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1.
Nucleic Acid Ther ; 32(6): 473-485, 2022 12.
Article in English | MEDLINE | ID: mdl-36355073

ABSTRACT

Nucleic acid-based phosphorothioate containing antisense oligonucleotides (PS-ASOs) have the potential to activate cellular innate immune responses, and the level of activation can vary quite dramatically with sequence. Minimizing the degree of proinflammatory effect is one of the main selection criteria for compounds intended to move into clinical trials. While a recently developed human peripheral blood mononuclear cell (hPBMC)-based assay showed excellent ability to detect innate immune active PS-ASOs, which can then be discarded from the developmental process, this assay is highly resource intensive and easily affected by subject variability. This compelled us to develop a more convenient high-throughput assay. In this study, we describe a new in vitro assay, utilizing a cultured human Bjab cell line, which was developed and validated to identify PS-ASOs that may cause innate immune activation. The assay was calibrated to replicate results from the hPBMC assay. The Bjab assay was designed to be high throughput and more convenient by using RT-qPCR readout of mRNA of the chemokine Ccl22. The Bjab assay was also shown to be highly reproducible and to provide a large dynamic range in determining the immune potential of PS-ASOs through comparison to known benchmark PS-ASO controls that were previously shown to be safe or inflammatory in clinical trials. In addition, we demonstrate that Bjab cells can be used to provide mechanistic information on PS-ASO TLR9-dependent innate immune activation.


Subject(s)
Burkitt Lymphoma , Oligonucleotides, Antisense , Humans , Oligonucleotides, Antisense/genetics , Burkitt Lymphoma/genetics , Burkitt Lymphoma/therapy , Leukocytes, Mononuclear , Toll-Like Receptor 9/genetics
2.
Nucleic Acid Ther ; 32(6): 457-472, 2022 12.
Article in English | MEDLINE | ID: mdl-35976085

ABSTRACT

A human peripheral blood mononuclear cell (PBMC)-based assay was developed to identify antisense oligonucleotide (ASO) with the potential to activate a cellular innate immune response outside of an acceptable level. The development of this assay was initiated when ISIS 353512 targeting the messenger ribonucleic acid for human C-reactive protein (CRP) was tested in a phase I clinical trial, in which healthy human volunteers unexpectedly experienced increases in interleukin-6 (IL-6) and CRP. This level of immune stimulation was not anticipated following rodent and nonhuman primate safety studies in which no evidence of exaggerated proinflammatory effects were observed. The IL-6 increase induced by ISIS 353512 was caused by activation of B cells. The IL-6 induction was inhibited by chloroquine pretreatment of PBMCs and the nature of ASOs suggested that the response is mediated by a Toll-like receptor (TLR), in all likelihood TLR9. While assessing the inter PBMC donor variability, two classes of human PBMC responders to ISIS 353512 were identified (discriminator and nondiscriminators). The discriminator donor PBMCs were shown to produce low level of IL-6 after 24 h in culture, in the absence of ASO treatment. The PBMC assay using discriminator donors was shown to be reproducible, allowing to assess reliably the immune potential of ASOs by comparison to known benchmark ASO controls that were previously shown to be either safe or inflammatory in clinical trials. Clinical Trial registration numbers: NCT00048321 NCT00330330 NCT00519727.


Subject(s)
Leukocytes, Mononuclear , Oligonucleotides, Antisense , Humans , Oligonucleotides, Antisense/genetics , Healthy Volunteers , Interleukin-6/genetics
3.
ESC Heart Fail ; 8(1): 652-661, 2021 02.
Article in English | MEDLINE | ID: mdl-33283485

