Nine dogs with hemophilia A were treated with adeno-associated viral (AAV) gene therapy and followed for up to 10 years. Administration of AAV8 or AAV9 vectors expressing canine factor VIII ...(AAV-cFVIII) corrected the FVIII deficiency to 1.9-11.3% of normal FVIII levels. In two of nine dogs, levels of FVIII activity increased gradually starting about 4 years after treatment. None of the dogs showed evidence of tumors or altered liver function. Analysis of integration sites in liver samples from six treated dogs identified 1,741 unique AAV integration events in genomic DNA and expanded cell clones in five dogs, with 44% of the integrations near genes involved in cell growth. All recovered integrated vectors were partially deleted and/or rearranged. Our data suggest that the increase in FVIII protein expression in two dogs may have been due to clonal expansion of cells harboring integrated vectors. These results support the clinical development of liver-directed AAV gene therapy for hemophilia A, while emphasizing the importance of long-term monitoring for potential genotoxicity.
Recent progress in engineering the genomes of large animals has spurred increased interest in developing better animal models for diseases where current options are inadequate. Here, we report the ...creation of Yucatan miniature pigs with targeted disruptions of the low-density lipoprotein receptor (LDLR) gene in an effort to provide an improved large animal model of familial hypercholesterolemia and atherosclerosis. Yucatan miniature pigs are well established as translational research models because of similarities to humans in physiology, anatomy, genetics, and size. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, male and female LDLR+/- pigs were generated. Subsequent breeding of heterozygotes produced LDLR-/- pigs. When fed a standard swine diet (low fat, no cholesterol), LDLR+/- pigs exhibited a moderate, but consistent increase in total and LDL cholesterol, while LDLR-/- pigs had considerably elevated levels. This severe hypercholesterolemia in homozygote animals resulted in atherosclerotic lesions in the coronary arteries and abdominal aorta that resemble human atherosclerosis. These phenotypes were more severe and developed over a shorter time when fed a diet containing natural sources of fat and cholesterol. LDLR-targeted Yucatan miniature pigs offer several advantages over existing large animal models including size, consistency, availability, and versatility. This new model of cardiovascular disease could be an important resource for developing and testing novel detection and treatment strategies for coronary and aortic atherosclerosis and its complications.
The rapid clearance of factor IX necessitates frequent intravenous administrations to achieve effective prophylaxis for patients with hemophilia B. Subcutaneous administration has historically been ...limited by low bioavailability and potency. Dalcinonacog alfa was developed using a rational design approach to be a subcutaneously administered, next-generation coagulation prophylactic factor IX therapy.
This study aimed to investigate the pharmacokinetic, pharmacodynamic, and safety profile of dalcinonacog alfa administered subcutaneously in hemophilia B dogs.
Two hemophilia B dogs received single-dose daily subcutaneous dalcinonacog alfa injections for six days. Factor IX antigen and activity, whole blood clotting time, and activated partial thromboplastin time were measured at various time points. Additionally, safety assessments for clinical adverse events and evaluations of laboratory test results were conducted.
There was an increase in plasma factor IX antigen with daily subcutaneous dalcinonacog alfa. Bioavailability of subcutaneous dalcinonacog alfa was 10.3% in hemophilia B dogs. Daily subcutaneous dosing of dalcinonacog alfa demonstrated the effects of bioavailability, time to maximal concentration, and half-life by reaching a steady-state activity sufficient to correct severe hemophilia to normal, after four days.
The increased potency of dalcinonacog alfa facilitated the initiation and completion of the Phase 1/2 subcutaneous dosing study in individuals with hemophilia B.
Colorectal cancer is a leading cause of cancer death, and a major risk factor is chronic inflammation. Despite the link between colitis and cancer, the mechanism by which inflammation leads to ...colorectal cancer is not well understood.
To investigate whether different forms of inflammation pose the same risk of cancer, we compared several murine models of colitis (dextran sodium sulfate DSS, 2,4,6-trinitrobenzene sulfonic acid, 4-ethoxylmethylene-2-phenyloxazol-5-one, Citrobacter rodentium, Fusobacterium nucleatum, and doxorubicin) with respect to their ability to lead to colonic tumorigenesis. We attempted to correlate the severity of colitis and inflammatory profile with the risk of tumorigenesis in both azoxymethane-dependent and Dclk1/APCfl/fl murine models of colitis-associated cancer.
