Dimerizer-mediated regulation of gene expression.

Several systems have been developed that allow transcription of a target gene to be chemically controlled, usually by an allosteric modulator of transcription factor activity. An alternative is to use chemical inducers of dimerization, or "dimerizers," to reconstitute active transcription factors from inactive fusion proteins. The most widely used system employs the natural product rapamycin, or a biologically inert analog, as the dimerizing drug. A key feature of this system is the tightness of regulation, with basal expression usually undetectable and induced expression levels comparable to constitutive promoters. In our experiments, the use of the minimal interleukin-2 (IL-2) promoter is an important determinant of this; substitution of a minimal simian virus 40 (SV40) or cytomegalovirus (CMV) promoter results in significantly higher levels of basal expression. The key factor dictating the successful use of the system is achieving high expression levels of the activation domain fusion protein. In the context of clinical gene therapies, the system has the advantage of being built exclusively from human proteins, potentially minimizing immunogenicity in the clinical setting. The dimerizer system has been successfully incorporated into diverse vector backgrounds and has been used to achieve long-term regulated gene expression in vitro and in vivo. This article provides guidance in designing constructs and experiments to achieve dimerizer-regulated expression of a target gene both in vitro and in vivo.

Dimerizer-mediated regulation of gene expression in vitro.

Several systems have been developed that allow transcription of a target gene to be chemically controlled, usually by an allosteric modulator of transcription factor activity. An alternative is to use chemical inducers of dimerization, or "dimerizers," to reconstitute active transcription factors from inactive fusion proteins. The most widely used system employs the natural product rapamycin, or a biologically inert analog, as the dimerizing drug. A key feature of this system is the tightness of regulation, with basal expression usually undetectable and induced expression levels comparable to constitutive promoters. In our experiments, the use of the minimal interleukin-2 (IL-2) promoter is an important determinant of this; substitution of a minimal simian virus 40 (SV40) or cytomegalovirus (CMV) promoter results in significantly higher levels of basal expression. The key factor dictating the successful use of the system is achieving high expression levels of the activation domain fusion protein. In the context of clinical gene therapies, the system has the advantage of being built exclusively from human proteins, potentially minimizing immunogenicity in the clinical setting. The dimerizer system has been successfully incorporated into diverse vector backgrounds and has been used to achieve long-term regulated gene expression in vitro and in vivo. This protocol describes the preparation of vectors for rapamycin- or rapalog-inducible gene expression, followed by induction of gene expression in vitro.

Dimerizer-mediated regulation of gene expression in vivo.

Several systems have been developed that allow transcription of a target gene to be chemically controlled, usually by an allosteric modulator of transcription factor activity. An alternative is to use chemical inducers of dimerization, or "dimerizers," to reconstitute active transcription factors from inactive fusion proteins. The most widely used system employs the natural product rapamycin, or a biologically inert analog, as the dimerizing drug. A key feature of this system is the tightness of regulation, with basal expression usually undetectable and induced expression levels comparable to constitutive promoters. In our experiments, the use of the minimal interleukin-2 (IL-2) promoter is an important determinant of this; substitution of a minimal simian virus 40 (SV40) or cytomegalovirus (CMV) promoter results in significantly higher levels of basal expression. The key factor dictating the successful use of the system is achieving high expression levels of the activation domain fusion protein. In the context of clinical gene therapies, the system has the advantage of being built exclusively from human proteins, potentially minimizing immunogenicity in the clinical setting. The dimerizer system has been successfully incorporated into diverse vector backgrounds and has been used to achieve long-term regulated gene expression in vitro and in vivo. This protocol describes how to achieve rapamycin- or rapalog-inducible gene expression in vivo.

Analysis of the pharmacodynamic activity of the mTOR inhibitor ridaforolimus (AP23573, MK-8669) in a phase 1 clinical trial.