ABSTRACT

AIMS: Amyloidogenic transthyretin (ATTR) amyloidosis is a fatal disease characterized by progressive cardiomyopathy and/or polyneuropathy. AKCEA-TTR-LRx (ION-682884) is a ligand-conjugated antisense drug designed for receptor-mediated uptake by hepatocytes, the primary source of circulating transthyretin (TTR). Enhanced delivery of the antisense pharmacophore is expected to increase drug potency and support lower, less frequent dosing in treatment. METHODS AND RESULTS: AKCEA-TTR-LRx demonstrated an approximate 50-fold and 30-fold increase in potency compared with the unconjugated antisense drug, inotersen, in human hepatocyte cell culture and mice expressing a mutated human genomic TTR sequence, respectively. This increase in potency was supported by a preferential distribution of AKCEA-TTR-LRx to liver hepatocytes in the transgenic hTTR mouse model. A randomized, placebo-controlled, phase 1 study was conducted to evaluate AKCEA-TTR-LRx in healthy volunteers (ClinicalTrials.gov: NCT03728634). Eligible participants were assigned to one of three multiple-dose cohorts (45, 60, and 90 mg) or a single-dose cohort (120 mg), and then randomized 10:2 (active : placebo) to receive a total of 4 SC doses (Day 1, 29, 57, and 85) in the multiple-dose cohorts or 1 SC dose in the single-dose cohort. The primary endpoint was safety and tolerability; pharmacokinetics and pharmacodynamics were secondary endpoints. All randomized participants completed treatment. No serious adverse events were reported. In the multiple-dose cohorts, AKCEA-TTR-LRx reduced TTR levels from baseline to 2 weeks after the last dose of 45, 60, or 90 mg by a mean (SD) of -85.7% (8.0), -90.5% (7.4), and -93.8% (3.4), compared with -5.9% (14.0) for pooled placebo (P < 0.001). A maximum mean (SD) reduction in TTR levels of -86.3% (6.5) from baseline was achieved after a single dose of 120 mg AKCEA-TTR-LRx . CONCLUSIONS: These findings suggest an improved safety and tolerability profile with the increase in potency achieved by productive receptor-mediated uptake of AKCEA-TTR-LRx by hepatocytes and supports further development of AKCEA-TTR-LRx for the treatment of ATTR polyneuropathy and cardiomyopathy.


Subject(s)
Amyloid Neuropathies, Familial , Oligonucleotides, Antisense , Amyloid Neuropathies, Familial/drug therapy , Amyloid Neuropathies, Familial/genetics , Animals , Ligands , Mice , Prealbumin/genetics
4.
Nucleic Acid Ther ; 30(2): 94-103, 2020 04.
Article in English | MEDLINE | ID: mdl-32043907

ABSTRACT

Inotersen, a 2'-O-methoxyethyl (2'-MOE) phosphorothioate antisense oligonucleotide, reduced disease progression and improved quality of life in patients with hereditary transthyretin amyloidosis with polyneuropathy (hATTR-PN) in the NEURO-TTR and NEURO-TTR open-label extension (OLE) trials. However, 300 mg/week inotersen treatment was associated with platelet count reductions in several patients. Mean platelet counts in patients in the NEURO-TTR-inotersen group remained ≥140 × 109/L in 50% and ≥100 × 109/L in 80% of the subjects. However, grade 4 thrombocytopenia (<25 × 109/L) occurred in three subjects in NEURO-TTR trial, and one of these suffered a fatal intracranial hemorrhage. The two others were treated successfully with corticosteroids and discontinuation of inotersen. Investigations in a subset of subjects in NEURO-TTR (n = 17 placebo; n = 31 inotersen) and OLE (n = 33) trials ruled out direct myelotoxicity, consumptive coagulopathy, and heparin-induced thrombocytopenia. Antiplatelet immunoglobulin G (IgG) antibodies were detected at baseline in 5 of 31 (16%) inotersen-treated subjects in NEURO-TTR, 4 of whom eventually developed grade 1 or 2 thrombocytopenia while on the drug. In addition, 24 subjects in the same group developed treatment-emergent antiplatelet IgG antibodies, of which 2 developed grade 2, and 3 developed grade 4 thrombocytopenia. Antiplatelet IgG antibodies in two of the three grade 4 thrombocytopenia subjects targeted GPIIb/IIIa. Plasma cytokines previously implicated in immune dysregulation, such as interleukin (IL)-23 and a proliferation-inducing ligand (APRIL) were often above the normal range at baseline. Collectively, these findings suggest an underlying immunologic dysregulation predisposing some individuals to immune-mediated thrombocytopenia during inotersen treatment.


Subject(s)
Amyloid Neuropathies, Familial/drug therapy , Oligonucleotides, Antisense/administration & dosage , Oligonucleotides/administration & dosage , Thrombocytopenia/blood , Adult , Aged , Amyloid Neuropathies, Familial/genetics , Amyloid Neuropathies, Familial/immunology , Amyloid Neuropathies, Familial/pathology , Female , Genetic Predisposition to Disease , Humans , Immune System Diseases/chemically induced , Immune System Diseases/immunology , Immune System Diseases/pathology , Immunoglobulin G , Intracranial Hemorrhages/chemically induced , Intracranial Hemorrhages/immunology , Intracranial Hemorrhages/pathology , Male , Middle Aged , Oligodeoxyribonucleotides, Antisense/administration & dosage , Oligonucleotides/adverse effects , Oligonucleotides, Antisense/adverse effects , Quality of Life , Thrombocytopenia/chemically induced , Thrombocytopenia/immunology , Thrombocytopenia/pathology
5.
Nucleic Acid Ther ; 27(5): 272-284, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28605247

