The importance of nef in the induction of human immunodeficiency virus type 1 replication from primary quiescent CD4 lymphocytes.

The viral regulatory gene, nef, is unique to the human immunodeficiency viruses (HIV) and their related primate lentiviruses. Expression of the nef gene has been shown to be essential to the maintenance of high levels of virus replication and the development of pathogenesis in the animal model of simian immunodeficiency virus (SIV) infection. In contrast to this in vivo model, the use of standard T cell culture systems to study nef function in vitro has produced a spectrum of contradictory results, and has failed to demonstrate a significant positive influence of nef on viral life cycle. We have developed a cell model to study regulation of HIV-1 replication that we believe reflects more accurately virus-cell interactions as they occur in vivo. Our experimental system used acute virus infection of purified, quiescent CD4 lymphocytes and subsequent induction of viral replication through T cell activation. With this cell model, NL4-3 virus clones with open and mutated nef reading frames were compared for replication competence. The clones with nef mutations showed reproducible and significant reductions in both rates of growth and maximal titers achieved. The degree of reduced replication was dependent on initial virus inoculum and the timing of T cell activation. The influence of nef was highly significant for induction of virus replication from a latent state within resting CD4 cells. Its effect was less apparent for virus infection of fully proliferating CD4 cells. This study demonstrates that nef confers a positive growth advantage to HIV-1 that becomes readily discernable in the primary cell setting of virus induction through T cell activation. The experimental cell model, which we describe here, provides not only a means to study nef function in vitro, but also provides important clues to the function of nef in HIV infection in vivo.

Phase I evaluation of ISIS 3521, an antisense oligodeoxynucleotide to protein kinase C-alpha, in patients with advanced cancer.

PURPOSE: To determine the maximum-tolerated dose (MTD) and pharmacologic behavior of ISIS 3521 (ISI 641A), an antisense phosphorothioate oligonucleotide to protein kinase C-alpha. PATIENTS AND METHODS: Thirty-six patients with advanced cancer received 99 cycles of ISIS 3521 (0.15 to 6.0 mg/kg/d) as a 2-hour intravenous infusion administered three times per week for 3 consecutive weeks and repeated every 4 weeks. Plasma and urine sampling was performed during the first week of treatment and subjected to capillary gel electrophoresis to determine full-length antisense oligonucleotide in addition to chain-shortened metabolites. RESULTS: Drug-related toxicities included mild to moderate nausea, vomiting, fever, chills, and fatigue. Hematologic toxicity was limited to thrombocytopenia (grade 1, four patients; grade 2, one patient; grade 3, one patient). There was no relationship between dose, maximum concentration of the drug (C(max)), or area under the plasma concentration versus time curve (AUC) and coagulation times or complement levels. Dose escalation was discontinued because of the attainment of peak plasma concentrations, which approached that associated with complement activation in primates. Two patients with non-Hodgkin's lymphoma who completed 17 and nine cycles of therapy achieved complete responses. The pharmacokinetic profile of ISIS 3521 revealed a short elimination half-life (18 to 92 minutes), as well as a dose-dependent decrease in clearance and dose-dependent increases in C(max), AUC, and elimination half-life. CONCLUSION: No dose-limiting toxicity of ISIS 3521 was identified, and clinical activity was observed. A short elimination half-life was identified, which suggests that alternate schedules with prolonged administration may be necessary for further clinical development.

Phase I clinical/pharmacokinetic and pharmacodynamic trial of the c-raf-1 antisense oligonucleotide ISIS 5132 (CGP 69846A).

