RNAi mediated silencing of STAT3/PD-L1 in tumor-associated immune cells induces robust anti-tumor effects in immunotherapy resistant tumors.

Malignant tumors are often associated with an immunosuppressive tumor microenvironment (TME), rendering most of them resistant to standard-of-care immune checkpoint inhibitors (CPIs). Signal transducer and activator of transcription 3 (STAT3), a ubiquitously expressed transcription factor, has well-defined immunosuppressive functions in several leukocyte populations within the TME. Since the STAT3 protein has been challenging to target using conventional pharmaceutical modalities, we investigated the feasibility of applying systemically delivered RNA interference (RNAi) agents to silence its mRNA directly in tumor-associated immune cells. In preclinical rodent tumor models, chemically stabilized acylated small interfering RNAs (siRNAs) selectively silenced Stat3 mRNA in multiple relevant cell types, reduced STAT3 protein levels, and increased cytotoxic T cell infiltration. In a murine model of CPI-resistant pancreatic cancer, RNAi-mediated Stat3 silencing resulted in tumor growth inhibition, which was further enhanced in combination with CPIs. To further exemplify the utility of RNAi for cancer immunotherapy, this technology was used to silence Cd274, the gene encoding the immune checkpoint protein programmed death-ligand 1 (PD-L1). Interestingly, silencing of Cd274 was effective in tumor models that are resistant to PD-L1 antibody therapy. These data represent the first demonstration of systemic delivery of RNAi agents to the TME and suggest applying this technology for immuno-oncology applications.

Nucleic Acid Polymers with Accelerated Plasma and Tissue Clearance for Chronic Hepatitis B Therapy.

REP 2139 is a nucleic acid polymer (NAP) currently under clinical development for chronic hepatitis B (HBV) therapy. This preclinical study investigated different REP 2139 analogs that would display reduced accumulation in the serum and tissues, while retaining an antiviral effect against HBV infection. REP 2139 analogs were evaluated in human plasma, CD-1 mice, cynomolgus monkeys, and Pekin ducks. Discrete ribose transformation to 2'OH in selected riboadenosines resulted in a slow degradation in acidified human plasma that plateaued after 48 hr. REP 2165, a REP 2139 analog containing three unmodified riboadenosines equally spaced throughout the polymer, showed similar plasma clearance and tissue distribution as REP 2139 in mice and cynomolgus monkeys after a single dose. Interestingly, after repeated administration, accumulation of REP 2165 in plasma and organs was reduced, indicating a dramatically faster rate of clearance from organs after therapy was ended in both species. Both REP 2139 and REP 2165 were well tolerated at clinically relevant doses, with no alterations in liver, kidney, or hematological function. In chronic duck HBV (DHBV) infection, REP 2165 displayed significantly reduced liver accumulation after repeated dosing but retained antiviral activity similar to REP 2139. These results indicate the therapeutic potential of REP 2165 against chronic HBV infection in patients is similar to REP 2139, but with significantly reduced drug accumulation and improved tissue clearance.

Recommendations of the Oligonucleotide Safety Working Group's Formulated Oligonucleotide Subcommittee for the Safety Assessment of Formulated Oligonucleotide-Based Therapeutics.

The use of lipid formulations has greatly improved the ability to effectively deliver oligonucleotides and has been instrumental in the rapid expansion of therapeutic development programs using oligonucleotide drugs. However, the development of such complex multicomponent therapeutics requires the implementation of unique, scientifically sound approaches to the nonclinical development of these drugs, based upon a hybrid of knowledge and experiences drawn from small molecule, protein, and oligonucleotide therapeutic drug development. The relative paucity of directly applicable regulatory guidance documents for oligonucleotide therapeutics in general has resulted in the generation of multiple white papers from oligonucleotide drug development experts and members of the Oligonucleotide Safety Working Group (OSWG). The members of the Formulated Oligonucleotide Subcommittee of the OSWG have utilized their collective experience working with a variety of formulations and their associated oligonucleotide payloads, as well as their insights into regulatory considerations and expectations, to generate a series of consensus recommendations for the pharmacokinetic characterization and nonclinical safety assessment of this unique class of therapeutics. It should be noted that the focus of Subcommittee discussions was on lipid nanoparticle and other types of particulate formulations of therapeutic oligonucleotides and not on conjugates or other types of modifications of oligonucleotide structure intended to facilitate delivery.

Development of pre-clinical models for evaluating the therapeutic potential of candidate siRNA targeting STAT6.

