor cells in this model, and all murine tissues are devoid of hCD45 and will not bind anti-hCD45 Ab, we also compared one-step and PRIT using an anti-murine (m)CD45 Ab where the target antigen is present on normal hematopoietic tissues. After 24 h, 27.3% +/- 2.8% of the injected dose of activity was delivered per gram (% ID/g) of lymph node using (131)I-A20-Ab compared with 40.0 +/- 5.4% ID/g for pretargeted (111)In-DOTA-biotin. These data suggest that pretargeted methods for delivering RIT may be superior to conventional RIT when targeting CD45 for the treatment of leukemia and may allow for the intensification of therapy, while minimizing toxicities.

Parallel synthesis and screening of polymers for nonviral gene delivery.

We describe the parallel synthesis and in vitro evaluation of a cationic polymer library for the discovery of nonviral gene delivery vectors. The library was synthesized based on the ring-opening polymerization reaction between epoxide groups of diglycidyl ethers and the amines of (poly)amines. Parallel screening of soluble library constituents led to the identification of lead polymers with high DNA-binding efficacies. Transfection efficacies of lead polymers were evaluated using PC3-PSMA human prostate cancer cells and murine osteoblasts in the absence and presence of serum. In vitro experiments resulted in the identification of a candidate polymer that demonstrated significantly higher transfection efficacies and lower cytotoxicities than poly(ethyleneimine) (pEI), the current standard for polymeric transfection agents. In addition, polymers that demonstrated moderately higher and comparable transfection efficacies with respect to pEI were also identified. Our results demonstrate that high-throughput synthesis and screening of polymers is a powerful approach for the identification of novel nonviral gene delivery agents.