Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors.

Hemophilia B, or factor IX deficiency, is an X-linked recessive disorder occurring in about 1 in 25,000 males. Affected individuals are at risk for spontaneous bleeding into many organs; treatment mainly consists of the transfusion of clotting factor concentrates prepared from human blood or recombinant sources after bleeding has started. Small- and large-animal models have been developed and/or characterized that closely mimic the human disease state. As a preclinical model for gene therapy, recombinant adeno-associated viral vectors containing the human or canine factor IX cDNAs were infused into the livers of murine and canine models of hemophilia B, respectively. There was no associated toxicity with infusion in either animal model. Constitutive expression of factor IX was observed, which resulted in the correction of the bleeding disorder over a period of over 17 months in mice. Mice with a steady-state concentration of 25% of the normal human level of factor IX had normal coagulation. In hemophilic dogs, a dose of rAAV that was approximately 1/10 per body weight that given to mice resulted in 1% of normal canine factor IX levels, the absence of inhibitors, and a sustained partial correction of the coagulation defect for at least 8 months.

Cloning and expression of the cDNA for human gamma-glutamyl carboxylase.

The cDNA for human gamma-glutamyl carboxylase, which accomplishes the post-translational modification required for the activity of all of the vitamin K-dependent proteins, was cloned. The enzyme is a 758-residue integral membrane protein and appears to have three transmembrane domains near its amino terminus. The hydrophilic COOH-terminal half of the carboxylase has 19.3 percent identity with soybean seed lipoxygenase. Expression of the cloned cDNA resulted in an increase in carboxylase activity in microsomes of transfected cells compared to mock-transfected cells.

A mutation in the propeptide of Factor IX leads to warfarin sensitivity by a novel mechanism.

The propeptide sequences of the vitamin K-dependent clotting factors serve as a recognition site for the enzyme gamma-glutamylcarboxylase, which catalyzes the carboxylation of glutamic acid residues at the NH2 terminus of the mature protein. We describe a mutation in the propeptide of Factor IX that results in warfarin sensitivity because of reduced affinity of the carboxylase for the Factor IX precursor. The proband has a Factor IX activity level of > 100% off warfarin and < 1% on warfarin, at a point where other vitamin K-dependent factors were at 30-40% activity levels. Direct sequence analysis of amplified genomic DNA from all eight exons and exon-intron junctions showed a single guanosine-->adenosine transition at nucleotide 6346 resulting in an alanine to threonine change at residue -10 in the propeptide. To define the mechanism by which the mutation resulted in warfarin sensitivity, we analyzed wild-type and mutant recombinant peptides in an in vitro carboxylation reaction. The peptides that were analyzed included the wild-type sequence, the Ala-10-->Thr sequence, and Ala-10-->Gly, a substitution based on the sequence in bone gamma-carboxyglutamic acid protein. Measurement of C02 incorporation at a range of peptide concentrations yielded a Vmax of 343 cpm/min/reaction for the wild-type peptide, and Vmax values of 638 and 726 for A-10T and A-10G respectively, a difference of only twofold. The Km values, on the other hand, showed a 33-fold difference between wild-type and the variants, with a value of 0.29 microM for wild-type, and 10.9 and 9.50 microM, respectively, for A-10T and A-10G. Similar kinetic experiments showed no substantial differences between wild-type and mutant peptides in kinetic parameters of the carboxylase-peptide complexes for reduced vitamin K. We conclude that the major defect resulting from the Factor IX Ala-l0-->Thr mutation is a reduction in affinity of the carboxylase for the mutant propeptide. These studies delineate a novel mechanism for warfarin sensitivity. In addition, the data may also explain the observation that bone Gla protein is more sensitive to warfarin than the coagulation proteins.

Factor XSanto Domingo. Evidence that the severe clinical phenotype arises from a mutation blocking secretion.

