Identification of a putative regulator of early T cell activation genes.

Molecules involved in the antigen receptor-dependent regulation of early T cell activation genes were investigated with the use of functional sequences of the T cell activation-specific enhancer of interleukin-2 (IL-2). One of these sequences forms a protein complex, NFAT-1, specifically with nuclear extracts of activated T cells. This complex appeared 10 to 25 minutes before the activation of the IL-2 gene. Studies with inhibitors of protein synthesis indicated that the time of synthesis of the activator of the IL-2 gene in Jurkat T cells corresponds to the time of appearance of NFAT-1. NFAT-1, or a very similar protein, bound functional sequences of the long terminal repeat (LTR) of the human immunodeficiency virus type 1; the LTR of this virus is known to be stimulated during early T cell activation. The binding site for this complex activated a linked promoter after transfection into antigen receptor-activated T cells but not other cell types. These characteristics suggest that NFAT-1 transmits signals initiated at the T cell antigen receptor.

Human c-Ki-ras2 proto-oncogene on chromosome 12.

A human colonic adenocarcinoma transforming gene, recently identified as a cellular homolog of the Kirsten sarcoma gene (v-ras), was used to assign the human cellular Kirsten ras2 gene to chromosome 12 by the Southern hybridization method. A single 640 base-pair Eco RI--Hind III fragment of the transforming gene, isolated by DNA transfection and molecular cloning, can detect a single Eco RI fragment (2.9 kilobase pairs) of DNA from phenotypically normal cells. The data suggest a constant chromosomal location of c-Ki-ras2.

The discovery and characterization of a novel nucleotide-based thrombin inhibitor.

Thrombin is a serine protease that plays a pivotal role in thrombosis and hemostasis, and is a major target for anticoagulation and cardiovascular disease therapy. Using a novel in vitro selection/amplification technique, we have identified a new class of thrombin inhibitors based on single-stranded DNA (ssDNA) oligodeoxyribonucleotides (oligos). These thrombin inhibitors are the first example of the use of this technique to obtain ssDNA oligos that bind a target protein that does not interact physiologically with nucleic acid. Here, we review how iterative selection and amplification were used to identify short ssDNA sequences that bind and inhibit thrombin (Bock et al., Nature 355 (1992) 564-566), and the tertiary structure of one aptamer sequence (Wang et al., Biochemistry 32 (1993) 1899-1904). Results from in vitro and in vivo studies are also summarized (Griffin et al., Blood 81 (1993) 3271-3276). The discovery of a new class of thrombin inhibitors using this technology demonstrates the power of this new approach for rapid drug discovery and development.

9-(2-Phosphonylmethoxyethyl)-N6-cyclopropyl-2,6-diaminopurine (cpr-PMEDAP) as a prodrug of 9-(2-phosphonylmethoxyethyl)guanine (PMEG).

9-(2-Phosphonylmethoxyethyl)-N6-cyclopropyl-2,6-diaminopurine (cpr-PMEDAP) is an acyclic nucleotide analog of the [9-(2-phosphonylmethoxyethyl)-] (PME) series containing a cyclopropyl substituent on the N6 position of the 2,6-diaminopurine (DAP) base. Growth inhibition assays in a broad range of tumor cell lines demonstrated that this analog had potent antiproliferative activity with IC50 values similar to those of the structurally related guanine analog 9-(2-phosphonylmethoxyethyl)guanine (PMEG). A substantially lower growth inhibitory effect was observed for the 2,6-diaminopurine analog, PMEDAP. To dissect the basis for these varying potencies, the metabolism of the three analogs was examined in a human pancreatic carcinoma cell line, BxPC-3. HPLC analysis of the intracellular metabolites demonstrated that the cpr-PMEDAP was deaminated to PMEG and subsequently phosphorylated to PMEG mono- and diphosphates (PMEGp and PMEGpp). The level of PMEGpp generated from cpr-PMEDAP-treated cells was 50% greater than the level generated from cells incubated with PMEG. The presence of PMEG in the DNA of cells incubated with cpr-PMEDAP confirmed that the cpr-PMEDAP was converted to PMEG. In contrast, PMEDAP was not deaminated to PMEG, but directly phosphorylated to PMEDAPp and PMEDAPpp. The adenylate deaminase inhibitor 2'-deoxycoformycin (dCF) inhibited the conversion of cpr-PMEDAP in a rat liver cytosolic extract and increased the IC50 value for growth inhibition by 40-fold. The antiproliferative activities of PMEG and PMEDAP were unaffected by dCF. Thus, it appears that cpr-PMEDAP, but not PMEDAP, is converted by an adenylate deaminase-like enzyme and functions as a prodrug of PMEG.