ABSTRACT

Antisense oligonucleotides (ASOs) are widely accepted therapeutic agents that suppress RNA transcription. While the majority of ASOs are well tolerated in vivo, few sequences trigger inflammatory responses in absence of conventional CpG motifs. In this study, we identified non-CpG oligodeoxy-nucleotide (ODN) capable of triggering an inflammatory response resulting in B cell and macrophage activation in a MyD88- and TLR9-dependent manner. In addition, we found the receptor for advance glycation end product (RAGE) receptor to be involved in the initiation of inflammatory response to suboptimal concentrations of both CpG- and non-CpG-containing ODNs. In contrast, dosing RAGE KO mice with high doses of CpG or non-CpG ODNs lead to a stronger inflammatory response than observed in wild-type mice. Together, our data provide a previously uncharacterized in vivo mechanism contingent on ODN-administered dose, where TLR9 governs the primary response and RAGE plays a distinct and cooperative function in providing a pivotal role in balancing the immune response.


Subject(s)
Immunity, Cellular/immunology , Inflammation/immunology , Oligonucleotides, Antisense/therapeutic use , Receptor for Advanced Glycation End Products/metabolism , Toll-Like Receptor 9/metabolism , Animals , B-Lymphocytes/immunology , Cytokines/blood , Humans , Macrophages/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myeloid Differentiation Factor 88/metabolism , Primary Cell Culture , RNA/genetics , RNA/metabolism , Receptor for Advanced Glycation End Products/genetics , Single-Cell Analysis , Toll-Like Receptor 9/genetics , Transcription, Genetic
6.
Nucleic Acid Ther ; 26(4): 236-49, 2016 08.
Article in English | MEDLINE | ID: mdl-27140858

ABSTRACT

The effects of repeated complement activation in cynomolgus monkeys after chronic antisense oligonucleotide (ASO) treatment were evaluated by using ISIS 104838, a representative 2'-O-methoxyethyl (2'-MOE) modified ASO. The treatment was up to 9 months with a total weekly dose of 30 mg/kg, given either as daily [4.3 mg/kg/day, subcutaneous (s.c.) injection] or once weekly [30 mg/kg, either as s.c. injection or 30-min intravenous (i.v.) infusion]. Acute elevations of complement split products (Bb and C3a) and a transient decrease in C3 occurred after the first dose and were drug plasma concentration dependent. However, with repeated complement activation after chronic ASO treatment, there were progressive increases in basal (predose) levels of Bb and C3a, and a sustained C3 reduction in all treated groups. There was also a progressive increase in C3d-bound circulating immune complex (CIC) that was considered secondary to the C3 depletion. Evidence of vascular inflammation was observed, mostly in the liver, kidney, and heart, and correlated with severe C3 depletion and increases in plasma IgG and IgM. Vascular inflammation was accompanied by increased C3 and IgM immunereactivity in the affected vasculatures and endothelial activation markers in serum. In summary, repeated complement activations in monkeys lead to a sustained decrease in circulating C3 over time. The concomitantly increased inflammatory signals and decreased CIC clearance due to impairment of complement function may lead to vascular inflammation after chronic ASO treatment in monkeys. However, based on the known sensitivity of monkeys to ASO-induced complement activation, these findings have limited relevance to humans.


Subject(s)
Complement Activation/drug effects , Oligonucleotides, Antisense/administration & dosage , Phosphorothioate Oligonucleotides/administration & dosage , Tumor Necrosis Factor-alpha/genetics , Animals , Dose-Response Relationship, Drug , Heart/drug effects , Humans , Kidney/drug effects , Liver/drug effects , Macaca fascicularis , Oligonucleotides, Antisense/chemistry , Phosphorothioate Oligonucleotides/chemistry , Tumor Necrosis Factor-alpha/antagonists & inhibitors
7.
J Med Chem ; 59(6): 2718-33, 2016 Mar 24.
Article in English | MEDLINE | ID: mdl-26914862

ABSTRACT

The comprehensive structure-activity relationships of triantennary GalNAc conjugated ASOs for enhancing potency via ASGR mediated delivery to hepatocytes is reported. Seventeen GalNAc clusters were assembled from six distinct scaffolds and attached to ASOs. The resulting ASO conjugates were evaluated in ASGR binding assays, in primary hepatocytes, and in mice. Five structurally distinct GalNAc clusters were chosen for more extensive evaluation using ASOs targeting SRB-1, A1AT, FXI, TTR, and ApoC III mRNAs. GalNAc-ASO conjugates exhibited excellent potencies (ED50 0.5-2 mg/kg) for reducing the targeted mRNAs and proteins. This work culminated in the identification of a simplified tris-based GalNAc cluster (THA-GN3), which can be efficiently assembled using readily available starting materials and conjugated to ASOs using a solution phase conjugation strategy. GalNAc-ASO conjugates thus represent a viable approach for enhancing potency of ASO drugs in the clinic without adding significant complexity or cost to existing protocols for manufacturing oligonucleotide drugs.


