CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis.

BACKGROUND: Transthyretin amyloidosis, also called ATTR amyloidosis, is a life-threatening disease characterized by progressive accumulation of misfolded transthyretin (TTR) protein in tissues, predominantly the nerves and heart. NTLA-2001 is an in vivo gene-editing therapeutic agent that is designed to treat ATTR amyloidosis by reducing the concentration of TTR in serum. It is based on the clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9) system and comprises a lipid nanoparticle encapsulating messenger RNA for Cas9 protein and a single guide RNA targeting TTR. METHODS: After conducting preclinical in vitro and in vivo studies, we evaluated the safety and pharmacodynamic effects of single escalating doses of NTLA-2001 in six patients with hereditary ATTR amyloidosis with polyneuropathy, three in each of the two initial dose groups (0.1 mg per kilogram and 0.3 mg per kilogram), within an ongoing phase 1 clinical study. RESULTS: Preclinical studies showed durable knockout of TTR after a single dose. Serial assessments of safety during the first 28 days after infusion in patients revealed few adverse events, and those that did occur were mild in grade. Dose-dependent pharmacodynamic effects were observed. At day 28, the mean reduction from baseline in serum TTR protein concentration was 52% (range, 47 to 56) in the group that received a dose of 0.1 mg per kilogram and was 87% (range, 80 to 96) in the group that received a dose of 0.3 mg per kilogram. CONCLUSIONS: In a small group of patients with hereditary ATTR amyloidosis with polyneuropathy, administration of NTLA-2001 was associated with only mild adverse events and led to decreases in serum TTR protein concentrations through targeted knockout of TTR. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.).

Gaining Efficiency in Clinical Trials With Cardiac Biomarkers: JACC Review Topic of the Week.

The momentum of cardiovascular drug development has slowed dramatically. Use of validated cardiac biomarkers in clinical trials could accelerate development of much-needed therapies, but biomarkers have been used less for cardiovascular drug development than in therapeutic areas such as oncology. Moreover, there are inconsistences in biomarker use in clinical trials, such as sample type, collection times, analytical methods, and storage for future research. With these needs in mind, participants in a Cardiac Safety Research Consortium Think Tank proposed the development of international guidance in this area, together with improved quality assurance and analytical methods, to determine what biomarkers can reliably show. Participants recommended the development of systematic methods for sample collection, and the archiving of samples in all cardiovascular clinical trials (including creation of a biobank or repository). The academic and regulatory communities also agreed to work together to ensure that published information is fully and clearly expressed.

CETP (Cholesteryl Ester Transfer Protein) Inhibition With Anacetrapib Decreases Production of Lipoprotein(a) in Mildly Hypercholesterolemic Subjects.

OBJECTIVE: Lp(a) [lipoprotein (a)] is composed of apoB (apolipoprotein B) and apo(a) [apolipoprotein (a)] and is an independent risk factor for cardiovascular disease and aortic stenosis. In clinical trials, anacetrapib, a CETP (cholesteryl ester transfer protein) inhibitor, causes significant reductions in plasma Lp(a) levels. We conducted an exploratory study to examine the mechanism for Lp(a) lowering by anacetrapib. APPROACH AND RESULTS: We enrolled 39 participants in a fixed-sequence, double-blind study of the effects of anacetrapib on the metabolism of apoB and high-density lipoproteins. Twenty-nine patients were randomized to atorvastatin 20 mg/d, plus placebo for 4 weeks, and then atorvastatin plus anacetrapib (100 mg/d) for 8 weeks. The other 10 subjects were randomized to double placebo for 4 weeks followed by placebo plus anacetrapib for 8 weeks. We examined the mechanisms of Lp(a) lowering in a subset of 12 subjects having both Lp(a) levels >20 nmol/L and more than a 15% reduction in Lp(a) by the end of anacetrapib treatment. We performed stable isotope kinetic studies using 2H3-leucine at the end of each treatment to measure apo(a) fractional catabolic rate and production rate. Median baseline Lp(a) levels were 21.5 nmol/L (interquartile range, 9.9-108.1 nmol/L) in the complete cohort (39 subjects) and 52.9 nmol/L (interquartile range, 38.4-121.3 nmol/L) in the subset selected for kinetic studies. Anacetrapib treatment lowered Lp(a) by 34.1% (P≤0.001) and 39.6% in the complete and subset cohort, respectively. The decreases in Lp(a) levels were because of a 41% reduction in the apo(a) production rate, with no effects on apo(a) fractional catabolic rate. CONCLUSIONS: Anacetrapib reduces Lp(a) levels by decreasing its production. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00990808.