Genetic engineering and coagulation factors.

It is unfortunate that we cannot report, in the area of coagulation, advances that have been seen in related fields such as thrombolytic therapy. The reported progress (Gold et al, 1984; Van de Werf et al, 1984) with human recombinant tissue plasminogen activator (Pennica et al, 1983) augers well for the application of recombinant technology to the problems faced by patients with coagulation defects. While plasminogen activator is being assessed in an acute therapeutic setting, its use signals a beginning of the application of the technology to abnormalities of the haemostatic mechanism. Chronic administration of coagulation factors for prophylaxis and replacement therapy would appear to be just one more step down the pathway illuminated by the biochemists, microbiologists and cell biologists who have preceded the clinicians in this promising area. There is no record of the use of genetically engineered materials in the treatment of coagulation defects, primarily because the body of knowledge and refined techniques have only recently been acquired. For this reason we have had to project developments in other areas onto the problems that exist for the haemostatically compromised patient. In describing the potential usefulness of these technologies, it is difficult to ascertain where the logical projection, from a fully investigated model system, diverges from flights of imaginative fancy. Cloning projects considered overly ambitious and grandiose at the beginning of this decade are already accomplished feats. The feasibility of gene therapy in the mammalian system has been demonstrated, and trade publications now discuss governmental approval for investigative use of this procedure in 1985. Panels of physicians, scientists and even politicians now seriously contemplate and promulgate views and regulations pertaining to the efficacy and ethics of the use of genetic engineering in the treatment of human disease. The haemophilias will certainly be among the first genetic diseases to be approached with the techniques of recombinant DNA technology. Diagnostic testing, using cloned DNA, is already underway and therapeutic trials are predicted for the near future. Every observer of this rapidly growing field has to define for himself when the future is. For the potential carrier of haemophilia B, the future is now. For the physician managing the patient with a haemophilic inhibitor, the future can't come soon enough. And, for those who are concerned with man's tampering with the gene pools of living things, from viruses to humans, the future comes too quickly to be dealt with in a rational and understanding way.

An abundant androgen-regulated mRNA in the mouse kidney.

We have identified an abundant 20,000 dalton protein (KAP) by in vitro translation of male mouse kidney mRNA. This protein is synthesized in reduced amounts from female kidney mRNA. A KAP cDNA fragment was purified and used for nucleic acid hybridization studies. Females and castrated males have 10 and 200 fold lower levels, respectively, of KAP mRNA relative to males. The administration of testosterone to females or castrated males results in the induction of KAP mRNA to normal male levels. Testicular feminized (Tfm) mice have 3 fold lower levels of KAP mRNA relative to normal males and are not induced by testosterone. KAP mRNA is not found in significant amounts in tissues other than the kidney, and the KAP gene renatures with kinetics similar to single-copy DNA. With the rapidly expanding knowledge of mouse genetics, KAP should prove useful in determining genetic factors which regulate the inducibility and tissue specificity of a hormonally regulated gene.

The application of a modified nucleotide in aptamer selection: novel thrombin aptamers containing 5-(1-pentynyl)-2'-deoxyuridine.