Developing siRNA therapeutics poses technical challenges including appropriate molecular design and testing in suitable pre-clinical models. We previously detailed sequence-selection and modification strategies for siRNA candidates targeting STAT6. Here, we describe methodology that evaluates the suitability of candidate siRNA for respiratory administration. Chemically-modified siRNA exhibited similar inhibitory activity (IC50) against STAT6 in vitro compared to unmodified siRNA and apical exposure testing with Caco-2 cell monolayers showed modification was not associated with cellular toxicity. Use of a modified RNA extraction protocol improved the sensitivity of a PCR-based bio-analytical assay (lower limit of siRNA strand quantification  =  0.01 pg/µl) which was used to demonstrate that lung distribution profiles for both siRNAs were similar following intra-tracheal administration. However, after 6 hours, modified siRNA was detected in lung tissue at concentrations >1000-fold higher than unmodified siRNA. Evaluation in a rat model of allergic inflammation confirmed the persistence of modified siRNA in vivo, which was detectable in broncho-alveolar lavage (BAL) fluid, BAL cells and lung tissue samples, 72 hours after dosing. Based upon the concept of respiratory allergy as a single airway disease, we considered nasal delivery as a route for respiratory targeting, evaluating an intra-nasal exposure model that involved simple dosing followed by fine dissection of the nasal cavity. Notably, endogenous STAT6 expression was invariant throughout the nasal cavities and modified siRNA persisted for at least 3 days after administration. Coupled with our previous findings showing upregulated expression of inflammatory markers in nasal samples from asthmatics, these findings support the potential of intranasal siRNA delivery. In summary, we demonstrate the successful chemical modification of STAT6 targeting siRNA, which enhanced bio-availability without cellular toxicity or reduced efficacy. We have established a robust, sensitive method for determining siRNA bio-distribution in vivo, and developed a nasal model to aid evaluation. Further work is warranted.

Expression of CD1c enhances human invariant NKT cell activation by α-GalCer.

Invariant natural killer T (iNKT) cells are innate T lymphocytes that specifically recognize α-linked glycosphingolipids (α-GSLs) as antigens presented by CD1d molecules. Activating iNKT cells by administering α-GSLs improves disease outcomes in murine cancer models and, thus, there is great interest in the clinical potential of these lipids for treating human cancers. However, humans possess several other CD1 isoforms that are not present in mice and it is not clear whether these CD1 molecules, which also bind lipids, affect human iNKT cell responses. We demonstrate here that CD1c, which is co-expressed with CD1d on blood dendritic cells and on a fraction of B cells, is able to present α-galactosylceramide (α-GalCer) as a weak agonist to human iNKT cells, and that the presence of CD1c synergistically enhances α-GalCerdependent activation of iNKT cells by CD1d. Primary human B cells expressing CD1c induced stronger iNKT cell responses to α-GalCer than the CD1c- subset, and an antibody against CD1c inhibited iNKT cell cytokine secretion. These results suggest that therapeutic activation of human iNKT cells by α-GSLs will be driven preferentially by CD1c+ cell types. Thus, B cell neoplasias that co-express CD1c and CD1d may be particularly susceptible to α-GSL therapy, and cancer vaccines using α-GSLs as adjuvants may be most effective when presented by CD1c+ antigen-presenting cells.

Mice engrafted with human fetal thymic tissue and hematopoietic stem cells develop pathology resembling chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is a significant roadblock to long-term hematopoietic stem cell (HSC) transplantation success. Effective treatments for cGVHD have been difficult to develop, in part because of a paucity of animal models that recapitulate the multiorgan pathologies observed in clinical cGVHD. Here we present an analysis of the pathology that occurs in immunodeficient mice engrafted with human fetal HSCs and implanted with fragments of human fetal thymus and liver. Starting at time points generally later than 100 days post-transplantation, the mice developed signs of illness, including multiorgan cellular infiltrates containing human T cells, B cells, and macrophages; fibrosis in sites such as lungs and liver; and thickened skin with alopecia. Experimental manipulations that delayed or reduced the efficiency of the HSC engraftment did not affect the timing or progression of disease manifestations, suggesting that pathology in this model is driven more by factors associated with the engrafted human thymic organoid. Disease progression was typically accompanied by extensive fibrosis and degradation of the thymic organoid, and there was an inverse correlation of disease severity with the frequency of FoxP3(+) thymocytes. Hence, the human thymic tissue may contribute T cells with pathogenic potential, but the generation of regulatory T cells in the thymic organoid may help to control these cells before pathology resembling cGVHD eventually develops. This model thus provides a new system to investigate disease pathophysiology relating to human thymic events and to evaluate treatment strategies to combat multiorgan fibrotic pathology produced by human immune cells.