Factor X (FX) is a vitamin K-dependent plasma protein required for the intrinsic and extrinsic pathways of blood coagulation. FXSanto Domingo is a hereditary FX deficiency which is characterized clinically by a severe bleeding diathesis. The proposita has a FX activity of less than 1% and a FX antigen of less than 5%. We have determined the molecular basis of the defect in the FXSanto Domingo gene by amplification of all eight exons with polymerase chain reaction and subsequent sequence analysis. The patient is homozygous for a G----A transition in exon I at codon -20 (numbering the alanine at the NH2 terminus of the mature protein as +1), resulting in the substitution of arginine for glycine in the carboxy-terminal part of the signal peptide. This amino acid change occurs near the presumed cleavage site of the signal peptidase. We hypothesized that the mutation might prevent cleavage by the signal peptidase which in turn would impair proper secretion of the FX protein. To test this hypothesis, we compared the expression of wild type and mutant FX cDNA in a human kidney cell line. Wild type and mutant constructs in the expression vector pCMV4 were introduced into the human embryonic kidney cell line 293 by calcium phosphate transfection. FX antigen levels in the supernatant of the cells harboring the wild type construct were 2.4 micrograms/10(7) cells per 24 h, whereas antigen levels in media from cells containing the FXSanto Domingo construct were undetectable. No FX antigen was detected in the cell lysates of cells transfected with the mutant construct. To insure that the difference in protein levels was not due to a difference in steady state levels of mRNA, Northern analysis was performed on RNA from the cell lysates of both constructs. The results showed a transcript of the same size, present in roughly equal amounts, in both cases. Thus, the defect in the signal sequence of FXSanto Domingo exerts its effect posttranscriptionally. FXSanto Domingo is the first described example of a bleeding diathesis due to a mutation in the signal sequence.

Replacing the first epidermal growth factor-like domain of factor IX with that of factor VII enhances activity in vitro and in canine hemophilia B.

Using the techniques of molecular biology, we made a chimeric Factor IX by replacing the first epidermal growth factor-like domain with that of Factor VII. The resulting recombinant chimeric molecule, Factor IXVIIEGF1, had at least a twofold increase in functional activity in the one-stage clotting assay when compared to recombinant wild-type Factor IX. The increased activity was not due to contamination with activated Factor IX, nor was it due to an increased rate of activation by Factor VIIa-tissue factor or by Factor XIa. Rather, the increased activity was due to a higher affinity of Factor IXVIIEGF1 for Factor VIIIa with a Kd for Factor VIIIa about one order of magnitude lower than that of recombinant wild-type Factor IXa. In addition, results from animal studies show that this chimeric Factor IX, when infused into a dog with hemophilia B, exhibits a greater than threefold increase in clotting activity, and has a biological half-life equivalent to recombinant wild-type Factor IX.

Polymorphisms in the VKORC1 gene are strongly associated with warfarin dosage requirements in patients receiving anticoagulation.

BACKGROUND: Warfarin is a mainstay of therapy for conditions associated with an increased risk of thromboembolic events. However, the use of this common agent is fraught with complications and little is known regarding inter-individual variation in warfarin response. OBJECTIVE: We tested for association between single nucleotide polymorphisms (SNPs) in VKORC1 and CYP2C9 and average weekly warfarin dose required to maintain patients at their desired anticoagulation target. METHODS: The sample consisted of 93 European-American patients from anticoagulation clinics at the University of North Carolina at Chapel Hill. Data on mean weekly warfarin dose were collected over a mean treatment period of 20.6 months. ANCOVA models were used and haplotype analysis was performed. RESULTS: Three of six VKORC1 SNPs were found to be very strongly associated with the average warfarin dose required to achieve the target international normalised ratio (INR; p<0.0001). The mean weekly dose by genotype ranged from approximately 27 to 47 mg. There was no evidence for an association between either of the two CYP2C9 polymorphisms studied, CYP2C9*2 and CYP2C9*3. CYP2C9*3 was significantly (p = 0.05) associated with average warfarin dosage after adjustment for VKORC1*1173. CONCLUSIONS: These results are of considerable clinical interest and confirm recently published results regarding the role of these two genes in modifying warfarin metabolism and maintenance dosage. The consistent findings regarding the role of VKORC1 and CYP2C9 in warfarin metabolism and maintenance dosage represent a clinically useful proof of principal for the use of pharmacogenomic information in medicine and may lead to improved understanding of warfarin's actions.