Combinatorial libraries of nucleic acids are developing into novel sources for lead compounds in drug development. In order to diversify the pool of ss DNA sequences, we have used a modified nucleotide, 5-(1-pentynyl)-2'-deoxyuridine, in place of thymidine in a random nucleic acid library and screened this library against human thrombin. Previously, we described this screening method to identify a novel structural inhibitor (an aptamer) of the coagulation protease thrombin (Bock, L. et. al. (1992) Nature 355 564-566). Using the modified nucleic acid library, we have now isolated a second pool of thrombin inhibitors with strikingly different sequence composition compared to the selection using natural bases. This second class of aptamers is dependent on the presence of the modified nucleotide for protein binding and clotting inhibition. Our method represents a potential strategy to enhance the diversity of libraries for in vitro selection, and thereby increasing the utility of this technique in the identification of molecules with novel biochemical properties.

Multiple genes coding for the androgen-regulated major urinary proteins of the mouse.

We have purified a cDNA fragment complementary to the mRNA coding for one of the major urinary proteins (MUPs) synthesized in the mouse liver. Using this cDNA as a hybridization probe, we have shown that the level of MUP mRNA is lower in the livers of females and castrated males than in those of males. The addition of testosterone to females and castrated males results in an increase in the concentration of the mRNA to levels found in males. There are approximately 15 gene per haploid genome coding for the MUPs; this allows a possible new interpretation of some of the genetic data concerning the regulation of levels of the different MUPs in the urine (Szoka and Paigen, 1978). Finally, we have shown that mouse MUP and rat alpha 2u-globulin mRNA share common sequences, but that there are surprising differences in gene number and regulation of the genes in these two closely related animals.

Triple helix formation inhibits transcription elongation in vitro.

We have identified a 15-nucleotide site within a G-free transcription cassette that forms triple helix with sequence-specific oligodeoxyribonucleotides. When oligodeoxynucleotides were added to template DNA prior to in vitro transcription, a significant fraction of transcripts were truncated at a site corresponding to the region of triple helix formation. Kinetic analysis of the transcription products demonstrated that these truncated transcripts could be elongated to full length upon prolonged incubation. When an alkylating base was incorporated into the oligodeoxynucleotide to form covalent triple helix, most of the transcripts remained truncated. We conclude that triple helix formation can stall or, in the case of covalent crosslinking, can block RNA polymerase II and thus may provide a method for the specific inhibition of gene expression.

Mutations in hepatitis B DNA polymerase associated with resistance to lamivudine do not confer resistance to adefovir in vitro.

To determine whether adefovir is active against lamivudine-resistant hepatitis B virus (HBV), the inhibition constants of adefovir diphosphate and lamivudine triphosphate for wild-type and mutant human HBV DNA polymerases, which contain amino acid substitutions associated with lamivudine resistance, were compared. Recombinant wild-type and mutant human HBV DNA polymerases were expressed and substantially purified using a baculovirus expression system and immunoaffinity chromatography. HBV DNA polymerase mutants M552I, M552V, and L528M/M552V showed resistance to lamivudine triphosphate with inhibition constants (Ki) increased by 8.0-fold, 19.6-fold, and 25.2-fold compared with that of wild-type HBV DNA polymerase. However, these mutants remained sensitive to adefovir diphosphate with the inhibition constants increasing by 1.3-fold and 2.2-fold or decreasing by 0.79-fold. The L528M single mutation, identified in patients with increasing HBV DNA levels during therapy with famciclovir, also remained sensitive to adefovir diphosphate with the inhibition constant increased by only 2.3-fold.

Biphasic clearance kinetics of hepatitis B virus from patients during adefovir dipivoxil therapy.