Subject(s)
Acetylgalactosamine/chemical synthesis , Acetylgalactosamine/pharmacology , Hepatocytes/drug effects , Oligonucleotides, Antisense/chemical synthesis , Oligonucleotides, Antisense/pharmacology , Animals , Apolipoprotein C-III/drug effects , Drug Delivery Systems , Factor XI/drug effects , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Scavenger Receptors, Class B/biosynthesis , Scavenger Receptors, Class B/genetics , Structure-Activity Relationship
8.
Nucleic Acids Res ; 44(5): 2093-109, 2016 Mar 18.
Article in English | MEDLINE | ID: mdl-26553810

ABSTRACT

High affinity antisense oligonucleotides (ASOs) containing bicylic modifications (BNA) such as locked nucleic acid (LNA) designed to induce target RNA cleavage have been shown to have enhanced potency along with a higher propensity to cause hepatotoxicity. In order to understand the mechanism of this hepatotoxicity, transcriptional profiles were collected from the livers of mice treated with a panel of highly efficacious hepatotoxic or non-hepatotoxic LNA ASOs. We observed highly selective transcript knockdown in mice treated with non-hepatotoxic LNA ASOs, while the levels of many unintended transcripts were reduced in mice treated with hepatotoxic LNA ASOs. This transcriptional signature was concurrent with on-target RNA reduction and preceded transaminitis. Remarkably, the mRNA transcripts commonly reduced by toxic LNA ASOs were generally not strongly associated with any particular biological process, cellular component or functional group. However, they tended to have much longer pre-mRNA transcripts. We also demonstrate that the off-target RNA knockdown and hepatotoxicity is attenuated by RNase H1 knockdown, and that this effect can be generalized to high affinity modifications beyond LNA. This suggests that for a certain set of ASOs containing high affinity modifications such as LNA, hepatotoxicity can occur as a result of unintended off-target RNase H1 dependent RNA degradation.


Subject(s)
Liver/drug effects , Oligonucleotides, Antisense/toxicity , Oligonucleotides/toxicity , RNA, Messenger/genetics , Ribonuclease H/genetics , Alanine Transaminase/blood , Alanine Transaminase/genetics , Animals , Gene Expression Profiling , Gene Expression Regulation , Liver/metabolism , Male , Mice , Mice, Inbred BALB C , Microarray Analysis , Oligonucleotides/genetics , Oligonucleotides/metabolism , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/metabolism , RNA Precursors/antagonists & inhibitors , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Ribonuclease H/antagonists & inhibitors , Ribonuclease H/metabolism , Transcriptome/drug effects
9.
Nucleic Acid Ther ; 23(3): 213-27, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23692080

ABSTRACT

ISIS 481464 is a constrained ethyl (cEt) modified phosphorothioate antisense oligonucleotide (ASO) targeting signal transducer and activator of transcription 3 (STAT3) studied in mice and monkey to support oncology clinical trials. Six-week toxicology studies were performed in mice and cynomolgus monkey (up to 70 and 30 mg/kg/week respectively). Reduction in STAT3 protein up to 90% of control was observed in monkey. Cynomolgus monkey was considered the most relevant species to human with respect to pharmacokinetic properties, but mice are useful in their relative sensitivity to the potential proinflammatory and hepatic effects of oligonucleotides. In monkeys, there was no impact on organ function at doses up to 30 mg/kg/week for 6 weeks. Minimal to slight proximal tubular epithelial cell degeneration and regeneration within the kidney was observed, which had no impact on renal function and showed reversibility at the end of the treatment-free period. Additionally, mild and transient activated partial thromboplastin time elevations and mild increases in complement Bb were observed at the higher doses by intravenous dosing only. In mice, the alterations at 70 mg/kg/week included spleen weight increase up to 1.4-fold relative to control, increases in alanine aminotransferase and aspartate aminotransferase up to 1.8-fold over control, interleukin-10 increases up to 3.7-fold, and monocyte chemoattractant protein-1 increase up to 1.9-fold over control. No significant clinical pathology or histopathology changes were seen in mice at 20 mg/kg/week or less. The toxicity profile of ISIS 481464 is consistent with effects observed with phosphorothioate ASOs containing 2'-O-methoxyethylribose modifications instead of cEt.