DSS colitis reproducibly led to colonic tumors in both mouse models of colitis-associated cancer. In contrast, all other forms of colitis did not lead to cancer. When compared with the colitis not associated with tumorigenesis, DSS colitis was characterized by significantly increased CD11b+F4/80+Ly6Chigh macrophages and CD11b+Ly6G+ neutrophils. Interestingly, depletion of the CD11b+F4/80+Ly6Chigh macrophages inhibited tumorigenesis, whereas depletion of CD11b+Ly6G+ neutrophils had no effect on tumorigenesis. Furthermore, the macrophage-derived cytokines interleukin-1β, tumor necrosis factor–α, and interleukin-6 were significantly increased in DSS colitis and promoted stemness of Dclk1+ tuft cells that serve as the cellular origin of cancer.
We have identified CD11b+F4/80+Ly6Chigh macrophages as key mediators of cancer initiation in colitis-associated cancer. Development of new therapies that target these cells may provide an effective preventative strategy for colitis-associated cancer.
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Despite proven benefits, prophylactic treatment for hemophilia A is hampered by the short half-life of factor VIII. A recombinant factor VIII-Fc fusion protein (rFVIIIFc) was constructed to determine ...the potential for reduced frequency of dosing. rFVIIIFc has an ∼ 2-fold longer half-life than rFVIII in hemophilia A (HemA) mice and dogs. The extension of rFVIIIFc half-life requires interaction of Fc with the neonatal Fc receptor (FcRn). In FcRn knockout mice, the extension of rFVIIIFc half-life is abrogated, and is restored in human FcRn transgenic mice. The Fc fusion has no impact on FVIII-specific activity. rFVIIIFc has comparable acute efficacy as rFVIII in treating tail clip injury in HemA mice, and fully corrects whole blood clotting time (WBCT) in HemA dogs immediately after dosing. Furthermore, consistent with prolonged half-life, rFVIIIFc shows 2-fold longer prophylactic efficacy in protecting HemA mice from tail vein transection bleeding induced 24-48 hours after dosing. In HemA dogs, rFVIIIFc also sustains partial correction of WBCT 1.5- to 2-fold longer than rFVIII. rFVIIIFc was well tolerated in both species. Thus, the rescue of FVIII by Fc fusion to provide prolonged protection presents a novel pathway for FVIII catabolism, and warrants further investigation.
In this issue of Blood, Chan et al have described mechanisms by which L5, the most electronegative of 5 recognized fractions of low density lipoprotein (LDL), activates both platelets and endothelium ...in a manner that supports thrombosis and could possibly produce ST-elevation myocardial infarctions (STEMI).
Treatment of hemophilia B requires frequent infusions of factor IX (FIX) to prophylax against bleeding episodes. Hemophilia B management would benefit from a FIX protein with an extended half-life. A ...recombinant fusion protein (rFIXFc) containing a single FIX molecule attached to the Fc region of immunoglobulin G was administered intravenously and found to have an extended half-life, compared with recombinant FIX (rFIX) in normal mice, rats, monkeys, and FIX-deficient mice and dogs. Recombinant FIXFc protein concentration was determined in all species, and rFIXFc activity was measured in FIX-deficient animals. The half-life of rFIXFc was approximately 3- to 4-fold longer than that of rFIX in all species. In contrast, in mice in which the neonatal Fc receptor (FcRn) was deleted, the half-life of rFIXFc was similar to rFIX, confirming the increased circulatory time was due to protection of the rFIXFc via the Fc/FcRn interaction. Whole blood clotting time in FIX-deficient mice was corrected through 144 hours for rFIXFc, compared with 72 hours for rFIX; similar results were observed in FIX-deficient dogs. Taken together, these studies show the enhanced pharmacodynamic and pharmacokinetic properties of the rFIXFc fusion protein and provide the basis for evaluating rFIXFc in patients with hemophilia B.