Analysis of the CD1 antigen presenting system in humanized SCID mice.

CD1 molecules are glycoproteins that present lipids and glycolipids for recognition by T cells. CD1-dependent immune activation has been implicated in a wide range of immune responses, however, our understanding of the role of this pathway in human disease remains limited because of species differences between humans and other mammals: whereas humans express five different CD1 gene products (CD1a, CD1b, CD1c, CD1d, and CD1e), muroid rodents express only one CD1 isoform (CD1d). Here we report that immune deficient mice engrafted with human fetal thymus, liver, and CD34(+) hematopoietic stem cells develop a functional human CD1 compartment. CD1a, b, c, and d isoforms were highly expressed by human thymocytes, and CD1a(+) cells with a dendritic morphology were present in the thymic medulla. CD1(+) cells were also detected in spleen, liver, and lungs. APCs from spleen and liver were capable of presenting bacterial glycolipids to human CD1-restricted T cells. ELISpot analyses of splenocytes demonstrated the presence of CD1-reactive IFN-γ producing cells. CD1d tetramer staining directly identified human iNKT cells in spleen and liver samples from engrafted mice, and injection of the glycolipid antigen α-GalCer resulted in rapid elevation of human IFN-γ and IL-4 levels in the blood indicating that the human iNKT cells are biologically active in vivo. Together, these results demonstrate that the human CD1 system is present and functionally competent in this humanized mouse model. Thus, this system provides a new opportunity to study the role of CD1-related immune activation in infections to human-specific pathogens.

Human NKT cells direct the differentiation of myeloid APCs that regulate T cell responses via expression of programmed cell death ligands.

NKT cells are innate lymphocytes that can recognize self or foreign lipids presented by CD1d molecules. NKT cells have been shown to inhibit the development of autoimmunity in murine model systems, however, the pathways by which they foster immune tolerance remain poorly understood. Here we show that autoreactive human NKT cells stimulate monocytes to differentiate into myeloid APCs that have a regulatory phenotype characterized by poor conjugate formation with T cells. The NKT cell instructed myeloid APCs show elevated expression of the inhibitory ligand PD-L2, and blocking PD-L1 and PD-L2 during interactions of the APCs with T cells results in improved cluster formation and significantly increased T cell proliferative responses. The elevated expression of PD-L molecules on NKT-instructed APCs appears to result from exposure to extracellular ATP that is produced during NKT-monocyte interactions, and blocking purinergic signaling during monocyte differentiation results in APCs that form clusters with T cells and stimulate their proliferation. Finally, we show that human monocytes and NKT cells that are injected into immunodeficient mice co-localize together in spleen and liver, and after 3 days in vivo in the presence of NKT cells a fraction of the myeloid cells have upregulated markers associated with differentiation into professional APCs. These results suggest that autoreactive human NKT cells may promote tolerance by inducing the differentiation of regulatory myeloid APCs that limit T cell proliferation through expression of PD-L molecules.

Virally delivered channelrhodopsin-2 safely and effectively restores visual function in multiple mouse models of blindness.

Previous work established retinal expression of channelrhodopsin-2 (ChR2), an algal cation channel gated by light, restored physiological and behavioral visual responses in otherwise blind rd1 mice. However, a viable ChR2-based human therapy must meet several key criteria: (i) ChR2 expression must be targeted, robust, and long-term, (ii) ChR2 must provide long-term and continuous therapeutic efficacy, and (iii) both viral vector delivery and ChR2 expression must be safe. Here, we demonstrate the development of a clinically relevant therapy for late stage retinal degeneration using ChR2. We achieved specific and stable expression of ChR2 in ON bipolar cells using a recombinant adeno-associated viral vector (rAAV) packaged in a tyrosine-mutated capsid. Targeted expression led to ChR2-driven electrophysiological ON responses in postsynaptic retinal ganglion cells and significant improvement in visually guided behavior for multiple models of blindness up to 10 months postinjection. Light levels to elicit visually guided behavioral responses were within the physiological range of cone photoreceptors. Finally, chronic ChR2 expression was nontoxic, with transgene biodistribution limited to the eye. No measurable immune or inflammatory response was observed following intraocular vector administration. Together, these data indicate that virally delivered ChR2 can provide a viable and efficacious clinical therapy for photoreceptor disease-related blindness.