Functional Study of the Vitamin K Cycle Enzymes in Live Cells.

Vitamin K-dependent carboxylation, an essential posttranslational modification catalyzed by gamma-glutamyl carboxylase, is required for the biological functions of proteins that control blood coagulation, vascular calcification, bone metabolism, and other important physiological processes. Concomitant with carboxylation, reduced vitamin K (KH2) is oxidized to vitamin K epoxide (KO). KO must be recycled back to KH2 by the enzymes vitamin K epoxide reductase and vitamin K reductase in a pathway known as the vitamin K cycle. Our current knowledge about the enzymes of the vitamin K cycle is mainly based on in vitro studies of each individual enzymes under artificial conditions, which are of limited usefulness in understanding how the complex carboxylation process is carried out in the physiological environment. In this chapter, we review the current in vitro activity assays for vitamin K cycle enzymes. We describe the rationale, establishment, and application of cell-based assays for the functional study of these enzymes in the native cellular milieu. In these cell-based assays, different vitamin K-dependent proteins were designed and stably expressed in mammalian cells as reporter proteins to accommodate the readily used enzyme-linked immunosorbent assay for carboxylation efficiency evaluation. Additionally, recently emerged genome-editing techniques TALENs and CRISPR-Cas9 were used to knock out the endogenous enzymes in the reporter cell lines to eliminate the background. These cell-based assays are easy to scale up for high-throughput screening of inhibitors of vitamin K cycle enzymes and have been successfully used to clarify the genotypes and their clinical phenotypes of enzymes of the vitamin K cycle.

Structural and functional insights into enzymes of the vitamin K cycle.

Vitamin K-dependent proteins require carboxylation of certain glutamates for their biological functions. The enzymes involved in the vitamin K-dependent carboxylation include: gamma-glutamyl carboxylase (GGCX), vitamin K epoxide reductase (VKOR) and an as-yet-unidentified vitamin K reductase (VKR). Due to the hydrophobicity of vitamin K, these enzymes are likely to be integral membrane proteins that reside in the endoplasmic reticulum. Therefore, structure-function studies on these enzymes have been challenging, and some of the results are notably controversial. Patients with naturally occurring mutations in these enzymes, who mainly exhibit bleeding disorders or are resistant to oral anticoagulant treatment, provide valuable information for the functional study of the vitamin K cycle enzymes. In this review, we discuss: (i) the discovery of the enzymatic activities and gene identifications of the vitamin K cycle enzymes; (ii) the identification of their functionally important regions and their active site residues; (iii) the membrane topology studies of GGCX and VKOR; and (iv) the controversial issues regarding the structure and function studies of these enzymes, particularly, the membrane topology, the role of the conserved cysteines and the mechanism of active site regeneration of VKOR. We also discuss the possibility that a paralogous protein of VKOR, VKOR-like 1 (VKORL1), is involved in the vitamin K cycle, and the importance of and possible approaches for identifying the unknown VKR. Overall, we describe the accomplishments and the remaining questions in regard to the structure and function studies of the enzymes in the vitamin K cycle.

Nucleotide sequence of two 3'-termini of rRNA in the sea urchin Lytechinus variegatus.

The 3'-terminus of 26 S rRNA from the sea urchin Lytechinus variegatus has been determined by oligonucleotide fingerprinting, S1 nuclease mapping and terminal nucleotide analysis. There are two species of 26 S rRNA of approximately equal abundance, one 19 nucleotides longer than than other.

Purification of sea urchin ribosomal RNA genes with a single-strand specific nuclease.

