Ionizable Lipid with Supramolecular Chemistry Features for RNA Delivery In Vivo.

Lipid nanoparticles (LNPs) and ribonucleic acid (RNA) technology are highly versatile tools that can be deployed for diagnostic, prophylactic, and therapeutic applications. In this report, supramolecular chemistry concepts are incorporated into the rational design of a new ionizable lipid, C3-K2-E14, for systemic administration. This lipid incorporates a cone-shaped structure intended to facilitate cell bilayer disruption, and three tertiary amines to improve RNA binding. Additionally, hydroxyl and amide motifs are incorporated to further enhance RNA binding and improve LNP stability. Optimization of messenger RNA (mRNA) and small interfering RNA (siRNA) formulation conditions and lipid ratios produce LNPs with favorable diameter (<150 nm), polydispersity index (<0.15), and RNA encapsulation efficiency (>90%), all of which are preserved after 2 months at 4 or 37 °C storage in ready-to-use liquid form. The lipid and formulated LNPs are well-tolerated in animals and show no deleterious material-induced effects. Furthermore, 1 week after intravenous LNP administration, fluorescent signal from tagged RNA payloads are not detected. To demonstrate the long-term treatment potential for chronic diseases, repeated dosing of C3-K2-E14 LNPs containing siRNA that silences the colony stimulating factor-1 (CSF-1) gene can modulate leukocyte populations in vivo, further highlighting utility.

Iterative Design of Ionizable Lipids for Intramuscular mRNA Delivery.

Lipid nanoparticles (LNPs) are the most clinically advanced delivery vehicles for RNA and have enabled the development of RNA-based drugs such as the mRNA COVID-19 vaccines. Functional delivery of mRNA by an LNP greatly depends on the inclusion of an ionizable lipid, and small changes to these lipid structures can significantly improve delivery. However, the structure-function relationships between ionizable lipids and mRNA delivery are poorly understood, especially for LNPs administered intramuscularly. Here, we show that the iterative design of a novel series of ionizable lipids generates key structure-activity relationships and enables the optimization of chemically distinct lipids with efficacy that is on-par with the current state of the art. We find that the combination of ionizable lipids comprising an ethanolamine core and LNPs with an apparent pKa between 6.6 and 6.9 maximizes intramuscular mRNA delivery. Furthermore, we report a nonlinear relationship between the lipid-to-mRNA mass ratio and protein expression, suggesting that a critical mass ratio exists for LNPs and may depend on ionizable lipid structure. Our findings add to the mechanistic understanding of ionizable lipids and demonstrate that hydrogen bonding, ionization behavior, and lipid-to-mRNA mass ratio are key design parameters affecting intramuscular mRNA delivery. We validate these insights by applying them to the rational design of new ionizable lipids. Overall, our iterative design strategy efficiently generates potent ionizable lipids. This hypothesis-driven method reveals structure-activity relationships that lay the foundation for the optimization of ionizable lipids in future LNP-RNA drugs. We foresee that this design strategy can be extended to other optimization parameters beyond intramuscular expression.

The engineering challenges and opportunities when designing potent ionizable materials for the delivery of ribonucleic acids.

INTRODUCTION: Ionizable lipids are critical components in lipid nanoparticles. These molecules sequester nucleic acids for delivery to cells. However, to build more efficacious delivery molecules, the field must continue to broaden structure-function studies for greater insight. While nucleic acid-binding efficiency, degradability and nanoparticle stability are vitally important, this review offers perspective on additional factors that must be addressed to improve delivery efficiency. AREAS COVERED: We discuss how administration route, cellular heterogeneity, uptake pathway, endosomal escape timing, age, sex, and threshold effects can change depending on the type of LNP ionizable lipid. EXPERT OPINION: Ionizable lipid structure-function studies often focus on the efficiency of RNA utilization and biodistribution. While these focus areas are critical, they remain high-level observations. As our tools for observation and system interrogation improve, we believe that the field should begin collecting additional data. At the cellular level, this data should include age (dividing or senescent cells), sex and phenotype, cell entry pathway, and endosome type. Additionally, administration route and dose are essential to track. This additional data will allow us to identify and understand heterogeneity in LNP efficacy across patient populations, which will help us provide better ionizable lipid options for different groups.

Triple-Negative Breast Cancer: A Review of Current Curative Intent Therapies.