Subject(s)
Kidney/drug effects , Liver/drug effects , Oligonucleotides, Antisense/toxicity , Phosphorothioate Oligonucleotides/toxicity , Spleen/drug effects , Alanine Transaminase/metabolism , Animals , Aspartate Aminotransferases/metabolism , Drug Administration Routes , Drug Administration Schedule , Drug Evaluation, Preclinical , Female , Humans , Kidney/metabolism , Kidney/pathology , Liver/metabolism , Liver/pathology , Macaca fascicularis , Male , Mice , Oligonucleotides, Antisense/chemical synthesis , Oligonucleotides, Antisense/pharmacokinetics , Organ Size/drug effects , Partial Thromboplastin Time , Phosphorothioate Oligonucleotides/chemical synthesis , Phosphorothioate Oligonucleotides/pharmacokinetics , STAT3 Transcription Factor/antagonists & inhibitors , Spleen/metabolism , Spleen/pathology
10.
J Pharmacol Exp Ther ; 342(1): 150-62, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22505629

ABSTRACT

Antisense oligonucleotides (ASO) containing 2'-O-methoxyethyl ribose (2'-MOE) modifications have been shown to possess both excellent pharmacokinetic properties and robust pharmacological activity in several animal models of human disease. 2'-MOE ASOs are generally well tolerated, displaying minimal to mild proinflammatory effect at doses far exceeding therapeutic doses. Although the vast majority of 2'-MOE ASOs are safe and well tolerated, a small subset of ASOs inducing acute inflammation in mice has been identified. The mechanism for these findings is not clear at this point, but the effects are clearly sequence-specific. One of those ASOs, ISIS 147420, causes a severe inflammatory response atypical of this class of oligonucleotides characterized by induction in interferon-ß (IFN-ß) at 48 h followed by acute transaminitis and extensive hepatocyte apoptosis and necrosis at 72 h. A large number of interferon-stimulated genes were significantly up-regulated in liver as early as 24 h. We speculated that a specific sequence motif might cause ISIS 147420 to be mistaken for viral RNA or DNA, thus triggering an acute innate immune response. ISIS 147420 toxicity was independent of Toll-like receptors, because there was no decrease in IFN-ß in Toll/interleukin-1 receptor-domain-containing adapter-inducing IFN-ß or Myd88-deficient mice. The involvement of cytosolic retinoic acid-inducible gene (RIG)-I-like pattern recognition receptors was also investigated. Pretreatment of mice with melanoma differentiation-associated gene 5 (MDA5) and IFN-ß promoter stimulator-1 ASOs, but not RIG-I or laboratory of genetics and physiology 2 (LGP2) ASOs, prevented the increase in IFN-ß and alanine aminotransferase induced by ISIS 147420. These results revealed a novel mechanism of oligonucleotide-mediated toxicity requiring both MDA5 and IPS-1 and resulting in the activation of the innate immune response.


Subject(s)
DEAD-box RNA Helicases/immunology , DNA/immunology , Immunity, Innate/immunology , Interferon Type I/immunology , Oligonucleotides, Antisense/immunology , Ribose/immunology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/immunology , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/immunology , Adaptor Proteins, Vesicular Transport/metabolism , Alanine Transaminase/genetics , Alanine Transaminase/immunology , Alanine Transaminase/metabolism , Animals , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , DNA/genetics , DNA/metabolism , Hepatocytes/immunology , Hepatocytes/metabolism , Immunity, Innate/genetics , Inflammation/genetics , Inflammation/immunology , Inflammation/metabolism , Interferon Type I/genetics , Interferon Type I/metabolism , Interferon-Induced Helicase, IFIH1 , Interferon-beta/genetics , Interferon-beta/immunology , Interferon-beta/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/immunology , Myeloid Differentiation Factor 88/metabolism , Oligonucleotides, Antisense/genetics , Receptors, Interleukin-1/genetics , Receptors, Interleukin-1/immunology , Receptors, Interleukin-1/metabolism , Ribose/genetics , Ribose/metabolism , Signal Transduction/genetics , Signal Transduction/immunology , Toll-Like Receptors/genetics , Toll-Like Receptors/immunology , Toll-Like Receptors/metabolism
11.
Mol Ther ; 17(3): 508-15, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19127252

ABSTRACT

Intravascular delivery (1.5 x 10(9) particles and higher) of recombinant adenovirus (rAd) induces myeloid cell mediated, self-limiting hemodynamic responses in normal mice. However, we observed anaphylactoid-type reactions and exacerbated hemodynamic events following rAd injection in mice bearing malignant 4T1 mammary carcinoma. Because 4T1 tumors induce significant CD11b(+)Gr-1(+) myeloid cell expansion and activation, we set to determine whether this causes rAd-induced exaggerated responses. When treated with a single intravenous dose (1 x 10(10) particles) of rAd, mice implanted with 4T1 carcinoma succumbed due to the anaphylactoid-type reactions. In contrast, normal mice and mice implanted with a related mammary carcinoma (66cl4) that does not induce CD11b(+)Gr-1(+) cell expansion, showed minimal responses. Depletion of phagocytic CD11b(+)Gr-1(+) cells prior to rAd delivery protected 4T1 tumor-bearing animals, whereas passive transfer of CD11b(+)Gr-1(+) cells from 4T1 tumor-bearing animals was sufficient to convey susceptibility to anaphylactoid-type reactions in normal animals. We further show that there is upregulation of nitric oxide and leukotriene signaling pathways in the 4T1 tumor-induced CD11b(+)Gr-1(+) myeloid cells and that pretreating mice with inhibitors of nitric oxide synthetase and leukotrienes can attenuate the anaphylactoid-type reactions. These data show that malignant tumor growth can alter CD11b(+)Gr-1(+) myeloid cells, rendering hosts susceptible to anaphylactoid-type reactions upon intravascular treatment with rAd.