Ribosomal RNA genes (rDNA) from Lytechinus variegatus were isolated by selective heat denaturation of main band DNA followed by single-strand specific nuclease (S1) treatment to remove the single-stranded DNA. After S1 nuclease treatment the partially purified fraction contained 10% rDNA, representing a 50-fold purification. Preparative CsCl centrifugation of this fraction resulted in highly purified rDNA with an average molecular weight of 1.9 - 10(7) and no single-strand breaks. High molecular weight sea urchin DNA was refractory to selective heat denaturation. DNA with an average molecular weight of greater than or equal to 2.9 - 10(7) was only 60-80% denatured after heating 13 degrees C above the Tm, whereas, DNA with an average molecular weight of less than or equal to 1.9 - 10(7) was 98% denatured. This phenomenon appears not to be due to time, buffer, or pH, but is dependent on size.

The region of transcriptional initiation in Lytechinus variegatus rRNA genes.

A sea urchin ribosomal DNA 1.9 kilobase BamHI fragment adjacent to the 5' end of the 18 S gene has been mapped with the restriction enzymes, XhoI, EcoRi, SmaI and HinfI. A 270 basepair fragment which most likely contains the 5' end of the presumed primary transcript of rRNA was identified by hybridization of [32P]DNA fragments to total nuclear RNA separated on methylmercury hydroxide gels and bound to diazobenzyloxymethyl paper. Under these denaturing conditions the size of Lytechinus variegatus precursor rRNA was determined to be 7.2 kilobases (33 S).

Identification of residues in the Gla-domain of human factor IX involved in the binding to conformation specific antibodies.

The binding of Ca2+ induces a conformational change in factor IX which can be monitored with conformation specific antibodies. Anti-FIX:Mg(II) antibodies recognize a conformational epitope (FIX') that can be induced by several metal ions such as Ca2+, Mg2+, Mn2+ and Ba2+, while anti-FIX:Ca(II) antibodies recognize a conformational epitope (FIX*) that can be only induced by Ca2+ and Sr2+ ions (Liebman et al., J. Biol. Chem., vol. 262 (1987) pp. 7605-7612). The latter conformation is essential for the function of factor IX. In this study we tried to identify residues in the Gla-domain of factor IX which are involved in binding to anti-factor IX:Mg(II) and anti-factor IX:Ca(II) antibodies. For this we substituted residues in recombinant human factor IX for those of factor X or factor VII. The substitution of residues 1-40 of factor IX by those of factor VII eliminated binding to both types of antibodies. Re-introduction of factor IX specific residues increased the binding to conformation specific anti-factor IX antibodies, but reduced the binding to conformation specific anti-factor VII antibodies, indicating that the structural integrity of the Gla-domain was not seriously affected by the mutations. We provide evidence that residues 33, 39 and 40 of human factor IX are important for binding to anti-factor IX:Mg(II) antibodies, while residues 1-11 are important for binding to anti-factor IX:Ca(II) antibodies.

The vitamin K cycle.

Post-translational modification of glutamate to gamma carboxyl glutamate is required for the activity of vitamin K-dependent proteins. Carboxylation is accomplished by the enzyme gamma glutamyl carboxylase (GGCX) which requires the propeptide-containing substrate and three co-substrates: reduced vitamin K, CO2, and O2. Most propeptides bind tightly to GGCX and all of the Glu residues that will be modified are modified during one binding event. Complete carboxylation is thus dependent upon the rate of carboxylation and the dissociation rate constant of the substrate from the GGCX enzyme. If the propeptide is released before carboxylation is complete, partially carboxylated vitamin K-dependent proteins are produced. The rate of carboxylation is mainly controlled by the level of reduced vitamin K available for the reactions while the dissociation rate constant is dependent upon both the propeptide and the Gla domain of the substrate. In addition, there are allosteric effects that increase the rate of dissociation of the fully carboxylated substrates. Carboxylation requires the abstraction of a proton from the 4-carbon of glutamate by reduced vitamin K and results in the conversion of vitamin K to vitamin K epoxide. The vitamin K epoxide must be recycled to vitamin K before it can be reused, a reaction catalyzed by the enzyme vitamin K epoxide reductase (VKOR). The gene for VKOR has recently been identified but the enzyme itself has not been purified to homogeneity. It appears, however, that most of the variability observed in patients response to warfarin may be attributed to variability in the VKOR gene.

