ALBERT

Description: Sickle cell anemia (SCD) is a hereditary blood disorder in which red blood cells (RBC) become sickle-shaped and block blood vessels, leading to painful vaso-occlusive episodes. Sickling occurs because of a point-mutation in the β-globin gene of hemoglobin. Fetal hemoglobin (HbF, α2γ2) is the main oxygen transport protein with greater oxygen binding affinity in the fetus during the last months of embryonic development and the first few months of life after birth. HbF inhibits sickling by interfering with the polymerization of hemoglobin S. Higher HbF levels in SCD correlate with better survival and because HbF production can be reactivated pharmacologically in adults, it can be used for the treatment of SCD as well as β-thalassemia. In β-thalassemia, there is reduced or absent synthesis of the β-globin gene, causing ineffective erythropoiesis. B-cell lymphoma/leukemia 11A (BCL11A) is a transcription factor in the zinc-finger protein family and is expressed in B cells and erythroid cells. BCL11A represses fetal hemoglobin expression by binding to the GGCCCGG motif in the β-globin promoter region. Erythroid Kruppel-like factor (KLF1) is an erythroid-specific transcription factor that regulates β-globin expression through direct interaction with its promoter and indirectly regulates γ-globin expression through the regulation of BCL11A. By reducing the expression of BCL11A and KLF1, we can promote production of HbF through the upregulation of γ-globin expression. To demonstrate upregulation of γ-globin mRNA expression in vitro, we used MEL-h-b-BAC line#7 cells, a murine erythroleukemic cell line harboring the entire human beta globin locus and expressing mouse BCL11A and KLF1 (Tim Townes, Univ. of Alabama at Birmingham). Antisense oligonucleotides (ASOs) targeting mouse BCL11A or mouse KLF1 were added to the cells in a dose-dependent manner. Seven days later, with free uptake of the ASOs into the cells, we observed dose-dependent reduction of mouse BCL11A mRNA (IC50 = 0.7 μM) and mouse KLF1 mRNA (IC50 = 3 μM). Consequently, we observed a 300 +/- 8% upregulation of human γ-globin mRNA expression after achieving ∼90% reduction in BCL11A mRNA expression after ASO treatment compared to the untreated control cells. Similarly, KLF1 ASO treatment caused a 500 +/- 58% up regulation of human γ-globin mRNA expression after achieving ∼80% mRNA reduction in KLF expression. These data indicate that targeting BCL11A and/or KLF1 with ASO treatment can cause an increase in γ-globin expression that is necessary for the upregulation of fetal hemoglobin and may be used for the treatment of sickle-cell anemia and β-thalassemia. Disclosures: Peralta: Isis Pharmaceuticals, Inc.: Employment. Low: Isis Pharmaceuticals, Inc.: Employment. Kim: Isis Pharmaceuticals, Inc.: Employment. Murray: Isis Pharmaceuticals, Inc.: Employment. Guo: Isis Pharmaceuticals, Inc.: Employment. Freier: Isis Pharmaceuticals, Inc.: Employment. Townes: University of Alabama at Birmingham: Employment. Hung: Isis Pharmaceuticals, Inc.: Employment.

Description: Background: Rising health care costs - especially those of new medications for cancer treatment - have caused a severe financial strain on patients, leading to significant alteration of lifestyles. Published information regarding financial distress is scant with data only from large tertiary care institutions that may not be relevant to smaller communities. We sought to analyze the extent of financial toxicity over time in a community cancer clinic staffed by two medical oncologists. Methods: Since 2010, a team of two oncology social workers prospectively collected data on financial assistance for patients treated at our community cancer clinic. Data regarding medication assistance is available for 2012-2014. Results: Approximately 500 new patients and 1600 unique follow-up patients are evaluated and treated at the clinic annually. From January 2010 through December 2014, 1,012 requests for assistance were processed. The number of requests increased from 134 in 2010 to 320 in 2014. Most common requests were for medications and financial aid. Trends are shown in the table. Though the number of patients applying for medication assistance increased in 2014, the amount requested was less than previous years because of the influx of specialty pharmacies in the market with their own assistance programs. Conclusions: Financial toxicity for cancer patients is a real issue in the community clinic setting.Requests for financial assistance have increased over time. The number of FDA approvals for new cancer medications seem to correlate with the increase. Studies are in progress to further clarify the financial impact of cancer on patients.Table.Trends in requests for assistance and aid and FDA approval for new medications.Requests for20102011201220132014Financial aid6411686101143Service311112733Equipment1311111823Transport312357Medications51534449114Patient cost of medications for which aid requested$731,891$1,554,684$1,253,827FDA approvals for new Oncology medications61181513 Disclosures No relevant conflicts of interest to declare.