PURPOSE: Raf-1 is a protein kinase that plays a broad role in oncogenic signaling and acts as a downstream effector of Ras in the mitogen-activated protein kinase pathway. The present study was designed to determine the maximum-tolerated dose (MTD), toxicity profile, pharmacokinetics, and antitumor activity of the c-raf-1 antisense oligodeoxynucleotide ISIS 5132 (CGP 69846A; ISIS Pharmaceuticals Inc, Carlsbad, CA). The effect of ISIS 5132 on c-raf-1 gene expression in peripheral-blood mononuclear cells (PBMCs) of treated patients was studied using a reverse transcriptase polymerase chain reaction assay. PATIENTS AND METHODS: Patients with refractory malignancies received ISIS 5132 as a 2-hour intravenous infusion three times weekly for 3 consecutive weeks. Pharmacokinetic sampling was performed during the first cycle in all patients; PBMCs for c-raf-1 mRNA analysis were collected at baseline and on days 3, 5, 8, and 15 of cycle 1 and on day 1 of each cycle thereafter. RESULTS: Thirty-one patients received ISIS 5132 at one of nine dose levels ranging from 0.5 mg/kg to 6.0 mg/kg. Clinical toxicities included fever and fatigue, but these were not dose limiting. A clinically defined MTD was not reached. The harmonic mean half-life of ISIS 5132 was 59.8 minutes (range, 35.5 to 107.3 minutes). The area under the concentration-time curve increased linearly with dose, and mean plasma clearance was 1.86 mL/kg/min (range, 1.21 to 2.41 mL/kg/min). Two patients experienced prolonged stable disease lasting more than 7 months, which was associated with persistent reduction in c-raf-1 expression in PBMCs. Significant decreases in c-raf-1 expression were identified at time points after the baseline value (P <.05) at doses >/= 2.5 mg/kg. CONCLUSION: ISIS 5132 is well tolerated at doses up to 6.0 mg/kg when administered as a thrice weekly 2-hour infusion for 3 consecutive weeks. The pharmacokinetic behavior of the drug is reproducible, and suppression of target gene expression is observed in circulating PBMCs.

A phase I trial of c-Raf kinase antisense oligonucleotide ISIS 5132 administered as a continuous intravenous infusion in patients with advanced cancer.

Raf proteins play a central role in the mitogen-activated protein kinase signaling pathway and hence are involved in oncogenic transformation and tumor cell proliferation. ISIS 5132 is a 20-base antisense phosphorothioate oligodeoxyribonucleotide that specifically down-regulates c-raf expression. We report here an initial study of the safety and tolerability of an i.v. infusion of ISIS 5132 in patients with advanced cancer. A continuous i.v. infusion of ISIS 5132 was administered for 21 days every 4 weeks to 34 patients with a variety of solid tumors refractory to standard therapy. The dose of ISIS 5132 was increased in sequential cohorts of patients, as toxicity allowed, until a final dose of 5.0 mg/kg body weight was reached. Toxicity was scored by common toxicity criteria, and tumor response was monitored. Pharmacokinetic studies were performed for 30 patients treated at doses of < or =4.0 mg/kg/day. The initial dose of ISIS 5132 was 0.5 mg/kg body weight and was successfully increased incrementally to 5.0 mg/kg body weight. Toxicities through the 4.0 mg/kg dose level were not dose limiting. Side effects were minimal and could not be specifically related to ISIS 5132. Two patients had prolonged stabilization of their disease, and one patient with ovarian carcinoma had a significant response with a 97% reduction in CA-125 levels. ISIS 5132, an antisense oligonucleotide against c-raf, was well tolerated at doses up to and including 4.0 mg/kg/day by 21-day continuous i.v. infusion and demonstrated antitumor activity at the doses tested.

Phase I study of an antisense oligonucleotide to protein kinase C-alpha (ISIS 3521/CGP 64128A) in patients with cancer.

Protein kinase C (PKC) is an attractive target in cancer therapy. It is overexpressed in a variety of cancers, and nonspecific inhibitors of PKC have demonstrated antitumor activity. Antisense oligonucleotides targeted against PKC-alpha, which have high specificity, can inhibit mRNA and protein expression as well as the growth of tumors in vitro and in vivo. This Phase I study sought to characterize the safety profile and to determine the maximum tolerated dose of antisense to PKC-alpha when administered by continuous infusion in patients. Patients with incurable malignancies received ISIS 3521, a 20-length phosphorothioate oligodeoxynucleotide specific for PKC-alpha. Treatment was delivered over a period of 21 days by continuous i.v. infusion followed by a 7-day rest period. Doses were increased from 0.5 to 3.0 mg/kg/day. Patients continued on the study until evidence of disease progression or unacceptable toxicity was detected. Between August 1996 and September 1997, 21 patients were treated in five patient cohorts. The maximum tolerated dose was 2.0 mg/kg/day. The dose-limiting toxicities were thrombocytopenia and fatigue at a dose of 3.0 mg/kg/day. Pharmacokinetic measurements showed rapid plasma clearance and dose-dependent steady-state concentrations of ISIS 3521. Evidence of tumor response lasting up to 11 months was observed in three of four patients with ovarian cancer. The recommended dose of ISIS 3521 for Phase II studies is 2.0 mg/kg/day when given over a period of 21 days. Side effects are modest and consist of thrombocytopenia and fatigue. Evidence of antitumor activity provides the rationale for Phase II studies in ovarian cancer and other malignancies.

