High-level transgene expression in induced pluripotent stem cell-derived megakaryocytes: correction of Glanzmann thrombasthenia.

Megakaryocyte-specific transgene expression in patient-derived induced pluripotent stem cells (iPSCs) offers a new approach to study and potentially treat disorders affecting megakaryocytes and platelets. By using a Gp1ba promoter, we developed a strategy for achieving a high level of protein expression in human megakaryocytes. The feasibility of this approach was demonstrated in iPSCs derived from two patients with Glanzmann thrombasthenia (GT), an inherited platelet disorder caused by mutations in integrin αIIbß3. Hemizygous insertion of Gp1ba promoter-driven human αIIb complementary DNA into the AAVS1 locus of iPSCs led to high αIIb messenger RNA and protein expression and correction of surface αIIbß3 in megakaryocytes. Agonist stimulation of these cells displayed recovery of integrin αIIbß3 activation. Our findings demonstrate a novel approach to studying human megakaryocyte biology as well as functional correction of the GT defect, offering a potential therapeutic strategy for patients with diseases that affect platelet function.

Platelet gene therapy improves hemostatic function for integrin alphaIIbbeta3-deficient dogs.

Activated blood platelets mediate the primary response to vascular injury. Although molecular abnormalities of platelet proteins occur infrequently, taken collectively, an inherited platelet defect accounts for a bleeding diathesis in ≈1:20,000 individuals. One rare example of a platelet disorder, Glanzmann thrombasthenia (GT), is characterized by life-long morbidity and mortality due to molecular abnormalities in a major platelet adhesion receptor, integrin αIIbß3. Transfusion therapy is frequently inadequate because patients often generate antibodies to αIIbß3, leading to immune-mediated destruction of healthy platelets. In the most severe cases allogeneic bone marrow transplantation has been used, yet because of the risk of the procedure it has been limited to few patients. Thus, hematopoietic stem cell gene transfer was explored as a strategy to improve platelet function within a canine model for GT. Bleeding complications necessitated the use of a mild pretransplant conditioning regimen; therefore, in vivo drug selection was used to improve engraftment of autologously transplanted cells. Approximately 5,000 αIIbß3 receptors formed on 10% of platelets. These modest levels allowed platelets to adhere to αIIbß3's major ligand (fibrinogen), form aggregates, and mediate retraction of a fibrin clot. Remarkably, improved hemostatic function was evident, with ≤135-fold reduced blood loss, and improved buccal bleeding times decreased to 4 min for up to 5 y after transplant. One of four transplanted dogs developed a significant antibody response to αIIbß3 that was attenuated effectively with transient immune suppression. These results indicate that gene therapy could become a practical approach for treating inherited platelet defects.

A single F153Sß3 mutation causes constitutive integrin αIIbß3 activation in a variant form of Glanzmann thrombasthenia.

This report identifies a novel variant form of the inherited bleeding disorder Glanzmann thrombasthenia, exhibiting only mild bleeding in a physically active individual. The platelets cannot aggregate ex vivo with physiologic agonists of activation, although microfluidic analysis with whole blood displays moderate ex vivo platelet adhesion and aggregation consistent with mild bleeding. Immunocytometry shows reduced expression of αIIbß3 on quiescent platelets that spontaneously bind/store fibrinogen, and activation-dependent antibodies (ligand-induced binding site-319.4 and PAC-1) report ß3 extension suggesting an intrinsic activation phenotype. Genetic analysis reveals a single F153Sß3 substitution within the ßI-domain from a heterozygous T556C nucleotide substitution of ITGB3 exon 4 in conjunction with a previously reported IVS5(+1)G>A splice site mutation with undetectable platelet messenger RNA accounting for hemizygous expression of S153ß3. F153 is completely conserved among ß3 of several species and all human ß-integrin subunits suggesting that it may play a vital role in integrin structure/function. Mutagenesis of αIIb-F153Sß3 also displays reduced levels of a constitutively activated αIIb-S153ß3 on HEK293T cells. The overall structural analysis suggests that a bulky aromatic, nonpolar amino acid (F,W)153ß3 is critical for maintaining the resting conformation of α2- and α1-helices of the ßI-domain because small amino acid substitutions (S,A) facilitate an unhindered inward movement of the α2- and α1-helices of the ßI-domain toward the constitutively active αIIbß3 conformation, while a bulky aromatic, polar amino acid (Y) hinders such movements and restrains αIIbß3 activation. The data collectively demonstrate that disruption of F153ß3 can significantly alter normal integrin/platelet function, although reduced expression of αIIb-S153ß3 may be compensated by a hyperactive conformation that promotes viable hemostasis.