Description: The ability to safely and effectively neutralize anticoagulant activity is of particular importance in the case of long acting drugs. While antisense oligonucleotides (ASOs) benefit from their slow clearance rate from liver (t1/2 approx 10 days in mice) which permits infrequent dosing, the potential necessity to reverse ASO drug activity presents a challenge. ISIS 401025 is a second generation ASO targeting prothrombin RNA that has previously been shown to reduce thrombin generation and prolong PT and aPTT as a result of depleting prothrombin mRNA levels in mice. To explore the possibility of developing an “antidote” strategy for ASO-mediated anticoagulation, we designed a sense oligonucleotide, ISIS 405277, complementary to the ISIS 401025 sequence and examined its ability to reverse the effects of prior treatment with ISIS 401025 in mice. A PT-INR of 3.0 was established after 3 weeks of treatment with ISIS 401025 at a weekly dose of 60 mg/kg, corresponding to a reduction of prothrombin mRNA transcript levels of 96%. A single injection of the sense oligonucleotide, ISIS 405277, resulted in a dose-dependent reversal of INR to 1.6, 1.1, and 0.9 three days after injection of 30, 60, and 90 mg/kg, respectively. Corresponding prothrombin transcript levels in liver tissue were returned to 18.4%, 27.8%, and 38.9% of normal levels, respectively. Results from a study designed to determine the kinetics of reversal of anticoagulation indicated that a 50% reduction of PT-INR 4.3, established following 3 weeks of treatment with ISIS 401025, required 11 days in the absence of subsequent treatment, while the reduction was achieved in 2.2 days following a single administration of 90 mg/kg of sense oligonucleotide. When mice were pretreated with an ASO targeting prothrombin that was not complementary to the sense oligonucleotide (ISIS 40527), sense oligonucleotide treatment was unable to reverse either target depletion or anticoagulation, indicating a sequence-specific antidote effect. These results demonstrate for the first time that subsequent administration of a sense strand oligonucleotide can neutralize ASO mediated target RNA degradation in animals, and demonstrates its potential utility in reversing ASO-based activity for anticoagulation.

Description: Numerous potential intervention points exist within coagulation pathways for development of novel anticoagulant agents. To help determine the most suitable target for the discovery of novel antithrombotic agents, we have implemented an antisense strategy to specifically deplete levels of various coagulation factors in mice. Here we report the effects of antisense oligonucleotides (ASOs) targeting factors II, VII, and IX on target RNA and protein levels in mice, as well as PT/aPTT. ISIS 401025 targeting factor II, ISIS 403102 targeting factor VII, and ISIS 402618 targeting factor IX dose-dependently suppressed their respective target transcript levels in a specific manner in primary mouse hepatocytes. Following subcutaneous administration to mice, each ASO reduced target RNA levels in liver and target protein levels in plasma in a specific and dose-dependent fashion (ED95 of approximate 25 mg/kg for each ASO). In conjunction with these effects, clotting times were also prolonged in a similar dose responsive fashion. Both PT and aPTT were increased following factor II inhibition and a PT-INR of 2.5 was achieved at 25 mg/kg, corresponding to a reduction in prothrombin transcript of 95%. As expected following factor VII ASO treatment, PT was significantly increased, while aPTT was unchanged. Conversely, following factor IX ASO treatment, aPTT was marginally but significantly prolonged, while PT was unchanged. These results demonstrate the capability of ASOs to dose-dependently and specifically modulate coagulation factor activity depletion and support the ASO approach as a novel strategy for the discovery of novel anticoagulation agents. Studies are in progress to assess relative safety and to expand the anticoagulation profile in mice for each of the factors under study.