PURPOSE: As part of a phase 1 dose-escalation trial, the pharmacodynamic activity of the mammalian target of rapamycin (mTOR) inhibitor ridaforolimus was assessed in multiple tissues by measuring levels of phosphorylated 4E binding protein-1 (p-4E-BP1) or S6, two downstream markers of mTOR activity. METHODS: 32 patients (pts) were dosed intravenously with ridaforolimus once daily for 5 consecutive days (QD × 5) every 2 weeks. The pharmacodynamic activity of ridaforolimus was assessed in peripheral blood mononuclear cells (PBMCs; 32 pts), skin (28 pts), and tumor specimens (3 pts) collected before and after dosing by measuring levels of p-4E-BP1 by immunoblot analysis or pS6 by immunohistochemistry. Levels of these markers were assessed in up to 19, 5, and 2 pre- and post-dose time points in PBMC, skin, and tumor specimens, respectively. RESULTS: In preclinical models, ridaforolimus induced a dose-dependent inhibition of p-4E-BP1 in PBMCs that was associated with antitumor activity. Rapid and potent inhibition of mTOR was observed in PBMCs from all 32 pts dosed, with a median level of inhibition of 96% observed within 1 h after the first dose. Inhibition of mTOR (>90%) was sustained during the entire QD × 5 dosing period, and substantial inhibition was still observed after the 9-day holiday between dosing courses. Evidence of mTOR inhibition was also obtained in skin in pts from all dose cohorts, although it did not persist through the break between courses. After two to three doses of ridaforolimus, inhibition of mTOR was detected in the tumor from one of three pts analyzed. CONCLUSIONS: Ridaforolimus was shown to inhibit its intended target, mTOR, in PBMCs, skin, and tumors. In PBMCs and skin, inhibition was observed at all dose levels tested, thus supporting but not driving the selection of a recommended phase 2 dose.

Ridaforolimus for patients with progressive or recurrent malignant glioma: a perisurgical, sequential, ascending-dose trial.

PURPOSE: This perisurgical phase 1 study evaluated the pharmacokinetics, pharmacodynamics, and safety of the mammalian target of rapamycin (mTOR) inhibitor ridaforolimus in patients (N = 10) with progressive or recurrent primary grade IV malignant glioma, who failed standard therapy. The primary objective of the study was to determine the maximum tolerated dose (MTD) of ridaforolimus. METHODS: Treatment was administered intravenously at doses of 12.5 mg (N = 7) or 15 mg (N = 3) once daily for 4 days prior to surgical resection, then resumed for 5 consecutive days every 2 weeks until disease progression or unacceptable toxicity, following a postsurgical recovery period. RESULTS: The MTD was not determined because the trial was suspended early due to slower than expected patient accrual and postsurgical drug administration challenges. Pharmacokinetic and pharmacodynamic analyses showed that ridaforolimus concentrations declined slowly during the 24-h dosing interval and remained detectable for 10 days after the last infusion in whole blood samples. In peripheral blood mononuclear cells, median levels of the mTOR downstream effector p4E-BP1 were reduced by >80% compared with baseline by 4 h after dosing. Resected brain specimens showed reduced levels of pS6, another mTOR downstream effector, while nuclear staining for p27(kip1), a protein that functions as a cell cycle inhibitor, increased after treatment. No dose-limiting toxicities were observed, and the reported adverse events were consistent with the previously established safety profile for ridaforolimus. One of 3 patients evaluable for efficacy had stable disease as best response. CONCLUSION: Results suggest that ridaforolimus can cross the blood-brain barrier in areas of tumor involvement, and may inhibit mTOR activity in advanced gliomas based on decreased pS6 levels. This perisurgical trial design should serve as a template for evaluating intratumoral pharmacokinetics and pharmacodynamics of other targeted agents in this patient population.

Phase II study of the mammalian target of rapamycin inhibitor ridaforolimus in patients with advanced bone and soft tissue sarcomas.

PURPOSE: Ridaforolimus is an inhibitor of mammalian target of rapamycin, an integral component of the phosphatidyl 3-kinase/AKT signaling pathway, with early evidence of activity in sarcomas. This multicenter, open-label, single-arm, phase II trial was conducted to assess the antitumor activity of ridaforolimus in patients with distinct subtypes of advanced sarcomas. PATIENTS AND METHODS: Patients with metastatic or unresectable soft tissue or bone sarcomas received ridaforolimus 12.5 mg administered as a 30-minute intravenous infusion once daily for 5 days every 2 weeks. The primary end point was clinical benefit response (CBR) rate (complete response or partial response [PR] or stable disease ≥ 16 weeks). Safety, progression-free survival (PFS), overall survival (OS), time to progression, and duration of response were also evaluated. RESULTS: A total of 212 patients were treated in four separate histologic cohorts. In this heavily pretreated population, 61 patients (28.8%) achieved CBR. Median PFS was 15.3 weeks; median OS was 40 weeks. Response Evaluation Criteria in Solid Tumors (RECIST) confirmed response rate was 1.9%, with four patients achieving confirmed PR (two with osteosarcoma, one with spindle cell sarcoma, and one with malignant fibrous histiocytoma). Archival tumor protein markers analyzed were not correlated with CBR. Related adverse events were generally mild or moderate and consisted primarily of stomatitis, mucosal inflammation, mouth ulceration, rash, and fatigue. CONCLUSION: Single-agent ridaforolimus in patients with advanced and pretreated sarcomas led to PFS results that compare favorably with historical metrics. A phase III trial based on these data will further define ridaforolimus activity in sarcomas.