Subject(s)
Adenoviridae/immunology , Anaphylaxis/metabolism , CD11b Antigen/immunology , Cell Differentiation , Genetic Therapy/adverse effects , Neoplasms/pathology , Receptors, Chemokine/metabolism , Adenoviridae/genetics , Anaphylaxis/physiopathology , Animals , Cardiovascular System/metabolism , Cell Line, Tumor , Disease Susceptibility , Female , Heart Rate , Humans , Leukotriene Antagonists/pharmacology , Liposomes , Mice , Myeloid Cells/metabolism , Neoplasm Transplantation , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/therapy , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Phagocytes , Receptors, Chemokine/immunology , Receptors, Leukotriene/metabolism
12.
Mol Ther ; 12(2): 254-63, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16043097

ABSTRACT

Intravascular administration of recombinant adenovirus (rAd) in cancer patients has been well tolerated. However, dose-limiting hemodynamic responses associated with suppression of cardiac output have been observed at doses of 7.5 x 10(13) particles. While analysis of hemodynamic responses induced by small-molecule pharmaceuticals is well established, little is known about the cardiovascular effects of rAd. Telemetric cardiovascular (CV) monitoring in mice was utilized to measure hemodynamic events following intravascular rAd administration. Electrocardiogram analysis revealed a block in the SA node 3-4 min postinfusion, resulting in secondary pacemaking initiated at the AV node. This was associated with acute bradycardia, reduced blood pressure, and hypothermia followed by gradual recovery. Adenovirus-primed murine sera with high neutralizing antibody (nAb) titers could inhibit CV responses, whereas human sera with equivalent nAb titers induced by natural infection were, surprisingly, not inhibitory. Interestingly, repeat dosing within 2-4 h of the primary injection resulted in desensitization, resembling tachyphylaxis, for subsequent CV responses. Last, depletion of Kupffer cells prior to rAd infusion precluded induction of CV responses. These inhibitory effects suggest that rAd interactions with certain cells of the reticular endothelial system are associated with induction of CV responses. Significantly, these studies may provide insight into management of acute adverse effects following rAd systemic delivery, enabling a broadening of therapeutic index.


Subject(s)
Adenoviridae/genetics , Genetic Therapy , Genetic Vectors/pharmacology , Heart Rate/drug effects , beta-Galactosidase/therapeutic use , Animals , Blood Pressure/drug effects , Carcinoma, Non-Small-Cell Lung , Cardiac Output/drug effects , Cardiovascular System/drug effects , Electrocardiography , Genetic Vectors/therapeutic use , Humans , Influenza A virus , Kupffer Cells/physiology , Mice , Mice, Inbred BALB C
13.
Urology ; 66(1): 224-9, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15992886

ABSTRACT

OBJECTIVES: To evaluate whether a recombinant replication-deficient adenovirus containing the secreted human interferon alpha-2b gene (rAd-IFN) could improve the tissue and urine levels of IFN protein by transducing the urothelium with the secreted human IFN-alpha gene. We also assessed whether varying the interval between rAd-IFN/Syn3 treatments would improve the duration and levels of gene expression. METHODS: The rats received intravesical administration of rAd-IFN at varying concentrations in a formulation containing Syn3, an agent identified that facilitates passage of the adenovirus through the protective barrier of the bladder. Urine was collected daily for 7 days, and human IFN was measured in the urine by enzyme-linked immunosorbent assay. For the redosing studies, the animals received a second dose at varying intervals ranging from 1 to 7 days after the first dose or at longer intervals (30, 60, or 90 days). RESULTS: Rats that received intravesical administration of rAd-IFN in a Syn3 formulation expressed levels of human IFN protein in their urine at peak concentrations of 50,000 to 100,000 pg/mL, but were undetectable by 7 days. Expression was localized to the bladder with only minimal systemic exposure to IFN. Short-term redosing marginally improved the IFN urine concentrations, with maximal levels achieved when a second dose was administered 3 days after a first dose. Although gene expression was attenuated when a second dose was given 5 to 7 days after the first treatment, the levels and duration of IFN expression recovered when the interval was increased to 90 days. CONCLUSIONS: Intravesical treatment with rAd-IFN facilitates high levels of IFN transgene exposure and may be a new approach to treating superficial bladder cancer.