Nucleotide sequence of the transcription initiation region for rRNA in the sea urchin Lytechinus variegatus.

The 1191-bp sequence which includes the 5' end of 18S X rDNA and its adjacent spacer have been determined for a cloned fragment of sea urchin rDNA. The 5' end of 33S precursor rRNA (pre-rRNA) has been located by S1 nuclease mapping and primer extension. Pre-rRNA appears to be initiated at an A, 658 bp upstream from the 5' terminus of 18S rRNA. The first nucleotide of 18S rRNA was also analyzed by S1 nuclease mapping and found to correspond to T in the nontranscribed DNA strand. Comparisons of the transcription initiation region in rDNA and the 5' end of 18S X rDNA with the corresponding regions in other eukaryotes reveal no significant nucleotide sequence homology in the precursor portion while the 5' end of 18S X rDNA is highly conserved.

Cloning and organization of genes for 5S ribosomal RNA in the sea urchin. Lytechinus variegatus.

A 1.35-kb EcoRI fragment of Lytechinus variegatus DNA containing a single 5S rRNA gene has been cloned into the plasmid vector pACYC184. Four clones from different transformation experiments contain 5S rDNA inserts of about the same size and have the same restriction enzyme digestion patterns for the enzymes HaeIII, HinfI, HhaI, and AluI. One EcoRI site near the HindIII site of the plasmid vector pACYC184 is missing in all the four clones. By DNA sequencing, the missing EcoRI ws found to be EcoRI site, d(AAATTN)d(TTTAAN) in pLu103, one of the four 5S rDNA clones. The structure of pLu103 was determined by restriction mapping and blot hybridization. Three restriction fragments, 1.0-kb HaeIII/HaeIII, 0.375-kb AluI/AluI and 0.249-kb MboII/MboII, which contain the 5S rRNA coding region, have been subcloned into the EcoRI site of the plasmid pACYC184. The organization of 5S rRNA genes in the sea urchin genome was also investigated. It was found that restriction endonuclease HaeIII has a single recognition site within each 5S rDNA repeat, and yields two fragment lengths, 1.2 and 1.3 kb. The behavior of these 5S rRNA genes when total L. variegatus DNA is partially digested with HaeIII is consistent with an arrangement of 5S rRNA genes in at least two tandemly repeated, non-interspersed families. Both the coding region and spacer region of the 5S rRNA gene in pLu103 hybridize to 1.2 and 1.3-kb rDNA families. This indicates that the cloned EcoRI fragment of 5S rDNA in pLu103 represents one single repeat of 5S rDNA in the genome.

Deduced amino acid sequence of mouse blood-coagulation factor IX.

A mouse fetal liver cDNA library was screened with a cDNA clone encoding human blood coagulation factor IX protein (hBCFIX). A bacteriophage lambda clone was isolated and the nucleotide sequence of a 2710-bp insert was determined. An open reading frame of 459 amino acids (aa) was identified within the sequence that has an 80% sequence similarity with hBCFIX. The cDNA contains a long 3'-untranslated sequence similar to that of BcfIX gene from human and canine sources. However, instead of a sequence that might form two hair-pins such as those found in hBcfIX, a (GA)16 repeat that has been reported to form H-DNA [Htun and Dahlberg, Science 241 (1988) 1791-1796; Johnston, Science 241 (1988) 1800-1804] was found in the 3'-untranslated region. The predicted aa sequence of mouse BCFIX serves as a comparative sequence for identifying key residues within hBCFIX where epitopes recognized by monoclonal antibodies produced from an immunized mouse are compared with respect to the human and mouse primary BCFIX sequence.