Ridaforolimus (AP23573; MK-8669), a potent mTOR inhibitor, has broad antitumor activity and can be optimally administered using intermittent dosing regimens.

The mTOR pathway is hyperactivated through oncogenic transformation in many human malignancies. Ridaforolimus (AP23573; MK-8669) is a novel rapamycin analogue that selectively targets mTOR and is currently under clinical evaluation. In this study, we investigated the mechanistic basis for the antitumor activity of ridaforolimus in a range of human tumor types, exploring potential markers of response, and determining optimal dosing regimens to guide clinical studies. Administration of ridaforolimus to tumor cells in vitro elicited dose-dependent inhibition of mTOR activity with concomitant effects on cell growth and division. We showed that ridaforolimus exhibits a predominantly cytostatic mode of action, consistent with the findings for other mTOR inhibitors. Potent inhibitory effects on vascular endothelial growth factor secretion, endothelial cell growth, and glucose metabolism were also observed. Although PTEN and/or phosphorylated AKT status have been proposed as potential mTOR pathway biomarkers, neither was predictive for ridaforolimus responsiveness in the heterogeneous panel of cancer cell lines examined. In mouse models, robust antitumor activity was observed in human tumor xenografts using a series of intermittent dosing schedules, consistent with pharmacodynamic observations of mTOR pathway inhibition for at least 72 hours following dosing. Parallel skin-graft rejection studies established that intermittent dosing schedules lack the immunosuppressive effects seen with daily dosing. Overall these findings show the broad inhibitory effects of ridaforolimus on cell growth, division, metabolism, and angiogenesis, and support the use of intermittent dosing as a means to optimize antitumor activity while minimizing systemic effects.

Phase IB study of the mTOR inhibitor ridaforolimus with capecitabine.

PURPOSE: Synergistic/additive cytotoxicity in tumor models and widespread applicability of fluoropyrimidines in solid tumors prompted the study of the combination of the mammalian target of rapamycin (mTOR) inhibitor, non-prodrug rapamycin analog ridaforolimus, with capecitabine. PATIENTS AND METHODS: Thirty-two adult patients were treated. Intravenous ridaforolimus was given once weekly for 3 weeks and capecitabine was given from days 1 to 14 every 4 weeks. Ridaforolimus was given at 25, 37.5, 50, or 75 mg with capecitabine at 1,650 mg/m(2) or 1,800 mg/m(2) divided into two daily doses. Pharmacokinetics of both drugs were determined during cycles 1 and 2. Pharmacodynamic studies in peripheral blood mononuclear cells (PBMCs) and wound tissue of the skin characterized pathways associated with the metabolism or disposition of fluoropyrimidines and mTOR and ERK signaling. RESULTS: Two recommended doses (RDs) were defined: 75 mg ridaforolimus/1,650 mg/m(2) capecitabine and 50 mg ridaforolimus/1,800 mg/m(2) capecitabine. Dose-limiting toxicities were stomatitis and skin rash. One patient achieved a partial response lasting 10 months and 10 of 29 evaluable patients had stable disease for ≥ 6 months. The only pharmacokinetic interaction was a ridaforolimus-induced increase in plasma exposure to fluorouracil. PBMC data suggested that prolonged exposure to capecitabine reduced the ridaforolimus inhibition of mTOR. Ridaforolimus influenced the metabolism of fluoropyrimidines and inhibited dihydropyrimidine dehydrogenase, behavior similar to that of rapamycin. Inhibition of the target thymidylate synthase by capecitabine was unaffected. mTOR and ERK signaling was inhibited in proliferating endothelial cells and was more pronounced at the RD with the larger amount of ridaforolimus. CONCLUSION: Good tolerability, feasibility of prolonged treatment, antitumor activity, and favorable pharmacologic profile support further investigation of this combination.