Regulatory polymorphisms and their contribution to interindividual differences in the expression of enzymes influencing drug and toxicant disposition.

The unexpected paucity of human protein encoding genes suggested that polymorphisms altering gene expression might be more important than initially thought. From an evolutionary perspective, traits such as xenobiotic metabolism and transport that require a dynamic response to environmental changes would evolve more efficiently through variation in regulatory sequences versus coding variants. Such variation would be manifest as co-dominant traits and selection pressures would operate more efficiently because of their ability to impact fitness in the heterozygous state. Our current understanding of regulatory polymorphisms impacting drug disposition is reviewed including specific discussion regarding knowledge gaps and future research opportunities.

Flavin-containing monooxygenase genetic polymorphism: impact on chemical metabolism and drug development.

The flavin-containing monooxygenases (FMOs) metabolize a broad range of therapeutics. Consisting of five gene products in humans (FMO1-5), the different FMO family members exhibit pronounced tissue- and temporal-specific expression patterns. Substantial interindividual differences are also observed, and the inability to modulate with exogenous agents is consistent with an important role for genetic variation. Several rare FMO3 alleles causative for trimethylaminuria have been well characterized. However, the identification and characterization of functional FMO1-5 polymorphisms has been more recent. Although none of these polymorphisms has been associated with an adverse drug reaction, the continued broadening of our therapeutic armamentarium makes such an event likely in the future. Furthermore, at least one example has been reported for a direct association between FMO3 polymorphism and therapeutic efficacy. Thus, it is anticipated that knowledge regarding functionally-relevant FMO genetic variability will become increasingly important for making drug development and patient therapeutic choices.

Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A.

It is essential to improve therapies for controlling excessive bleeding in patients with haemorrhagic disorders. As activated blood platelets mediate the primary response to vascular injury, we hypothesize that storage of coagulation Factor VIII within platelets may provide a locally inducible treatment to maintain haemostasis for haemophilia A. Here we show that haematopoietic stem cell gene therapy can prevent the occurrence of severe bleeding episodes in dogs with haemophilia A for at least 2.5 years after transplantation. We employ a clinically relevant strategy based on a lentiviral vector encoding the ITGA2B gene promoter, which drives platelet-specific expression of human FVIII permitting storage and release of FVIII from activated platelets. One animal receives a hybrid molecule of FVIII fused to the von Willebrand Factor propeptide-D2 domain that traffics FVIII more effectively into α-granules. The absence of inhibitory antibodies to platelet-derived FVIII indicates that this approach may have benefit in patients who reject FVIII replacement therapies. Thus, platelet FVIII may provide effective long-term control of bleeding in patients with haemophilia A.

Identification and functional analysis of common human flavin-containing monooxygenase 3 genetic variants.

Flavin-containing monooxygenases (FMOs) are important for the disposition of many therapeutics, environmental toxicants, and nutrients. FMO3, the major adult hepatic FMO enzyme, exhibits significant interindividual variation. Eighteen FMO3 single-nucleotide polymorphism (SNP) frequencies were determined in 202 Hispanics (Mexican descent), 201 African Americans, and 200 non-Latino whites. Using expressed recombinant enzyme with methimazole, trimethylamine, sulindac, and ethylenethiourea, the novel structural variants FMO3 E24D and K416N were shown to cause modest changes in catalytic efficiency, whereas a third novel variant, FMO3 N61K, was essentially devoid of activity. The latter variant was present at an allelic frequency of 5.2% in non-Latino whites and 3.5% in African Americans, but it was absent in Hispanics. Inferring haplotypes using PHASE, version 2.1, the greatest haplotype diversity was observed in African Americans followed by non-Latino whites and Hispanics. Haplotype 2A and 2B, consisting of a hypermorphic promoter SNP cluster (-2650C>G, -2543T>A, and -2177G>C) in linkage with synonymous structural variants was inferred at a frequency of 27% in the Hispanic population, but only 5% in non-Latino whites and African Americans. This same promoter SNP cluster in linkage with one or more hypomorphic structural variant also was inferred in multiple haplotypes at a total frequency of 5.6% in the African-American study group but less than 1% in the other two groups. The sum frequencies of the hypomorphic haplotypes H3 [15,167G>A (E158K)], H5B [-2650C>G, 15,167G>A (E158K), 21,375C>T (N285N), 21,443A>G (E308G)], and H6 [15,167G>A (E158K), 21,375C>T (N285N)] was 28% in Hispanics, 23% in non-Latino whites, and 24% in African Americans.