Identification of small interfering RNA targeting Signal Transducer and Activator of Transcription 6: Characterisation and selection of candidates for pre-clinical development.

The interleukin (IL)-13 pathway and its associated transcription factor, signal transducer and activator of transcription 6 (STAT6), have been clearly implicated in the pathogenesis of bronchial asthma. We have developed a system to effectively screen the STAT6 gene for targeting with small interfering (si) RNA molecules. By incorporating an in silico and in vitro screening system we were able to identify fourteen siRNA molecules suitable for pre-clinical drug development. Furthermore, we were able to demonstrate that modification of certain siRNAs, designed to improve in vivo longevity, was possible without significant loss of target knockdown efficacy and that the siRNA produced by our selection process did not induce demonstrable interferon responses. These data suggest that several STAT6-targeting siRNA suitable for pre-clinical development are available for potential use in the treatment of asthma.

RNAi-based treatment for neovascular age-related macular degeneration by Sirna-027.

PURPOSE: To assess the safety, tolerability, pharmacokinetics, and dose-limiting toxicity of single intravitreal injection of Sirna-027, a small interfering RNA targeting vascular endothelial growth factor receptor-1, in patients with choroidal neovascularization (CNV) resulting from neovascular age-related macular degeneration (AMD). Secondary objectives included assessment of anatomic changes in retinal thickness, size of CNV, and changes in visual acuity. DESIGN: Prospective, open-label, single-dose, dose-escalation phase 1 study. METHODS: Twenty-six eyes of 26 patients with a median age of 82 years and CNV resulting from AMD who had previous treatments with other therapies were treated at 2 academic retinal practices. Patients received a single dose of Sirna-027 (100, 200, 400, 800, 1200, or 1600 microg/eye). Blood was sampled for pharmacokinetic analysis at 1, 4, and 24 hours after injection and on day 7. Patients underwent ophthalmic examinations including visual acuity, fluorescein angiography, and optical coherence tomography at screening and days 7, 14, 28, and 84. The main outcome measures were adverse reactions and dose-limiting toxicities. RESULTS: Intravitreal injection of a single dose of Sirna-027 from 100 to 1600 microg was well tolerated in patients with AMD, with no dose-limiting toxicity found. Adverse events were mild to moderate in severity. Adjusted mean foveal thickness decreased within 2 weeks after study treatment. The decrease was most pronounced in the 100- and 200-microg doses. CONCLUSIONS: A single intravitreal dose of Sirna-027 up to 1600 microg/eye was well tolerated in patients with CNV resulting from neovascular AMD that had been refractory to other therapies. Stabilization or improvement in visual acuity and foveal thickness was observed. No dose-response or dose-limiting effects were noted.

Recognition of lyso-phospholipids by human natural killer T lymphocytes.

Natural killer T (NKT) cells are a subset of T lymphocytes with potent immunoregulatory properties. Recognition of self-antigens presented by CD1d molecules is an important route of NKT cell activation; however, the molecular identity of specific autoantigens that stimulate human NKT cells remains unclear. Here, we have analyzed human NKT cell recognition of CD1d cellular ligands. The most clearly antigenic species was lyso-phosphatidylcholine (LPC). Diacylated phosphatidylcholine and lyso-phosphoglycerols differing in the chemistry of the head group stimulated only weak responses from human NKT cells. However, lyso-sphingomyelin, which shares the phosphocholine head group of LPC, also activated NKT cells. Antigen-presenting cells pulsed with LPC were capable of stimulating increased cytokine responses by NKT cell clones and by freshly isolated peripheral blood lymphocytes. These results demonstrate that human NKT cells recognize cholinated lyso-phospholipids as antigens presented by CD1d. Since these lyso-phospholipids serve as lipid messengers in normal physiological processes and are present at elevated levels during inflammatory responses, these findings point to a novel link between NKT cells and cellular signaling pathways that are associated with human disease pathophysiology.

Liquid chromatography electrospray ionization mass spectrometry analysis of the ocular metabolites from a short interfering RNA duplex.