In a recent phase II clinical study, 13 chronic hepatitis B-infected patients treated daily with 30 mg adefovir dipivoxil for 12 weeks displayed a median 4.1-log10 decrease in plasma hepatitis B virus (HBV)-DNA levels. The decline of viral load during therapy displayed a biphasic kinetic profile that was modeled to determine the efficacy of inhibition of viral production, as well as kinetic constants for the clearance of free virus and the loss of infected cells. Viral production was suppressed with an efficacy of 0.993 +/- 0.008, indicating that only 0.7% of viral production persisted during therapy. The initial, faster phase of viral load decline reflects the clearance of HBV particles from plasma with a half-life of 1.1 +/- 0.3 days, translating to a 48% daily turnover of the free virus. The second, slower phase of viral load decline closely mirrors the rate-limiting process of infected cell loss, with a half-life of 18 +/- 7 days. The duration of therapy required to completely eliminate the virus from plasma or suppress it to levels sufficient to induce seroconversion is a function of the half-life of the free virus, the half-life of infected cells, and the efficacy of inhibition of virus production from infected cells. These quantitative analyses provide a more detailed picture of the dynamics of HBV infection and therapy, and can be used to compare the efficacy of various doses and inhibitors of HBV replication for the treatment of HBV infections.

Translation and developmental regulation of RNA encoded by the eukaryotic transposable element copia.

copia-specific RNA was isolated from Drosophila melanogaster tissue culture cells by hybridization of cytoplasmic polyadenylylated RNA to copia DNA immobilized on cellulose. The purified RNA was translated in reticulocyte lysates. One major polypeptide of approximately 51,000 daltons was synthesized in addition to several others between 18,000 and 38,000 daltons. The 51,000-dalton polypeptide and several of the others are encoded by mRNAs of about 2000 nucleotides. The approximate locations on the copia element of the coding sequences for the 51,000-dalton polypeptide and several other proteins were determined by hybrid-arrested translation with copia restriction fragments. The relative abundance of copia-specific RNA was determined at various stages of the Drosophila life cycle. The level of copia-specific RNA is modulated during development of the organism, with the highest level occurring during the larval stages.

A large region (approximately equal to 95 kDa) of human factor VIII is dispensable for in vitro procoagulant activity.

Factor VIII (antihemophilic factor) is a high molecular weight plasma glycoprotein that participates in the blood clotting cascade. The recent cloning and sequence analysis of the cDNA encoding human factor VIII revealed an obvious domain structure for the protein, which can be represented as A1-A2-B-A3-C1-C2. We now report the DNA sequence analysis of porcine exons encoding the entire B domain and part of the A2 and A3 domains. We found an unusually high degree of porcine-human amino acid sequence divergence in the B region compared with the limited sequence available for other regions of the porcine factor VIII molecule. In addition to sequence divergence, there are numerous gaps in the porcine B domain totalling over 200 amino acids. Recombinant DNA techniques were used to effect the removal of large segments of DNA encoding the B domain from the full-length human factor VIII cDNA. These constructs directed the synthesis of biologically active factor VIII when introduced into mammalian cells despite the deletion of up to 38% of the factor VIII molecule.

Selection of single-stranded DNA molecules that bind and inhibit human thrombin.

Aptamers are double-stranded DNA or single-stranded RNA molecules that bind specific molecular targets. Large randomly generated populations can be enriched in aptamers by in vitro selection and polymerase chain reaction. But so far single-stranded DNA has not been investigated for aptamer properties, nor has a target protein been considered that does not interact physiologically with nucleic acid. Here we describe the isolation of single-stranded DNA aptamers to the protease thrombin of the blood coagulation cascade and report binding affinities in the range 25-200 nM. Sequence data from 32 thrombin aptamers, selected from a pool of DNA containing 60 nucleotides of random sequence, displayed a highly conserved 14-17-base region. Several of these aptamers at nanomolar concentrations inhibited thrombin-catalysed fibrin-clot formation in vitro using either purified fibrinogen or human plasma.

A novel nucleotide-based thrombin inhibitor inhibits clot-bound thrombin and reduces arterial platelet thrombus formation.