Effects of Rifampin, a potent inducer of drug-metabolizing enzymes and an inhibitor of OATP1B1/3 transport, on the single dose pharmacokinetics of anacetrapib.

Anacetrapib is a novel cholesteryl ester transfer protein (CETP) inhibitor in development for treatment of dyslipidemia. This open-label, fixed-sequence, 3-period study was intended to evaluate the potential of anacetrapib to be a victim of OATP1B1/3 inhibition and strong CYP3A induction using acute and chronic dosing of rifampin, respectively, as a probe. In this study, 16 healthy subjects received 100 mg anacetrapib administered without rifampin (Day 1, Period 1), with single-dose (SD) 600 mg rifampin (Day 1, Period 2), and with multiple-dose (MD) 600 mg rifampin for 20 days (Day 14, Period 3). Log-transformed anacetrapib AUC0-∞ and Cmax were analyzed by a linear mixed effects model. The GMRs and 90% CIs for anacetrapib AUC0-∞ and Cmax were 1.25 (1.04, 1.51) and 1.43 (1.13, 1.82) for SD rifampin (Period 2/Period 1) and 0.35 (0.29, 0.42) and 0.26 (0.21, 0.32) for MD rifampin (Period 3/Period 1), respectively. Anacetrapib was generally well tolerated in both the absence/presence of SD and MD rifampin. In conclusion, treatment with SD rifampin, which inhibits the OATP1B1/3 transporter system, did not substantially influence the SD pharmacokinetics of anacetrapib, while chronic (20 days) administration of rifampin, which strongly induces CYP3A isozymes, reduced mean systemic exposure to SD anacetrapib by 65%.

Quantification of circulating D-dimer by peptide immunoaffinity enrichment and tandem mass spectrometry.

D-dimer is a product of the coagulation cascade and is associated with venous thromboembolism, disseminated intravascular coagulation, and additional clinical conditions. Despite its importance, D-dimer measurement has limited clinical utility due in part to the lack of reliable assays. The difficulty in developing an immunoassay that is specific for D-dimer arises from the inherent heterogeneity in its structure. In this report, we describe a highly specific method for the quantification of D-dimer level in human plasma. In our method, the reciprocally cross-linked peptide resulting from factor XIIIa-catalyzed dimerization of fibrin γ chains was selected to represent the D-dimer antigen. Using an antipeptide antibody, we enriched the cross-linked peptide from trypsin-digested plasma prior to quantitative analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The assay has a quantitative range of 500 pmol/L to 100 nmol/L in human plasma. In further characterization of the assay, we found that it exhibited good correlation with fibrinolytic activity in human donors and with thrombin generation and clot strength in an in vitro thromboelastography assay. These observations thus establish the biological relevance of the assay and suggest it may be a valuable biomarker in characterization and treatment of blood coagulation disorders.

Connexin-43 prevents hematopoietic stem cell senescence through transfer of reactive oxygen species to bone marrow stromal cells.