Phase I Trial of ISIS 5132, an antisense oligonucleotide inhibitor of c-raf-1, administered by 24-hour weekly infusion to patients with advanced cancer.

Raf-1 is a serine/threonine kinase that functions as a critical effector of Ras-mediated signal transduction via the mitogen-activated protein kinase pathway. Constitutive activation of this pathway directly contributes to malignant transformation in many human tumors. A 20-base phosphorothioate oligonucleotide complementary to c-raf-1 mRNA (ISIS 5132; CGP 69846A) has been shown to specifically suppress Raf-1 expression both in vitro and in vivo. This Phase I trial, involving 22 patients with advanced cancer, was designed to evaluate the safety, feasibility, and maximum tolerated dose of ISIS 5132 administration as a weekly 24-h i.v. infusion. Pharmacokinetic analysis was performed, and c-raf-1 mRNA levels in peripheral blood mononuclear cells were assessed using quantitative reverse transcription-PCR. This trial defined a maximum tolerated dose of 24 mg/kg/week on this schedule. Two of four patients treated at 30 mg/kg/week had serious adverse events after the first dose of ISIS 5132, including acute hemolytic anemia and acute renal failure and anasarca. There were no major responses documented. Dose-dependent complement activation was demonstrated on this schedule, but not on previously evaluated schedules, of ISIS 5132 administration. In contrast to other trials of ISIS 5132, there appeared to be no consistent suppression of peripheral blood mononuclear cell c-raf-1 mRNA level on this schedule at any of the dose levels analyzed. These data suggest that the efficacy and toxicity profiles of antisense oligonucleotides may be highly dependent on the schedule of administration and support the analysis of the putative molecular target in the evaluation of novel therapeutics.

Stabilization of a degradable protein by its overexpression in Escherichia coli.

Synthesis of proteins in Escherichia coli using recombinant DNA methodology has become an important tool for isolating and studying proteins. However, the E. coli protein degradation systems can interfere with the expression of cloned genes. To examine the effect of protein degradation, we have cloned the X90 allele of the E. coli lacZ gene. The X90 allele, an ochre mutant, codes for beta-galactosidase lacking approx. 12 amino acids from the carboxyl terminus. The X90 protein is rapidly degraded in wild-type E. coli. Randomly sheared DNA fragments from lambda placZ-X90 were inserted into the EcoRI site of the plasmid pOP203-UV5-3, a derivative of pMB9 containing the lactose operator-promoter region. Recombinant plasmids that carry the lacZ-X90 gene were identified by the Lac+ phenotype of their transformants in an ochre-suppressor-containing host and the Lac- phenotype in Su degrees or supE hosts. One recombinant plasmid, p41, with an insert of 7.6 kb codes for the synthesis of the X90 promoter at a quantity equal to or greater than 50% of the total cellular protein of several strains. In contrast to the normal situation, the X90 molecules synthesized in great excess from the plasmid are stable in Su degrees hosts and can be recovered primarily from the 10 000 X g pellets of sonication lysates. The surprising stability of the overproduced X90 protein may be due to the formation of proteinaceous aggregates.

In vitro and in vivo manipulations of bacteriophage Mu DNA: cloning of Mu ends and construction of mini-Mu's carrying selectable markers.

Recombinant plasmids carrying one or both ends of the bacteriophage Mu genome were constructed by molecular cloning. Transposable mini-Mu's with selectable markers (ampicillin resistance, kanamycin resistance or the entire lac operon of Escherichia coli) inserted between the Mu ends were also constructed. As a source of lac operon DNA, a pBR322 derivative with a 27 kb insert containing the lac operon was constructed. The plasmids with both ends of Mu (mini-Mu's) conferred full Mu immunity upon the host cells. However, the same mini-Mu's containing kan or lac inserts were defective in immunity. A summary of the construction and physical characterization, including restriction endonuclease cleavage maps and some of the biological properties of the plasmids, is presented.

Toward antisense oligonucleotide therapy for cancer: ISIS compounds in clinical development.

Antisense oligonucleotides offer the promise of therapeutic effect with few toxic effects, by virtue of their high selectivity. Preclinical studies have provided evidence of antisense effects in vitro and in vivo, and phase I clinical trials have demonstrated safety, feasibility and activity of antisense oligonucleotides for the treatment of cancer. This review summarizes the status of development of three anticancer antisense oligonucleotides from ISIS Pharmaceuticals.

Target amplification systems in nucleic acid-based diagnostic approaches.