Developmental expression of aryl, estrogen, and hydroxysteroid sulfotransferases in pre- and postnatal human liver.

Aryl- (SULT1A1), estrogen- (SULT1E1), and hydroxysteroid- (SULT2A1) sulfotransferases (SULTs) are active determinants of xenobiotic detoxication and hormone metabolism in the adult human liver. To investigate the role of these conjugating enzymes in the developing human liver, the ontogeny of immunoreactive SULT1A1, SULT1E1, and SULT2A1 expression was characterized in a series of 235 pre- and postnatal human liver cytosols ranging in age from early gestation to a postnatal age of 18 years. Interindividual variability in expression levels was apparent for all three SULTs in pre- and postnatal liver samples. Expression of the three SULTs displayed distinctly different developmental profiles. Semiquantitative Western blot analyses indicated that SULT1A1 and SULT2A1 immunoreactive protein levels were readily detectable in the majority of developmental human liver cytosols throughout the prenatal period. Whereas SULT1A1 expression did not differ significantly among the various developmental stages, SULT2A1 expression increased during the third trimester of gestation and continued to increase during postnatal life. By contrast, SULT1E1, a cardinal estrogen-inactivating enzyme, achieved the highest levels of expression during the earliest periods of gestation in prenatal male livers, indicating a requisite role for estrogen inactivation in the developing male. The present analysis suggests that divergent regulatory mechanisms are responsible for the differential patterns of hepatic SULT1A1, SULT1E1, and SULT2A1 immunoreactive protein levels that occur during pre- and postnatal human development, and implicates a major role for sulfotransferase expression in the developing fetus.

Enzyme-mediated protein haptenation of dapsone and sulfamethoxazole in human keratinocytes: II. Expression and role of flavin-containing monooxygenases and peroxidases.

Arylamine compounds, such as sulfamethoxazole (SMX) and dapsone (DDS), are metabolized in epidermal keratinocytes to arylhydroxylamine metabolites that auto-oxidize to arylnitroso derivatives, which in turn bind to cellular proteins and can act as antigens/immunogens. Previous studies have demonstrated that neither cytochromes P450 nor cyclooxygenases mediate this bioactivation in normal human epidermal keratinocytes (NHEKs). In this investigation, we demonstrated that methimazole (MMZ), a prototypical substrate of the flavin-containing monooxygenases (FMOs), attenuated the protein haptenation observed in NHEKs exposed to SMX or DDS. In addition, recombinant FMO1 and FMO3 were able to bioactivate both SMX and DDS, resulting in covalent adduct formation. Western blot analysis confirmed the presence of FMO3 in NHEKs, whereas FMO1 was not detectable. In addition to MMZ, 4-aminobenzoic acid hydrazide (ABH) also attenuated SMX- and DDS-dependent protein haptenation in NHEKs. ABH did not alter the bioactivation of these drugs by recombinant FMO3, suggesting its inhibitory effect in NHEKs was due to its known ability to inhibit peroxidases. Studies confirmed the presence of peroxidase activity in NHEKs; however, immunoblot analysis and reverse transcription-polymerase chain reaction indicated that myeloperoxidase, lactoperoxidase, and thyroid peroxidase were absent. Thus, our results suggest an important role for FMO3 and yet-to-be identified peroxidases in the bioactivation of sulfonamides in NHEKs.

Discovery of novel flavin-containing monooxygenase 3 (FMO3) single nucleotide polymorphisms and functional analysis of upstream haplotype variants.

The flavin-containing monooxygenases (FMOs) are important for xenobiotic metabolism. FMO3, the predominant FMO enzyme in human adult liver, exhibits significant interindividual variation that is poorly understood. This study was designed to identify common FMO3 genetic variants and determine their potential for contributing to interindividual differences in FMO3 expression. FMO3 single nucleotide polymorphism (SNP) discovery was accomplished by resequencing DNA samples from the Coriell Polymorphism Discovery Resource. Population-specific SNP frequencies were determined by multiplexed, single-base extension using DNA from 201 Hispanic American (Mexican descent), 201 African American, and 200 White (northern European descent) subjects. Haplotypes were inferred and population frequencies estimated using PHASE version 2.1. Multiple site-directed mutagenesis was used to introduce inferred upstream haplotypes into an FMO3/luciferase construct for functional analysis in HepG2 cells. Sequence analysis revealed seven FMO3 upstream SNPs, 11 exon SNPs, and 22 intron SNPs. Five of the latter fell within consensus splice sites. A g.72G>T variant (E24D) is predicted to impact the structure of the Rossmann fold involved in FAD binding, whereas a g.11177C>A variant (N61K) is predicted to disrupt the secondary structure of a conserved membrane interaction domain. Seven common (>1%) promoter region haplotypes were inferred in one or more of the study populations that differed in estimated frequency among the groups. Haplotype 2 resulted in an 8-fold increase in promoter activity, whereas haplotypes 8 and 15 exhibited a near complete loss of activity. In conclusion, FMO3 promoter haplotype variants modulate gene function and probably contribute to interindividual differences in FMO3 expression.