Description: Sickle cell anemia (SCD) is a hereditary blood disorder in which red blood cells (RBC) become sickle-shaped and block blood vessels, leading to painful vaso-occlusive episodes. Sickling occurs because of a point-mutation in the β-globin gene of hemoglobin. Fetal hemoglobin (HbF, α2γ2) is the main oxygen transport protein in the fetus during the last months of embryonic development and the first few months of life after birth. HbF has a slightly greater oxygen binding affinity than adult hemoglobin (HbA, α2β2) and inhibits sickling by interfering with the polymerization of hemoglobin S. Higher HbF levels in SCD correlate with better survival and because HbF production can be reactivated pharmacologically in adults, it can be used for the treatment of SCD. Erythroid Kruppel-like factor (KLF1) is an erythroid-specific transcription factor that regulates β-globin expression through direct interaction with its promoter and indirectly regulates γ-globin expression through the regulation of BCL11A. By reducing the expression of KLF1, we can promote production of HbF through the upregulation of γ-globin expression. Since rodents don’t express γ-globin, we have employed both human and engineered mouse cell lines to demonstrate upregulation of γ-globin mRNA expression in vitro. We used MEL-h-b-BAC line#7 cells, a murine erythroleukemic cell line harboring the entire human beta globin locus and expressing mouse KLF1, and treated with antisense oligonucleotides (ASOs) targeting mouse KLF1. After 7 days of free uptake with the ASOs, we observed a 6-fold increase of human γ-globin mRNA expression after achieving 65% mRNA reduction of mouse KLF1 compared to the untreated control. We were also able to demonstrate significant upregulation of human γ-globin protein expression in these cells by western blot. We have shown similar results in a human erythroleukemia cell line, K562, using ASOs targeting human KLF1. K562 cells were electroporated with the KLF1 ASOs and 4 days later, we observed a 5-fold increase of human γ-globin mRNA expression after achieving 40% mRNA reduction of human KLF1 compared to the untreated control. These data indicate that targeting mouse or human KLF1 with ASO treatment can cause an increase in human γ-globin expression in vitrothat is necessary for the upregulation of fetal hemoglobin. We have also shown that we are able to target the bone marrow in both mice and rats through subcutaneous administration of our KLF1 ASOs. In wild type mice, at a dose of 100 mpk/wk for 4 weeks, we observed KLF1 target reduction of 88% and a β-globin reduction of 58% compared to the saline control in whole bone marrow. In Sprague-Dawley rats, at a dose of 50 mpk/wk for 4 weeks, we observed KLF1 target reduction of 83% and a β-globin reduction of 77% compared to the saline control in whole bone marrow. Therefore, we are able to achieve significant β-globin mRNA reduction in the bone marrow in both mice and rats after subcutaneous administration of KLF ASOs. These data indicate that reducing KLF1 with antisense oligonucleotides is a viable option for the treatment of sickle cell anemia. Disclosures Peralta: Isis Pharmaceuticals, Inc.: Employment. Low:Isis Pharmaceuticals, Inc.: Employment. Booten:Isis Pharmaceuticals, Inc.: Employment. Zhou:Univeristy of Alabama at Birmingham: Employment. Kim:Isis Pharmaceuticals, Inc.: Employment. Freier:Isis Pharmaceuticals, Inc.: Employment. Guo:Isis Pharmaceuticals, Inc.: Employment. Murray:Isis Pharmaceuticals, Inc.: Employment. Townes:University of Alabama at Birmingham: Employment. Hung:Isis Pharmaceuticals, Inc.: Employment.