Subject(s)
Interferon-alpha/administration & dosage , Interferon-alpha/genetics , Urinary Bladder , Adenoviridae/genetics , Animals , Dose-Response Relationship, Drug , Drug Carriers , Female , Interferon alpha-2 , Rats , Rats, Sprague-Dawley , Recombinant Proteins
14.
Virology ; 328(1): 52-61, 2004 Oct 10.
Article in English | MEDLINE | ID: mdl-15380358

ABSTRACT

Replication competent adenoviruses with various E1 modifications designed to restrict their replication to tumor cells are being evaluated as oncolytic agents in clinical trials. In mouse models, we observed that such oncolytic adenoviruses showed greater hepatotoxicity than E1-deleted adenovirus vectors following intravenous administration. Additional studies in congenic BALB/c, nude, and beige/Scid mice demonstrated dose-dependent hepatotoxicity and indicated that beige/Scid was the most sensitive strain. Comparison of E1-containing viruses showed that hepatotoxicity correlated with expression of wild-type E1a in the liver. Pharmacokinetic analysis showed rapid increases in viral DNA levels in the liver with a virus containing wild-type E1a. This was correlated with rapid induction of TNF-alpha to high levels and with rapid elevation of serum ALT. Hepatotoxicity was significantly reduced for an adenovirus with deletions in the region E1a (dl01/07) or a virus lacking E1a. The results suggest a mechanism for hepatotoxicity involving virus-induced production of local TNF-alpha release and E1a-mediated sensitization of hepatocyte killing.


Subject(s)
Adenoviridae/genetics , Adenovirus E1A Proteins/metabolism , Genetic Vectors/pharmacokinetics , Hepatitis/virology , Liver/virology , Tumor Necrosis Factor-alpha/biosynthesis , Adenovirus E1A Proteins/analysis , Adenovirus E1A Proteins/genetics , Alanine Transaminase/blood , Animals , DNA, Viral/analysis , DNA, Viral/biosynthesis , Female , Genetic Vectors/genetics , Genetic Vectors/physiology , Hepatitis/metabolism , Hepatitis/pathology , Immunocompromised Host , Injections, Intravenous , Liver/metabolism , Liver/pathology , Mice , Mice, Inbred BALB C , Mice, Nude , Mice, SCID , Models, Animal , Mutation , Species Specificity , Time Factors , Tumor Necrosis Factor-alpha/analysis , Viruses/genetics
15.
Bioconjug Chem ; 15(4): 831-40, 2004.
Article in English | MEDLINE | ID: mdl-15264871

ABSTRACT

Linear and branched poly(ethylenimines), lPEI and bPEI, respectively, grafted with beta-cyclodextrin are prepared to give CD-lPEI and CD-bPEI, respectively, and are investigated as in vitro and in vivo nonviral gene delivery agents. The in vitro toxicity and transfection efficiency are sensitive to the level of cyclodextrin grafting. The cyclodextrin-containing polycations, when combined with adamantane-poly(ethylene glycol) (AD-PEG) conjugates, form particles that are stable at physiological salt concentrations. PEGylated CD-lPEI-based particles give in vitro gene expression equal to or greater than lPEI as measured by the percentage of EGFP expressing cells. Tail vein injections into mice of 120 microg of plasmid DNA formulated with CD-lPEI and AD-PEG do not reveal observable toxicities, and both nucleic acid accumulation and expression are observed in liver.


Subject(s)
Cyclodextrins/chemistry , Drug Delivery Systems/methods , Polyethyleneimine/chemistry , Polymers/chemical synthesis , Animals , Cell Line, Tumor , Drug Delivery Systems/instrumentation , Genes, p53/genetics , Humans , Hydrogen-Ion Concentration , Inhibitory Concentration 50 , Liver/metabolism , Mice , Mice, Inbred BALB C , Microscopy, Electron , Molecular Structure , Plasmids/administration & dosage , Plasmids/chemistry , Plasmids/genetics , Plasmids/toxicity , Polymers/administration & dosage , Polymers/chemistry , Polymers/toxicity , Salts/pharmacology , Solvents , Titrimetry , Transfection
16.
Cancer Res ; 63(14): 4003-8, 2003 Jul 15.
Article in English | MEDLINE | ID: mdl-12873998

ABSTRACT

Central to the development of oncolytic virotherapies for cancer will be a better understanding of the parameters that influence the outcome of virotherapy to treat disseminated cancer by i.v. administration versus regional disease by local treatment. Intratumoral administration of 01/PEME, an oncolytic adenovirus, required approximately 1000-fold less dose than i.v. administration to induce similar tumor growth inhibition. Despite the short (<10 min) circulating half-life of the virus DNA, we could monitor virus distribution to the tumor site and observed virus replication by >1000-fold increase in virus DNA copies over time. There were doses of 01/PEME for which the virus DNA concentration in the tumor increased over time but did not result in antitumor efficacy. Oncolytic virus replication at a tumor site may not be a relevant indication of antitumor efficacy. Efficient distribution to the tumor site may be one of the most critical parameters for antitumor efficacy with oncolytic virotherapy.