Hematopoietic stem cell (HSC) aging has become a concern in chemotherapy of older patients. Humoral and paracrine signals from the bone marrow (BM) hematopoietic microenvironment (HM) control HSC activity during regenerative hematopoiesis. Connexin-43 (Cx43), a connexin constituent of gap junctions (GJs) is expressed in HSCs, down-regulated during differentiation, and postulated to be a self-renewal gene. Our studies, however, reveal that hematopoietic-specific Cx43 deficiency does not result in significant long-term competitive repopulation deficiency. Instead, hematopoietic Cx43 (H-Cx43) deficiency delays hematopoietic recovery after myeloablation with 5-fluorouracil (5-FU). 5-FU-treated H-Cx43-deficient HSC and progenitors (HSC/P) cells display decreased survival and fail to enter the cell cycle to proliferate. Cell cycle quiescence is associated with down-regulation of cyclin D1, up-regulation of the cyclin-dependent kinase inhibitors, p21(cip1.) and p16(INK4a), and Forkhead transcriptional factor 1 (Foxo1), and activation of p38 mitogen-activated protein kinase (MAPK), indicating that H-Cx43-deficient HSCs are prone to senescence. The mechanism of increased senescence in H-Cx43-deficient HSC/P cells depends on their inability to transfer reactive oxygen species (ROS) to the HM, leading to accumulation of ROS within HSCs. In vivo antioxidant administration prevents the defective hematopoietic regeneration, as well as exogenous expression of Cx43 in HSC/P cells. Furthermore, ROS transfer from HSC/P cells to BM stromal cells is also rescued by reexpression of Cx43 in HSC/P. Finally, the deficiency of Cx43 in the HM phenocopies the hematopoietic defect in vivo. These results indicate that Cx43 exerts a protective role and regulates the HSC/P ROS content through ROS transfer to the HM, resulting in HSC protection during stress hematopoietic regeneration.

Connexin-43 in the osteogenic BM niche regulates its cellular composition and the bidirectional traffic of hematopoietic stem cells and progenitors.

Connexin-43 (Cx43), a gap junction protein involved in control of cell proliferation, differentiation and migration, has been suggested to have a role in hematopoiesis. Cx43 is highly expressed in osteoblasts and osteogenic progenitors (OB/P). To elucidate the biologic function of Cx43 in the hematopoietic microenvironment (HM) and its influence in hematopoietic stem cell (HSC) activity, we studied the hematopoietic function in an in vivo model of constitutive deficiency of Cx43 in OB/P. The deficiency of Cx43 in OB/P cells does not impair the steady state hematopoiesis, but disrupts the directional trafficking of HSC/progenitors (Ps) between the bone marrow (BM) and peripheral blood (PB). OB/P Cx43 is a crucial positive regulator of transstromal migration and homing of both HSCs and progenitors in an irradiated microenvironment. However, OB/P Cx43 deficiency in nonmyeloablated animals does not result in a homing defect but induces increased endosteal lodging and decreased mobilization of HSC/Ps associated with proliferation and expansion of Cxcl12-secreting mesenchymal/osteolineage cells in the BM HM in vivo. Cx43 controls the cellular content of the BM osteogenic microenvironment and is required for homing of HSC/Ps in myeloablated animals.

Transgenic expression of a dominant negative K(ATP) channel subunit in the mouse endothelium: effects on coronary flow and endothelin-1 secretion.

K(ATP) channels are involved in regulating coronary function, but the contribution of endothelial K(ATP) channels remains largely uncharacterized. We generated a transgenic mouse model to specifically target endothelial K(ATP) channels by expressing a dominant negative Kir6.1 subunit only in the endothelium. These animals had no obvious overt phenotype and no early mortality. Histologically, the coronary endothelium in these animals was preserved. There was no evidence of increased susceptibility to ergonovine-induced coronary vasospasm. However, isolated hearts from these animals had a substantially elevated basal coronary perfusion pressure. The K(ATP) channel openers, adenosine and levcromakalim, decreased the perfusion pressure whereas the K(ATP) channel blocker glibenclamide failed to produce a vasoconstrictive response. The inducible endothelial nitric oxide pathway was intact, as evidenced by vasodilation caused by bradykinin. In contrast, basal endothelin-1 release was significantly elevated in the coronary effluent from these hearts. Treatment of mice with bosentan (endothelin-1 receptor antagonist) normalized the coronary perfusion pressure, demonstrating that the elevated endothelin-1 release was sufficient to account for the increased coronary perfusion pressure. Pharmacological blockade of K(ATP) channels led to elevated endothelin-1 levels in the coronary effluent of isolated mouse and rat hearts as well as enhanced endothelin-1 secretion from isolated human coronary endothelial cells. These data are consistent with a role for endothelial K(ATP) channels to control the coronary blood flow by modulating the release of the vasoconstrictor, endothelin-1.