A phase I trial to determine the safety, tolerability, and maximum tolerated dose of deforolimus in patients with advanced malignancies.

PURPOSE: This was a phase I trial to determine the maximum tolerated dose and toxicity of deforolimus (AP23573, MK-8669), an inhibitor of mammalian target of rapamycin (mTOR). The pharmacokinetics, pharmacodynamics, and antineoplastic effects were also studied. EXPERIMENTAL DESIGN: Deforolimus was administered intravenously over 30 min every 7 days according to a flat dosing schedule. Dose was escalated according to an accelerated titration design. Patients remained on study until disease progression as long as they tolerated the drug without significant toxicities. RESULTS: Forty-six patients were enrolled on the study. Common side effects included fatigue, anorexia, and mucositis. The maximum tolerated dose was 75 mg and mucositis was the dose-limiting toxicity. Similar to other mTOR inhibitors, deforolimus exhibited nonlinear pharmacokinetics and a prolonged half-life. Among 34 patients evaluable for response, 1 patient had a partial response, 21 patients had stable disease, and 12 had progressed. Percent change in tumor size was significantly associated with AUC (P=0.015). A significant association was also detected for maximum change in cholesterol within the first two cycles of therapy and change in tumor size (r=-0.38; P=0.029). CONCLUSIONS: Deforolimus was well tolerated on the schedule tested in this trial with toxicity and pharmacokinetic profiles that were similar to that of other mTOR inhibitors. Additional phase II studies are needed to determine if deforolimus is superior to other mTOR inhibitors in terms of efficacy. The change in serum cholesterol as a potential biomarker of activity should be studied further.

Dimerizer regulation of AADC expression and behavioral response in AAV-transduced 6-OHDA lesioned rats.

Recombinant AAV vectors containing a dimerizer-inducible system of transcriptional activation provide a strategy for control of therapeutic gene expression in the CNS. Here we explored this system for regulated expression of human aromatic L-amino acid decarboxylase (hAADC) in a rodent model of Parkinson disease. Expression of hAADC, the enzyme that converts L-dopa to dopamine, was dependent on reconstitution of a functional transcription factor (TF) by the dimerizer rapamycin. Two vectors, AAV-CMV-TF and AAV-Z12-hAADC, were infused into striata of 6-OHDA-lesioned rats. Rapamycin-induced increases in expression of hAADC repeatedly produced robust rotational behavior in response to low doses of L-dopa. Seven weeks after vector infusion, AADC expression in brain was quantitated by both stereology and Western blot analysis following the final rapamycin treatment. While a low level of hAADC was observed in rats that were not induced with rapamycin, this basal expression was not significant enough to elicit a rotational response to L-dopa. This study demonstrated a robust behavioral response of parkinsonian rats to regulated hAADC expression. Recombinant AAV vectors controlled by rapamycin or its analogs show promise as candidates for CNS therapies in which regulation of the transgene is desired.

Detection and frequency estimation of rare variants in pools of genomic DNA from large populations using mutational spectrometry.

DNA variants underlying the inheritance of risk for common diseases are expected to have a wide range of population allele frequencies. The detection and scoring of the rare alleles (at frequencies of <0.01) presents significant practical problems, including the requirement for large sample sizes and the limitations inherent in current methodologies for allele discrimination. In the present report, we have applied mutational spectrometry based on constant denaturing capillary electrophoresis (CDCE) to DNA pools from large populations in order to improve the prospects of testing the role of rare variants in common diseases on a large scale. We conducted a pilot study of the cytotoxic T lymphocyte-associated antigen-4 gene (CTLA4) in type 1 diabetes (T1D). A total of 1228 bp, comprising 98% of the CTLA4 coding sequence, all adjacent intronic mRNA splice sites, and a 3' UTR sequence were scanned for unknown point mutations in pools of genomic DNA from a control population of 10,464 young American adults and two T1D populations, one American (1799 individuals) and one from the United Kingdom (2102 individuals). The data suggest that it is unlikely that rare variants in the scanned regions of CTLA4 represent a significant proportion of T1D risk and illustrate that CDCE-based mutational spectrometry of DNA pools offers a feasible and cost-effective means of testing the role of rare variants in susceptibility to common diseases.