A novel thrombin inhibitor based on single-stranded (ss) deoxynucleotides with the sequence GGTTGGTGTGGTTGG (thrombin aptamer) has been recently discovered. In this study, we tested its efficacy in inhibiting clot-bound thrombin activity and platelet thrombus formation in an ex vivo whole artery angioplasty model. The thrombin aptamer showed a specific dose-dependent inhibition of thrombin-induced platelet aggregation (0.5 U/mL) in human platelet-rich plasma, with an IC50 of approximately 70 to 80 nmol/L. In an in vitro clot-bound thrombin assay system, heparin, used at clinically relevant concentrations of 0.2 U/mL and 0.4 U/mL, was ineffective in inhibiting clot-bound thrombin (6.5% and 34.9% inhibition at 0.2 U/mL and 0.4 U/mL, respectively). In contrast, the thrombin aptamer at an equivalent anticoagulant concentration inhibited clot-bound thrombin (79.7% inhibition). In an ex vivo whole artery angioplasty model, the thrombin aptamer markedly suppressed the generation of fibrinopeptide A (FPA), whereas heparin at 2 U/mL was ineffective. Compared with a scrambled ssDNA control, the thrombin aptamer reduced platelet deposition by 34.5% +/- 5% (mean +/- SEM, n = 4, P = .09) at low shear rates (approximately 200 s-1) and 61.3% +/- 11% (mean +/- SEM, n = 4, P = .05) at high shear rates (approximately 850 s-1). Thrombin aptamers based on ssDNA molecules represent a new class of thrombin inhibitors with potent anticoagulant and antithrombotic properties.

Characterization of a human colon/lung carcinoma oncogene.

DNA sequences capable of inducing oncogenic transformation of NIH3T3 mouse cells are found in a number of human tumour cell lines. When DNAs of these cell lines are applied to monolayer cultures of the mouse fibroblasts, foci of transformed cells are observed 2-3 weeks later. DNA from cells of such primary foci can be used in turn to induce foci in a second cycle of gene transfer. The human DNA sequences responsible for transformation have been called oncogenes, the best characterized of which is closely related to the Harvey murine sarcoma virus oncogene. Here we present a characterization of an oncogene which we found originally to be present in DNA of the SW480 colon carcinoma cell line. We indicate its structural outlines and demonstrate, in extension of reported results, its presence in an activated form in the genome of several types of human tumour cell lines as well as in biopsy tissue from an adenocarcinoma of the large bowel. We identify this tumour oncogene with c-Ki-ras2, one of two known members of the Kirsten ras family of human proto-oncogenes, extending a series of recent reports which have demonstrated homologies between human oncogenes and those of Harvey and Kirsten murine sarcoma viruses. The c-Ki-ras2 oncogene of several tumour cell lines is shown to be amplified.

The single-stranded DNA aptamer-binding site of human thrombin.

A new class of thrombin inhibitors based on sequence-specific single-stranded DNA oligonucleotides (thrombin aptamer) has recently been identified. The aptamer-binding site on thrombin was examined by a solid-phase plate binding assay and by chemical modification. Binding assay results demonstrated that the thrombin aptamer bound specifically to alpha-thrombin but not to gamma-thrombin and that hirudin competed with aptamer binding, suggesting that thrombin's anion-binding exosite was important for aptamer-thrombin interactions. To identify lysine residues of thrombin that participated in the binding of the thrombin aptamer, thrombin was modified with fluorescein 5'-isothiocyanate in the presence or absence of the thrombin aptamer, reduced, carboxymethylated, and digested with endoproteinase Arg-C. The digestion products were analyzed by reversed-phase high performance liquid chromatography and the peptide maps compared. Four peptides with significantly decreased modification in the presence of the aptamer were identified and subjected to N-terminal sequence analysis. Results indicated that B chain Lys-21 and Lys-65, both located within the anion-binding exosite, are situated within or in close proximity to the aptamer-binding site of human alpha-thrombin. The thrombin aptamer binds to the anion-binding exosite and inhibits thrombin's function by competing with exosite binding substrates fibrinogen and the platelet thrombin receptor.