Nucleotide sequence of a cDNA for a member of the human 90-kDa heat-shock protein family.

This paper describes the isolation and sequence of a human cDNA homologous to a class of proteins commonly referred to as 90-kDa heat-shock proteins. The complete nucleotide sequence of 2563 bp and the deduced amino acid sequence are presented. A single long open reading frame encodes a protein of 83,303 Da, the amino acid composition of which correlates well with that determined for the human 90-kDa heat-shock or 'stress' protein [Welch, W.J. and Feramisco, J.R., J. Biol. Chem. 257 (1982) 14949-14959]. Moreover, sequence analysis of this gene reveals extensive homology with the Drosophila 83-kDa and yeast 90-kDa heat-shock proteins. A comparison of the translated product of the human cDNA to the published yeast 90-kDa heat-shock protein reveals more than 60% homology at both the nucleotide and amino acid levels. Several regions of 50 aa or more show greater than 90% identity. This cDNA also hybridizes with an RNA species which increases upon heat shock of HeLa cells.

Identification of a vitamin K-dependent carboxylase in the venom duct of a Conus snail.

Peptides from the venom ducts of cone snails (genus Conus) contain gamma-carboxyglutamate residues. The gamma-glutamyl carboxylase responsible for this post-translational modification is localized in the microsomal fraction, strictly dependent on vitamin K, activated by ammonium sulfate, and is associated with endogenous substrate. The K(m) of the enzyme for vitamin K is comparable to that for the bovine carboxylase. However, a propeptide containing substrate related to the blood coagulation protein factor IX, a highly efficient substrate for the bovine enzyme, was poorly carboxylated by the Conus enzyme, suggesting differences in gamma-carboxylase recognition signal sequences and/or structural requirements at the carboxylation site.

Complete and sustained phenotypic correction of hemophilia B in mice following hepatic gene transfer of a high-expressing human factor IX plasmid.

Therapeutic correction of hemophilia B was achieved by rapid infusion of a large-volume solution containing a high-expressing human factor IX (hFIX) plasmid into the tail vein of hemophilia B mice. hFIX circulated at therapeutic levels (1-5 micro g mL-1) in all animals for more than 1 year as determined by both species-specific antigen assay and an activated partial thromboplastin time (APTT)-based clotting assay. There was acute, transient hepatic tissue damage by the infusion procedure and no significant inhibitory anti-hFIX antibodies developed. No bleeding episode was observed during or after treatment. Immunohistochemical studies indicated that the hFIX gene was exclusively expressed in hepatocytes, and that transduced cells had readily detectable hFIX protein at 4 h postinfusion, and stainable protein persisted for up to 1 year. Repeated infusions of hFIX plasmids boosted the hFIX expression to higher levels. These results demonstrate that hemophilia B can be treated by gene transfer of naked hFIX plasmids.

Identification of a gene encoding a typical gamma-carboxyglutamic acid domain in the tunicate Halocynthia roretzi.

We report the identification of a gene capable of encoding a novel Gla (gamma-carboxyglutamic acid) protein from the tunicate Halocynthia roretzi, a primitive member of the phylum Chordata. We call this new hypothetical protein Gla-RTK; it has a Gla domain typical of human vitamin K-dependent coagulation factors, a transmembrane domain, and a receptor tyrosine kinase domain. The receptor tyrosine kinase domain is very similar to the ARK (adhesion-related kinase) family of receptor tyrosine kinases. The ARK family includes Axl, Tyro3, and c-Mer. This gene also encodes a propeptide that binds to the human gamma-glutamyl carboxylase within a range of affinities observed for mammalian propeptides. The cDNA for this putative protein is found distributed throughout the oocyte and embryo but the cDNA is apparently not transcribed except during oogenesis. One of the most interesting aspects of this hypothetical protein is that its Gla domain is highly homologous to the Gla domain of Gas6, a ligand for Axl, while its receptor tyrosine kinase domain is highly homologous to Axl.