Proliferation of adult sertoli cells following conditional knockout of the Gap junctional protein GJA1 (connexin 43) in mice.

GJA1 (also known and referred to here as connexin 43 and abbreviated CX43) is the predominant testicular gap junction protein, and CX43 may regulate Sertoli cell maturation and spermatogenesis. We hypothesized that lack of CX43 would inhibit Sertoli cell differentiation and extend proliferation. To test this, a Sertoli cell-specific Cx43 knockout (SC-Cx43 KO) mouse was generated using Cre-lox technology. Immunohistochemistry indicated that CX43 was not expressed in the Sertoli cells of SC-Cx43 KO mice, but was normal in organs such as the heart. Testicular weight was reduced by 41% and 76% in SC-Cx43 KO mice at 20 and 60 days, respectively, vs. wild-type (wt) mice. Seminiferous tubules of SC-Cx43 KO mice contained only Sertoli cells and actively proliferating early spermatogonia. Sertoli cells normally cease proliferation at 2 wk of age in mice and become terminally differentiated. However, proliferating Sertoli cells were present in SC-Cx43 KO but not wt mice at 20 and 60 days of age. Thyroid hormone receptor alpha (THRA) is high in proliferating Sertoli cells, then declines sharply in adulthood. Thra mRNA expression was increased in 20-day SC-Cx43 KO vs. wt mice, and it showed a trend toward an increase in 60-day mice, indicating that loss of CX43 in Sertoli cells inhibited their maturation. In conclusion, we have generated mice lacking CX43 in Sertoli cells but not other tissues. Our data indicate that CX43 in Sertoli cells is essential for spermatogenesis but not spermatogonial maintenance/proliferation. SC-Cx43 KO mice showed continued Sertoli cell proliferation and delayed maturation in adulthood, indicating that CX43 plays key roles in Sertoli cell development.

Bone marrow connexin-43 expression is critical for hematopoietic regeneration after chemotherapy.

Contact between bone marrow (BM) hematopoietic stem cells (HSC) and osteoblast/stromal (OS) cells has been shown to be crucial in the regulation of hematopoiesis. However, very little is known about the regulatory mechanisms of direct cell-to-cell communication in the hematopoietic microenvironment. Gap junction channels (connexons) are formed by polypeptides (connexins) arranged in hexamers and represent the best described intercellular communication system. Connexin-43 (Cx43) is expressed by BM OS cells and has been associated with the cadherin/beta -catenin signaling pathway, recently reported as relevant in the OS/HSC interaction at the stem cell niche. Here, we employed an inducible gene-targeted murine approach to study the role of Cx43 in HSC proliferation and differentiation in vivo. Mx-Cre/Cx43+/+ and Mx-Cre/Cx43flox/flox littermates have been analyzed after gene deletion induced in vivo by the interferon-inducer poly (I)-poly (C), generating control (Cx43+) and Cx43-deficient (Cx43-/-) mice. After one week, Cx43+ and Cx43-/- mice were treated with 5-fluorouracil (5-FU). Cx43 expression in Cx43-/- BM was markedly reduced (> 90%) as analyzed on day +14 post-5-FU treatment. Cx43 deficiency did not induce a significant change in peripheral blood counts before 5-FU treatment, but the hematopoiesis recovery after 5-FU treatment was severely impaired as demonstrated by absence of recovery of peripheral blood counts, including profound neutropenia, anemia with reticulocytopenia, thrombocytopenia and a 5- to 8-fold decrease of cellularity and hematopoietic progenitor content (granulomacrophagic colony-forming-units (CFU-GM-), erythroid burst forming units (BFU-E) and mixed colony forming units (CFU-mix-) in BM and spleen on day +14 post-5-FU treatment. However, the femoral content of Lin-/c-kit+/Sca1+ cells in Cx43-/- BM was maintained when compared to Cx43+ BM. Short-term competitive repopulation ability of Cx43-/- BM cells was diminished as compared to Cx43+ mice, specifically for myeloid and B lymphoid cells, but showed spared long-term competitive repopulation ability with roughly normal hematopoietic differentiation. These data suggest that hematopoietic regeneration after cycle-specific chemotherapy is blocked in Cx43-deficient mice at the long-term HSC repopulating level. Cx43 expression within the BM appears to be crucial in the development of an efficient response to hematopoietic stress.