Currently, PCR is the standard method for target amplification because it is the oldest and most developed procedure. However, several new alternative approaches for target amplification have recently been developed. Although these new methods are at a relatively early stage of development, each has some advantages over PCR, such as greater amplification per cycle (TAS, 3SR, Q beta), isothermal reaction (3SR), or coupled amplification-mutation detection (LAR/LAS). As a result, each may eventually gain widespread use after further development.

The nucleotide sequence of the chicken thymidine kinase gene and the relationship of its predicted polypeptide to that of the vaccinia virus thymidine kinase.

The entire DNA nucleotide sequence of a 3.0 kilobase pair Hind III fragment containing the chicken cytoplasmic thymidine kinase gene was determined. Oligonucleotide linker insertion mutations distributed throughout this gene and having known effects upon gene activity ( Kwoh , T.J., Zipser , D., and Wigler , M. 1983. J. Mol. Appl. Genet. 2, 191-200), were used to access regions of the Hind III fragment for sequencing reactions. The complete nucleotide sequence, together with the positions of the linker insertion mutations within the sequence, allows us to propose a structure for the chicken thymidine kinase gene. The protein coding sequence of the gene is divided into seven small segments (each less than 160 base pairs) by six small introns (each less than 230 base pairs). The proposed 244 amino acid polypeptide encoded by this gene bears strong homology to the vaccinia virus thymidine kinase. No homology with the thymidine kinases of the herpes simplex viruses was found.

Amino acid sequence of the precursor of rat liver alpha 2 micro-globulin.

The amino acid sequence of the signal peptide of the rat liver protein alpha 2 micro-globulin has been determined using a combination of nucleic acid and protein sequencing techniques. The NH2-terminal portion of pre-alpha 2 micro-globulin is: (formula see text) As is the case with most secreted proteins, the majority (13 of 19) of the amino acid residues in this signal are hydrophobic. However, there are no obvious similarities between this signal sequence and that determined for preproalbumin, a protein presumably synthesized and secreted by the same cells in the liver.

Mutational analysis of the cloned chicken thymidine kinase gene.

We have analyzed the transcription and coding unit of the chicken thymidine kinase (tk) gene. We have constructed a library of mutant chicken tk genes by the in vitro linker insertion method of Heffron et al. A total of 125 mutations within a 3.0 kbp HindIII fragment containing the gene were isolated and mapped. The effect of each mutation upon the thymidine kinase gene was determined by measurement of the transfection efficiency in mouse Ltk- cells. The chicken tk mRNA is about 2 kb and polyadenylated. The direction of transcription was also determined. From these results, we propose a structure for the gene in which at least three small introns separate the amino acid coding region into at least four segments.

Analysis of substrate specificity of the PaeR7 endonuclease: effect of base methylation on the kinetics of cleavage.

In murine cells expressing the PaeR7 endonuclease and methylase genes, the recognition sites (CTCGAG) of these enzymes can be methylated at the adenine residue by the PaeR7 methylase and at the internal cytosine by the mouse DNA methyltransferase. Using nonadecameric duplex deoxyoligonucleotide substrates, the specificity of the PaeR7 endonuclease for unmethylated, hemi-methylated, and fully methylated N6-methyladenine (m6A) and C5-methylcytosine (m5C) versions of these substrates has been studied. The Km, Kcat, and Ki values for these model substrates have been measured and suggest that fully or hemi-m6A-methylated PaeR7 sites in the murine genome are completely protected. However, the reactivity of fully or hemi-m5C-methylated PaeR7 sites is depressed 2900- and 100-fold respectively, compared to unmodified PaeR7 sites. The implications of the kinetic constants of the PaeR7 endonuclease for these methylated recognition sites as they occur in murine cells expressing this endonuclease gene are discussed.

Introduction and expression of the bacterial PaeR7 restriction endonuclease gene in mouse cells containing the PaeR7 methylase.

To study the factors essential for a functional restriction system, the PaeR7 restriction-modification system has been introduced and expressed in murine cells. Transfer of this system was accomplished in two steps. First, cells containing sufficient PaeR7 methylase to completely methylate the mouse genome were constructed. In the second step, the mouse metallothionein promoter-regulated, endonuclease expression vector linked to the hygromycin B resistance selection marker was used to transfect the high methylase-expressing cells. Sixty percent of the clones isolated contained PaeR7 endonuclease enzymatic activity. Transfected cells expressing both methylase and endonuclease were incapable of blocking infection by DNA viruses, and possible explanations are discussed.

Introduction and expression of the bacterial PaeR7 methylase gene in mammalian cells.