Glucocorticoids have anti-inflammatory and immunosuppressive functions and have commonly been used for preventing liver toxicity after the systemic application of a high dose of adeno-associated ...virus (AAV) vector for gene therapy. Clinical studies have reported that glucocorticoids have rescued factor IX (FIX) expression in patients with hemophilia B who showed a reduced FIX expression at 6 to 10 weeks post-AAV vector administration. In this study, we explored whether glucocorticoids could affect transgene expression in AAV targeted livers in animal models. When dexamethasone was applied before AAV9/FIX vector administration in the wild-type C57BL/6 mice, FIX expression was much higher than that of the control mice at any time point. More importantly, FIX expression transiently increased after dexamethasone was administered at week 6 or later post-AAV injection regardless of the various dexamethasone treatments applied. The transient enhancement in transgene expression was observed once there were one to several consecutive dexamethasone treatments completed. A similar result was also achieved in other wild-type BALB/c and hemophilia B mice that were treated with AAV9/FIX and dexamethasone. This mechanism study demonstrated that the administration of dexamethasone did not change either AAV genome copy number or transgene expression at the transcription level but transiently decreased interferon beta (IFN-β) and tumor necrosis factor alpha (TNF-α) expression in the livers of mice at a later time after AAV injection. Next, we studied the effect of dexamethasone on late transgene expression in hemophilia B dogs. Dexamethasone was administered 1 year after AAV9/FIX injection. Inconsistent with the results in mice, no significant change of FIX expression was observed in hemophilia B dogs. In summary, the results from this study indicate that dexamethasone may have various effects on transgene expression in AAV-transduced livers in different species, which provides valuable information about the rational application of dexamethasone in future clinical studies.
Biotic interactions underlie ecosystem structure and function, but predicting interaction outcomes is difficult. We tested the hypothesis that biotic interaction strength increases toward the ...equator, using a global experiment with model caterpillars to measure predation risk. Across an 11,660-kilometer latitudinal gradient spanning six continents, we found increasing predation toward the equator, with a parallel pattern of increasing predation toward lower elevations. Patterns across both latitude and elevation were driven by arthropod predators, with no systematic trend in attack rates by birds or mammals. These matching gradients at global and regional scales suggest consistent drivers of biotic interaction strength, a finding that needs to be integrated into general theories of herbivory, community organization, and life-history evolution.
Small‐cell lung cancer (SCLC) is a heterogeneous disease, consisting of intratumoral and intertumoral neuroendocrine (ASCL1 and/or NEUROD1), mesenchymal‐like, and YAP‐driven transcriptional states. ...Lysine‐specific demethylase 1 (LSD1; also known as KDM1A) inhibitors have recently been progressed to clinical trials in SCLC based on a promising preclinical antitumor activity. A potential clinical limitation of LSD1 inhibitors is the heterogeneous drug responses that have been observed in SCLC cell lines and patient‐derived models. Based on these observations, we studied molecular and transcriptional signatures that predict patient response to this class of drug. Employing SCLC patient‐derived transcriptional signatures, we define that SCLC cell lines sensitive to LSD1 inhibitors are enriched in neuroendocrine transcriptional markers, whereas cell lines enriched in a mesenchymal‐like transcriptional program demonstrate intrinsic resistance to LSD1 inhibitors. We have identified a reversible, adaptive resistance mechanism to LSD1 inhibitors through epigenetic reprogramming to a TEAD4‐driven mesenchymal‐like state. Our data suggest that only a segment of SCLC patients, with a defined neuroendocrine differentiation state, will likely benefit from LSD1 inhibitors. It provides novel evidence for the selection of a TEAD4‐driven mesenchymal‐like subpopulation resistant to LSD1 inhibitors in SCLC patients that may require effective drug combinations to sustain effective clinical responses.
Here, we identify intratumor and intertumor heterogeneity limits the activity of LSD1 inhibitors in SCLC. Activity of LSD1 inhibitors in SCLC is confided in cells enriched in neuroendocrine transcriptional programs. Transdifferentiation of SCLC into a TEAD4‐driven mesenchymal‐like state represents an acquired resistance mechanism to LSD1 inhibitors. Drug combinations targeting YAP‐TEAD pathways with LSD1 inhibitors may be an effective strategy to target intrinsic‐ and adaptive‐resistant populations and sustain effective clinical responses.
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