In vivo anticoagulant properties of a novel nucleotide-based thrombin inhibitor and demonstration of regional anticoagulation in extracorporeal circuits.

Using a novel in vitro selection/amplification technique, we have recently identified a new class of thrombin inhibitors based on single-stranded DNA oligonucleotides. One oligonucleotide, GGTTGGTGTGGTTGG (thrombin, aptamer), showed potent anticoagulant activity in vitro. We have initiated pharmacologic studies in cynomolgus monkeys to study the thrombin aptamer's in vivo anticoagulant properties. Upon infusion of the thrombin aptamer, anticoagulation was rapidly achieved, with a plateau reached within 10 minutes. There was a linear dose-response relationship between thrombin aptamer infusion rate and prolongation of plasma prothrombin time. Ten minutes after the infusion was stopped, no prolongation of prothrombin time was observed, indicating that the thrombin aptamer has an extremely short in vivo half-life, estimated to be 108 +/- 14 seconds. In addition, inhibition of thrombin-induced platelet aggregation in platelet-rich plasma was observed ex vivo without an effect on collagen-induced aggregation, indicating that the inhibition was specific for thrombin and not due to a nonspecific inhibitory effect on platelets. To exploit the short in vivo half-life of the thrombin aptamer, its ability to achieve regional anticoagulation in an extracorporeal hemofiltration circuit in sheep was tested. Doubling of the prothrombin time in the circuit was observed, whereas the systemic prothrombin time was minimally prolonged. We conclude that the thrombin aptamer is a potent anticoagulant in vivo, and specifically inhibits thrombin-induced platelet aggregation ex vivo. The rapid onset of action and short half-life in vivo suggest that the thrombin aptamer may be useful in anticoagulation with extracorporeal circuits and may have distinct advantages in certain acute clinical settings.

Inhibition of duck hepatitis B virus replication by 9-(2-phosphonylmethoxyethyl)adenine, an acyclic phosphonate nucleoside analogue.

The use of regimens that use nucleoside analogues for the treatment of chronic hepatitis B virus infection is often limited because of their high relapse rates. This is thought to be due to the persistence of virus in nonhepatocyte reservoirs and/or the viral covalently closed circular (CCC) DNA species in the nucleus of infected hepatocytes. We have evaluated the novel nucleoside analogue 9-(2-phosphonylmethoxyethyl)adenine (PMEA) in the duck model of hepatitis B. Eight Pekin-Aylesbury ducks congenitally infected with the duck hepatitis B virus (DHBV) were treated with PMEA at a dosage of 15 mg/kg of body weight/day via the intraperitoneal route for 4 weeks. At the end of the treatment period, four animals were killed and the remainder were monitored for a further 4-week drug-free period before analysis. The results were compared with those for eight age-matched, untreated controls. The levels of viremia, the total intrahepatic DHBV load, and CCC DNA, viral RNA, and protein levels were measured by Southern hybridization, Northern hybridization, and immunoblotting of the appropriate specimen, respectively. Viral proteins and DNA were also measured by immunohistochemistry (IHC) and in situ hybridization (ISH) of sections of liver and pancreatic tissue. PMEA treatment reduced the viremia to undetectable levels, while the total viral DNA load in the liver was reduced by 95% compared to the control level. Viral RNA and protein levels decreased by approximately 30%. ISH and IHC confirmed the PMEA-related intrahepatic changes and established that the amount of virus in bile duct epithelial cells (BDEC) was reduced by 70% during therapy. During the follow-up period all parameters of active virological replication returned to those for the age-matched controls. PMEA had no significant effect upon the number of virus-infected islet or acinar cells in the pancreas. PMEA at a dosage of 15 mg/kg/day has potent activity against DHBV found within hepatocytes and BDEC and inhibits DHBV replication in BDEC.