An approach is devised for studying the role of DNA methylation in eukaryotic gene expression. The approach is based on the expression of site-specific bacterial methylase genes in animal cells. A model system using the cloned PaeR7 (an isoschizomer of Xho I) methylase gene was constructed to test the feasibility of this approach. Expression plasmids for the PaeR7 methylase gene were introduced into mouse Ltk- cells by cotransfection with the cloned chicken thymidine kinase (tk) gene. Several of the cell strains derived from Tk+ colonies were found to express the PaeR7 gene as judged by four criteria: the cellular DNA of these strains showed increased resistance to cleavage by Xho I; these strains contained cellular proteins that comigrated with pure PaeR7 methylase protein, as visualized by immunoblotting; PaeR7 methylase activity was found in vitro in crude extracts of total cellular protein from these strains; and murine adenovirus genomes grown on cells expressing PaeR7 methylase showed resistance to cleavage to PaeR7 endonuclease. The potential applications of this approach for the study of cellular and viral gene regulation, DNA repair, and restriction modification are discussed.

Optimal infectivity in vitro of human immunodeficiency virus type 1 requires an intact nef gene.

The replication competence of human immunodeficiency virus type 1 genomes containing mutations in the nef open reading frame was evaluated in continuous cell lines. Mutants that contained a deletion in the nef open reading frame, premature termination codons, or missense mutations in the N-terminal myristoylation signal were constructed. The replication of these mutants was tested in three ways. First, plasmid genomes were used to transfect T-lymphoblastoid cells. Second, low-passage posttransfection supernatants were used to infect cells with a relatively low virus input. Third, high-titer virus stocks were used to infect cells with a relatively high virus input. These experiments demonstrated a 100- to 10,000-fold decrement in p24 production by the nef mutants compared with that by the wild-type virus. The greatest difference was obtained after infection with the lowest virus input. The myristoylation signal was critical for this positive effect of nef. To investigate the mechanism of the positive influence of nef, nef-positive and nef-minus viruses were compared during a single cycle of replication. These single-cycle experiments were initiated both by infection with high-titer virus stocks and by transfection with viral DNA. Single-cycle infection yielded a three- to fivefold decrement in p24 production by nef-minus virus. Single-cycle transfection yielded equal amounts of p24 production. These results implied that nef does not affect replication after the provirus is established. In support of these results, viral production from cells chronically infected with nef-positive or nef-minus viruses was similar over time. To determine whether the effect of nef was due to infectivity, end point titrations of nef-positive and nef-minus viruses were performed. nef-positive virus had a greater infectivity per picogram of HIV p24 antigen than nef-minus virus. These data indicated that the positive influence of nef on viral growth rate is due to an infectivity advantage of virus produced with an intact nef gene.

A Phase I trial of H-ras antisense oligonucleotide ISIS 2503 administered as a continuous intravenous infusion in patients with advanced carcinoma.

BACKGROUND: Abnormal expression of Ras proteins frequently is found with oncogenic transformation making ras a promising therapeutic target. ISIS 2503 is a 20-base antisense phosphorothioate oligodeoxyribonucleotide that specifically downregulates H-ras expression and inhibits tumor cell growth in preclinical studies. Here, the authors report an initial clinical study of the safety and tolerability of an intravenous infusion of ISIS 2503 in patients with advanced cancer. METHODS: A continuous intravenous infusion of ISIS 2503 was administered for 14 days every 3 weeks to 23 patients with a variety of solid tumors refractory to standard therapy. The dose of ISIS 2503 was increased in sequential cohorts of patients, as toxicity allowed, until a final dose of 10.0 mg/kg/day of body weight was reached. Toxicity was scored by the National Cancer Institute's Common Toxicity Criteria, and tumor response was monitored after every two treatment cycles. Pharmacokinetic studies were performed in some of the patients up to, and including, the final dose of 10 mg/kg/day/day of body weight. Levels of H-ras mRNA expression also were determined in the circulating lymphocytes of some patients by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: A total of 23 patients received 63 cycles of ISIS 2503 at escalating doses to 10.0 mg/kg/day without dose-limiting toxicity and only minimal side effects. Four patients had stabilization of their disease for 6-10 cycles. No consistent decreases in H-ras mRNA levels were observed in peripheral blood lymphocytes. CONCLUSIONS: ISIS 2503, an antisense oligonucleotide against H-ras, was well tolerated as a single agent at doses up to 10.0 mg/kg/day by 14-day continuous intravenous infusion. Several patients had stabilization of disease, suggesting that ISIS 2503 had some tumor growth inhibitory effects and future trials of ISIS 2503 in combination with chemotherapy should be considered.