Human hepatic flavin-containing monooxygenases 1 (FMO1) and 3 (FMO3) developmental expression.

The flavin-containing monooxygenases (FMOs) are important for the metabolism of numerous therapeutics and toxicants. Six mammalian FMO genes (FMO1-6) have been identified, each exhibiting developmental and tissue- and species-specific expression patterns. Previous studies demonstrated that human hepatic FMO1 is restricted to the fetus whereas FMO3 is the major adult isoform. These studies failed to describe temporal expression patterns, the precise timing of the FMO1/FMO3 switch, or potential control mechanisms. To address these questions, FMO1 and FMO3 were quantified in microsomal fractions from 240 human liver samples representing ages from 8 wk gestation to 18 y using Western blotting. FMO1 expression was highest in the embryo (8-15 wk gestation; 7.8 +/- 5.3 pmol/mg protein). Low levels of FMO3 expression also were detectable in the embryo, but not in the fetus. FMO1 suppression occurred within 3 d postpartum in a process tightly coupled to birth, but not gestational age. The onset of FMO3 expression was highly variable, with most individuals failing to express this isoform during the neonatal period. FMO3 was detectable in most individuals by 1-2 y of age and was expressed at intermediate levels until 11 y (12.7 +/- 8.0 pmol/mg protein). These data suggest that birth is necessary, but not sufficient for the onset of FMO3 expression. A gender-independent increase in FMO3 expression was observed from 11 to 18 y of age (26.9 +/- 8.6 pmol/mg protein). Finally, 2- to 20-fold interindividual variation in FMO1 and FMO3 protein levels were observed, depending on the age bracket.

Human hepatic CYP2E1 expression during development.

Human hepatic CYP2E1 expression developmental changes likely have an impact on the effects of xenobiotics metabolized by the encoded enzyme. To resolve previous conflicting results, CYP2E1 content was determined in human hepatic microsomes from samples spanning fetal (n = 73, 8-37 weeks) and postnatal (n = 165, 1 day-18 years) ages. Measurable immunodetectable CYP2E1 was seen in 18 of 49 second-trimester (93-186 gestational days) and 12 of 15 third-trimester (>186 days) fetal samples (medians = 0.35 and 6.7 pmol/mg microsomal protein, respectively). CYP2E1 in neonatal samples was low and less than that of infants 31 to 90 days of age, which was less than that of older infants, children, and young adults [median (range) = 8.8 (0-70); 23.8 (10-43); 41.4 (18-95) pmol/mg microsomal protein, respectively; each P < 0.001, analysis of variance, post hoc]. Among those older than 90 days of age, CYP2E1 content was similar. A 4-fold or greater intersubject variation was observed among samples from each age group, with the greatest variation, 80-fold, seen among neonatal samples. Among subjects of known gestational and postnatal age (n = 29) increasing protein content was associated with increasing postnatal age (P < 0.001, linear regression), but only equivocally with increasing gestational age (P = 0.07). Individuals from the third trimester through 90 days postnatal age with one or more CYP2E1*1D alleles had lower CYP2E1 protein content than similar-aged subjects who were homozygous CYP2E1*1C. In summary, CYP2E1 was clearly expressed in human fetal liver. Furthermore, the postnatal data suggest that infants less than 90 days old would have decreased clearance of CYP2E1 substrates compared with older infants, children, and adults.

Genetic variability at the human FMO1 locus: significance of a basal promoter yin yang 1 element polymorphism (FMO1*6).