Subject(s)
Adenoviridae/genetics , Prostatic Neoplasms/therapy , Prostatic Neoplasms/virology , Adenoviridae/metabolism , Animals , Genes, p53 , Humans , Injections, Intralesional , Injections, Intravenous , Male , Mice , Xenograft Model Antitumor Assays
17.
Mol Ther ; 7(4): 558-64, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12727120

ABSTRACT

We conducted a series of experiments to determine if intraperitoneal (IP) delivery of recombinant adenovirus (rAd)-based therapies is improved through carrier vehicle selection, and compared an icodextrin solution (a high molecular weight dextrin with a prolonged peritoneal cavity residence time) with a standardized phosphate buffered saline (PBS) delivery solution. In vitro, comparative adenovirus particle concentration determination (27 h) and bioactivity assay (24h) indicated equivalent compatibility with icodextrin or PBS. In vivo, rabbits treated IP (100 ml) with rAd-betagal 1 x 10(9) P/ml in icodextrin showed improved transgene expression throughout the peritoneal wall compared to rAd-betagal in PBS. In PC-3 tumor-bearing mice treated IP with 5 x 10(9) P/0.5 ml or 1 x 10(10) P/0.5 ml rAd-betagal, transgene expression was significantly enhanced (p < 0.01) with icodextrin compared to PBS in both tumor specimens and peritoneal wall. In subsequent studies we compared prolongation of survival in intraperitoneal PC-3 and MDAH-2774 human xenograft tumor models in nude mice using rAd-p53 in icodextrin or PBS in multi-dose ranging (1 x 10(8) to 1 x 10(10) P) experiments. The icodextrin formulation alone significantly increased rAd-p53 mediated survival (p < 0.05). In animals, these results show that IP rAd gene therapy can be improved with the use of icodextrin, and suggest that prolonged retention and distribution in the peritoneal cavity is an important factor.


Subject(s)
Adenoviridae/genetics , Glucans/administration & dosage , Glucose/administration & dosage , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/therapy , Animals , Female , Gene Expression Regulation , Genetic Therapy , Genetic Vectors/administration & dosage , Glucans/therapeutic use , Glucose/therapeutic use , Humans , Icodextrin , Injections, Intraperitoneal , Mice , Mice, Nude , Peritoneum/enzymology , Rabbits , Rats , Rats, Sprague-Dawley , Transgenes/genetics , beta-Galactosidase/metabolism
18.
Mol Ther ; 6(1): 50-6, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12095303

ABSTRACT

Inducible synthesis and secretion of therapeutic proteins following gene transfer could be a viable strategy to deliver biopharmaceuticals that currently require parenteral administration. Evaluating the protein pharmacokinetics and biological responses generated by different delivery modalities will provide a better understanding of the advantages and disadvantages of each strategy. The interferon-alpha (IFN-alpha) family of proteins, used clinically for infectious and malignant diseases, has a short half-life, and IFN-alpha therapy requires frequent administration of the drug by injection. Subcutaneous xenograft tumors were inhibited by weekly administration of polyethylene glycol modified (PEGylated) IFN-alpha protein or by a single administration of an adenovirus constitutively expressing IFN-alpha (IACB). Both treatment modalities inhibited tumor growth in a dose-dependent manner, suggesting that increasing exposure to IFN-alpha could result in effective tumor control. A single adenovirus that encodes the components necessary for tetracycline induction (IADR) expressed IFN-alpha in a ligand-dependent manner. Adding doxycycline to the drinking water of mice treated intravenously with the inducible adenovirus IADR inhibited tumor growth by 85% compared with mice that were not given doxycycline. The correlation between serum IFN-alpha concentration and the degree of tumor growth inhibition did not depend on the delivery technology used. It is likely that it will be feasible to control expression of IFN-alpha by oral administration of small molecule drugs after gene delivery to induce therapeutic concentrations of proteins.


Subject(s)
Adenoviridae/metabolism , Gene Expression Regulation, Viral/physiology , Interferon-alpha/metabolism , Polyethylene Glycols/metabolism , Animals , Doxycycline/metabolism , Genetic Vectors/administration & dosage , Genetic Vectors/metabolism , Interferon-alpha/administration & dosage , Interferon-alpha/blood , Interferon-alpha/genetics , Interferon-alpha/pharmacology , Mice , Neoplasms, Experimental/therapy
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