The ocular metabolism of an siRNA duplex, SIRNA-027, was examined by ion-pair reversed-phase liquid chromatography (IP-RP-LC) coupled to electrospray ionization mass spectrometry (ESI-MS). The RNA duplex was injected intraocularly into the eyes of New Zealand white rabbits. Rabbits were sacrificed at different timepoints and the vitreous and retina/choroid tissue analyzed for siRNA by IP-RP-LC-MS. The method used a hexafluoroisopropanol (HFIP)/triethylamine (TEA) ion-pairing buffer with a methanol gradient. Using electrospray ionization, the duplex was preserved in the gas phase for analysis by a triple quadrupole mass spectrometer. With this methodology metabolites from rabbit ocular vitreous humor and retina/choroid tissue were identified and a pattern of siRNA degradation was established. Results showed that the duplex was metabolized predominantly from one end. This end of the siRNA duplex was calculated to have the weakest binding energy of the two ends indicating that the ability of the siRNA to split into single strands is a factor in its degradation.

Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs.

The efficacy of lipid-encapsulated, chemically modified short interfering RNA (siRNA) targeted to hepatitis B virus (HBV) was examined in an in vivo mouse model of HBV replication. Stabilized siRNA targeted to the HBV RNA was incorporated into a specialized liposome to form a stable nucleic-acid-lipid particle (SNALP) and administered by intravenous injection into mice carrying replicating HBV. The improved efficacy of siRNA-SNALP compared to unformulated siRNA correlates with a longer half-life in plasma and liver. Three daily intravenous injections of 3 mg/kg/day reduced serum HBV DNA >1.0 log(10). The reduction in HBV DNA was specific, dose-dependent and lasted for up to 7 d after dosing. Furthermore, reductions were seen in serum HBV DNA for up to 6 weeks with weekly dosing. The advances demonstrated here, including persistence of in vivo activity, use of lower doses and reduced dosing frequency are important steps in making siRNA a clinically viable therapeutic approach.

A phase I clinical trial of a ribozyme-based angiogenesis inhibitor targeting vascular endothelial growth factor receptor-1 for patients with refractory solid tumors.

PURPOSE: This study intended to determine the maximum tolerated dose, safety, pharmacokinetic variables, clinical response, and pharmacodynamic markers of daily s.c. administration of Angiozyme. PATIENTS AND METHODS: Patients with refractory solid tumors were enrolled in a dose escalation and expanded cohort design. Dose escalation involved cohorts of patients at doses of 10, 30, 100, or 300 mg/m(2)/d for 29 days. A second component enrolled 15 additional patients at a daily dose of 100 mg/m(2). Patients were eligible to continue on therapy until disease progression. RESULTS: Thirty-one patients were enrolled and 28 were evaluable (range, 29-505 days; median, 89.5 days). A maximum tolerated dose was not defined by toxicity but rather by the maximal deliverable dose of 300 mg/m(2)/d. Grade 1 to 2 injection site reactions were the most common toxicities. One patient in the 300 mg/m(2) group experienced a reversible grade 3 injection site reaction. Angiozyme showed dose-dependent plasma concentrations with good bioavailability. Surrogate markers showed Angiozyme localization in tumor biopsies and a significant increase in serum von Willebrand factor antigen, a marker for endothelial cell dysfunction. Although Angiozyme-reactive antibody production was noted for some patients, no antibody-related adverse events were noted. Seven of 28 (25%) evaluable patients had stable disease for >or =6 months, with the longest treatment duration of > or =16 months. Two patients (nasopharyngeal carcinoma and melanoma) showed minor responses. CONCLUSION: Angiozyme was well tolerated with satisfactory pharmacokinetic variables for daily s.c. dosing. Results have provided the basis for subsequent clinical trials of this first-of-class biologically targeted therapeutic.

Activity of stabilized short interfering RNA in a mouse model of hepatitis B virus replication.

To develop synthetic short interfering RNA (siRNA) molecules as therapeutic agents for systemic administration in vivo, chemical modifications were introduced into siRNAs targeted to conserved sites in hepatitis B virus (HBV) RNA. These modifications conferred significantly prolonged stability in human serum compared with unmodified siRNAs. Cell culture studies revealed a high degree of gene silencing after treatment with the chemically modified siRNAs. To assess activity of the stabilized siRNAs in vivo initially, an HBV vector-based model was used in which the siRNA and the HBV vector were codelivered via high-volume tail vein injection. More than a 3 log10 decrease in levels of serum HBV DNA and hepatitis B surface antigen, as well as liver HBV RNA, were observed in the siRNA-treated groups compared with the control siRNA-treated and saline groups. Furthermore, the observed decrease in serum HBV DNA was 1.5 log10 more with stabilized siRNA compared with unmodified siRNA, indicating the value of chemical modification in therapeutic applications of siRNA. In subsequent experiments, standard systemic intravenous dosing of stabilized siRNA 72 hours after injection of the HBV vector resulted a 0.9 log10 reduction of serum HBV DNA levels after 2 days of dosing. In conclusion, these experiments establish the strong impact that siRNAs can have on the extent of HBV infection and underscore the importance of stabilization of siRNA against nuclease degradation.