The flavin-containing monooxygenases (FMOs) are important for the disposition of a variety of toxicants, therapeutics, and dietary components. Although FMO1 is the dominant isoform in fetal liver and adult kidney and intestine and despite up to a 10-fold intersubject variation in expression, a paucity of information is available on FMO1 genetic variability. To address this issue, 24 samples from the Coriell DNA Polymorphism Discovery Resource Panel were sequenced revealing 10 common single nucleotide polymorphisms (SNPs): four located upstream of the structural gene; three within exonic sequences; one within the intron 1 splice donor site; and two with the 3'-untranslated region. Six of these variants are novel. Compared with other FMO loci within the chromosome 1q23-25 cluster, FMO1 seems more highly conserved. Of the identified FMO1 SNPs, only a C>A transversion 9536 base pairs upstream of the exon 2 ATG start codon (g.-9536C>A) would likely affect function, because it lies within the conserved core binding sequence for the yin yang 1 (YY1) transcription factor. Electrophoretic mobility shift assays demonstrated that the g.-9536C>A transversion eliminated YY1 binding. Furthermore, data from transient expression assays in HepG2 cells suggested this SNP could account for a 2- to 3-fold loss of FMO1 promoter activity. Genotype analysis revealed a g.-9,536A allele (FMO1*6) frequency of 13 and 11% in African- and northern European-Americans, respectively, but a significantly higher frequency of 30% in Hispanic-Americans. Thus, the FMO1*6 variant may account for some of the observed interindividual variation in FMO1 expression.

Developmental expression of the major human hepatic CYP3A enzymes.

The human cytochrome P4503A forms show expression patterns subject to developmental influence. CYP3A7 and CYP3A4 are generally classified as the major fetal and adult liver forms, respectively. However, characterization of CYP3A4, -3A5, and -3A7 developmental expression has historically been confounded by the lack of CYP3A isoform-specific antibodies or marker enzyme activities. Therefore, the objective of this study was to characterize the developmental expression of hepatic CYP3A forms from early gestation to 18 years of age using up to 212 fetal and pediatric liver samples. Based on immunoquantitation, CYP3A5 protein expression was found to be highly variable, generally independent of age, and more frequently observed for African-American individuals. For differentiation of CYP3A4 and -3A7 levels, dehydroepiandrosterone metabolite patterns for expressed CYP3A forms were characterized and used for simultaneous quantitation of protein levels within liver microsome samples. The major metabolite formed by CYP3A4, 7beta-hydroxy-dehydroepiandrosterone, was identified based on cochromatography and mass spectra matching with the authentic standard. Kinetic analysis showed a 34-fold greater intrinsic clearance of 7beta-hydroxy-dehydroepiandrosterone by CYP3A4 versus -3A7, whereas CYP3A7 showed the highest 16alpha-hydroxy-dehydroepiandrosterone intrinsic clearance. Metabolite profiles for the expressed enzymes were fit to a multiple response model and CYP3A4 and -3A7 levels in fetal and pediatric liver microsome samples were calculated. Fetal liver microsomes showed extremely high CYP3A7 levels (311-158 pmol/mg protein) and significant expression through 6 months postnatal age. Low CYP3A4 expression was noted for fetal liver (< or =10 pmol/mg), with mean levels increasing with postnatal age.

Developmental expression of human hepatic CYP2C9 and CYP2C19.

The CYP2C subfamily is responsible for metabolizing many important drugs and accounts for about 20% of the cytochrome p450 in adult liver. To determine developmental expression patterns, liver microsomal CYP2C9 and -2C19 were measured (n = 237; ages, 8 weeks gestation-18 years) by Western blotting and with diclofenac or mephenytoin, respectively, as probe substrates. CYP2C9-specific content and catalytic activity were consistent with expression at 1 to 2% of mature values (i.e., specific content, 18.3 pmol/mg protein and n = 79; specific activity, 549.5 pmol/mg/min and n = 72) during the first trimester, with progressive increases during the second and third trimesters to levels approximately 30% of mature values. From birth to 5 months, CYP2C9 protein values varied 35-fold and were significantly higher than those observed during the late fetal period, with 51% of samples exhibiting values commensurate with mature levels. Less variable CYP2C9 protein and activity values were observed between 5 months and 18 years. CYP2C19 protein and catalytic activities that were 12 to 15% of mature values (i.e., specific content, 14.6 pmol/mg and n = 20; specific activity, 18.5 pmol/mg/min and n = 19) were observed as early as 8 weeks of gestation and were similar throughout the prenatal period. CYP2C19 expression did not change at birth, increased linearly over the first 5 postnatal months, and varied 21-fold from 5 months to 10 years. Adult CYP2C19 protein and activity values were observed in samples older than 10 years. The ontogeny of CYP2C9 and -2C19 were dissimilar among both fetal and 0- to 5-months postnatal samples, implying different developmental regulatory mechanisms.