Modulation of cardiac gap junction expression and arrhythmic susceptibility.

Connexin43 (Cx43), the predominant ventricular gap junction protein, is critical for maintaining normal cardiac electrical conduction, and its absence in the mouse heart results in sudden arrhythmic death. The mechanisms linking reduced Cx43 abundance in the heart and inducibility of malignant ventricular arrhythmias have yet to be established. In this report, we investigate arrhythmic susceptibility in a murine model genetically engineered to express progressively decreasing levels of Cx43. Progressively older cardiac-restricted Cx43 conditional knockout (CKO) mice were selectively bred to produce a heart-specific Cx43-deficient subline ("O-CKO" mice) in which the loss of Cx43 in the heart occurs more gradually. O-CKO mice lived significantly longer than the initial series of CKO mice but still died suddenly and prematurely. At 25 days of age, cardiac Cx43 protein levels decreased to 59% of control values (P<0.01), but conduction velocity was not significantly decreased and no O-CKO mice were inducible into sustained ventricular tachyarrhythmias. By 45 days of age, cardiac Cx43 abundance had decreased in a heterogeneous fashion to 18% of control levels, conduction velocity had slowed to half of that observed in control hearts, and 80% of O-CKO mice were inducible into lethal tachyarrhythmias. Enhanced susceptibility to induced arrhythmias was not associated with altered invasive hemodynamic measurements or changes in ventricular effective refractory period. Thus, moderately severe reductions in Cx43 abundance are associated with slowing of impulse propagation and a dramatic increase in the susceptibility to inducible ventricular arrhythmias.

Exercise training normalizes altered calcium-handling proteins during development of heart failure.

The cardiac sarcoplasmic reticulum calcium-ATPase (SERCA2a), Na+/Ca2+ exchanger (NCX1), and ryanodine receptor (RyR2) are proteins involved in the regulation of myocyte calcium. We tested whether exercise training (ET) alters those proteins during development of chronic heart failure (CHF). Ten dogs were chronically instrumented to permit hemodynamic measurements. Five dogs underwent 4 wk of cardiac pacing (210 beats/min for 3 wk and 240 beats/min for the 4th wk), whereas five dogs underwent the same pacing regimen plus daily ET (5.1 +/- 0.3 km/h, 2 h/day). Paced animals developed CHF characterized by hemodynamic abnormalities and reduced ejection fraction. ET preserved resting hemodynamics and ejection fraction. Left ventricular samples were obtained from all dogs and another five normal dogs for mRNA (Northern analysis, band intensities normalized to glyceraldehyde-3-phosphate dehydrogenase) and protein level (Western analysis, band intensities normalized to tubulin) measurements. In failing hearts, SERCA2a was decreased by 33% (P < 0.05) and 65% (P < 0.05) in mRNA and protein level, respectively, compared with normal hearts; there was only an 8.6% reduction in mRNA and a 32% reduction in protein in exercised animals (P < 0.05 from CHF). mRNA expression of NCX1 increased by 44% in paced-only dogs compared with normal (P < 0.05) but only by 22% in trained dogs (P < 0.05 vs. CHF); protein level of NCX1 was elevated in paced-only dogs (71%, P < 0.05) but partially normalized by ET (33%, P < 0.05 from CHF). RyR2 was not altered in any of the dogs. In conclusion, long-term ET may ameliorate cardiac deterioration during development of CHF, in part via normalization of myocardial calcium-handling proteins.