Safety and pharmacokinetic study of RPI.4610 (ANGIOZYME), an anti-VEGFR-1 ribozyme, in combination with carboplatin and paclitaxel in patients with advanced solid tumors.

PURPOSE: RPI.4610 (ANGIOZYME) is a chemically stabilized ribozyme targeting vascular endothelial growth factor receptor 1. The purpose of this study was to evaluate the safety and pharmacokinetics of RPI.4610 in combination with carboplatin and paclitaxel in patients with advanced solid tumors. METHODS: The study used a sequential treatment design evaluating a single dose level for all three drugs: paclitaxel 175 mg m-2 and carboplatin AUC=6 on day 1 of a 21-day cycle, and RPI.4610 100 mg m-2 day-1 beginning on day 8 and continuing daily thereafter. Pharmacokinetic samples were drawn on day 1 of courses 1 (chemotherapy alone) and 2 (chemotherapy+RPI.4610), and on day 8 of course 1 (RPI.4610 alone). Ratios were generated by comparing the pharmacokinetic parameters for the combination of carboplatin with paclitaxel when administered alone or together with RPI.4610. RESULTS: Twelve patients were enrolled in this trial and received two to six courses of treatment each. The most common grade 3-4 toxicities were neutropenia (three patients), thrombocytopenia (three patients), pain (three patients), anemia (two patients) and fatigue (two patients). The ratio of the mean maximum plasma concentration (Cmax) for carboplatin when administered with paclitaxel alone versus when administered with paclitaxel and RPI.4610 was 1.07 (90% confidence interval, 0.77-1.37). Similarly, the ratio of the mean AUC0-last for carboplatin was 1.04 (0.73-1.35). For paclitaxel the ratio of the mean Cmax when administered with carboplatin alone versus with carboplatin and RPI.4610 was 1.17 (1.03-1.31), and the ratio of the mean AUC0-last was 1.17 (1.04-1.30). Objective tumor responses were observed and included one patient with a complete response (bladder cancer) and one patient with a partial response (esophageal cancer). CONCLUSIONS: These results indicate that RPI.4610, carboplatin, and paclitaxel can be administered safely in combination without substantial pharmacokinetic interactions.

Development and validation of a sensitive, specific, and rapid hybridization-ELISA assay for determination of concentrations of a ribozyme in biological matrices.

Ribozymes are RNA or modified RNA polymers capable of catalyzing cleavage reactions in target strands RNA, and are under development as human therapeutics. Previous methods used for quantitation of nucleic acid polymers in serum or plasma required extraction of the polymer followed by capillary electrophoresis, HPLC, or gel electrophoresis. These methods are time consuming and lack sensitivity. A bioanalytical method has been developed that does not require extraction of the ribozyme analyte from serum. This technique relies on hybridization of the ribozyme molecule to two complementary biotin and digoxigenin labeled oligonucleotide probes. Serum containing the ribozyme is mixed with the labeled probes, and the mixture is heated at 75 degrees C for 5 min to disrupt the ribozyme secondary structure. Samples are then cooled to permit probe annealing and are added to a streptavidin-coated 96-well plate. The bound complex is detected with an anti-digoxigenin alkaline phosphatase (AP) conjugate using PNPP (p-nitrophenyl phosphate) as a substrate. The amount of colored product is measured on a microtiter plate reader at a wavelength of 405 nm. Concentrations of unknown ribozyme samples are estimated based on a standard curve (0.37-270 ng/ml) prepared in serum. The validated lower and upper limits of quantification are 5.0 and 120 ng/ml, respectively. The assay can be completed in approximately 5h and does not require extraction procedures or electrophoretic/chromatographic separation. It is therefore a simple, sensitive and rapid technique. This assay has been validated and has been used for quantitation of serum levels of the HEPTAZYME ribozyme in mouse, monkey, and human pharmacokinetic studies.