Breast cancer is the most commonly diagnosed malignancy in women, with triple-negative breast cancer (TNBC) accounting for 10-20% of cases. Historically, fewer treatment options have existed for this subtype of breast cancer, with cytotoxic chemotherapy playing a predominant role. This article aims to review the current treatment paradigm for curative-intent TNBC, while also reviewing potential future developments in this landscape. In addition to chemotherapy, recent advances in the understanding of the molecular biology of TNBC have led to promising new studies of targeted and immune checkpoint inhibitor therapies in the curative-intent setting. The appropriate selection of TNBC patient subgroups with a higher likelihood of benefit from treatment is critical to identify the best treatment approach.

Evolution of an invasive ductal carcinoma to a small cell carcinoma of the breast: A case report.

RATIONALE: Small cell carcinoma (SCC) is a rare subtype of breast cancer and presents a complex diagnostic and treatment challenge, due to paucity of data. To the best of our knowledge, most cases of breast SCC reported in the literature describe a de novo breast primary. Our case is unique as it describes the evolution of an invasive ductal carcinoma after treatment into a SCC of the breast. PATIENT CONCERNS AND DIAGNOSIS: We report a case of a 53-year-old female, lifelong non-smoker, who initially presented with breast mass noted on self examination. Breast and axillary lymph node biopsy demonstrated a hormone receptor positive invasive ductal carcinoma with a metastatic T3 lesion. INTERVENTION: She was treated with first-line palbociclib/letrozole with initial clinical response, and at progression was switched to capecitabine with no response. Repeat biopsy of the axillary lesion showed evolution of the tumor into a triple negative breast cancer. She was then treated with third-line paclitaxel and radiation therapy with good initial response. She eventually had further disease progression and presented with a new mediastinal lymphadenopathy causing SVC syndrome. Biopsy of this showed a small cell variant of breast neuroendocrine carcinoma. Due to the evolution of histology in this case, a retrospective review of her initial breast specimen as well as the second biopsy from the axilla was conducted which confirmed that the mediastinal lymphadenopathy was metastatic from the original breast tumor. OUTCOMES AND LESSONS: We speculate that the initial treatment allowed a minority of treatment-resistant neuroendocrine cells to grow and become the dominant face of the tumor. Our patient had an excellent response to carboplatin/etoposide and consolidative locoregional radiotherapy but presented with an early intracranial recurrence. This is a similar pattern of metastases as seen in lung SCC and highlights a potential role for prophylactic cranial irradiation in breast SCC. Further studies are needed to better understand the biology and treatment of breast SCC which continues to present a challenge for clinicians.

Frataxin deficiency promotes endothelial senescence in pulmonary hypertension.

The dynamic regulation of endothelial pathophenotypes in pulmonary hypertension (PH) remains undefined. Cellular senescence is linked to PH with intracardiac shunts; however, its regulation across PH subtypes is unknown. Since endothelial deficiency of iron-sulfur (Fe-S) clusters is pathogenic in PH, we hypothesized that a Fe-S biogenesis protein, frataxin (FXN), controls endothelial senescence. An endothelial subpopulation in rodent and patient lungs across PH subtypes exhibited reduced FXN and elevated senescence. In vitro, hypoxic and inflammatory FXN deficiency abrogated activity of endothelial Fe-S-containing polymerases, promoting replication stress, DNA damage response, and senescence. This was also observed in stem cell-derived endothelial cells from Friedreich's ataxia (FRDA), a genetic disease of FXN deficiency, ataxia, and cardiomyopathy, often with PH. In vivo, FXN deficiency-dependent senescence drove vessel inflammation, remodeling, and PH, whereas pharmacologic removal of senescent cells in Fxn-deficient rodents ameliorated PH. These data offer a model of endothelial biology in PH, where FXN deficiency generates a senescent endothelial subpopulation, promoting vascular inflammatory and proliferative signals in other cells to drive disease. These findings also establish an endothelial etiology for PH in FRDA and left heart disease and support therapeutic development of senolytic drugs, reversing effects of Fe-S deficiency across PH subtypes.

Nanoparticle-encapsulated siRNAs for gene silencing in the haematopoietic stem-cell niche.

Bone-marrow endothelial cells in the haematopoietic stem-cell niche form a network of blood vessels that regulates blood-cell traffic as well as the maintenance and function of haematopoietic stem and progenitor cells. Here, we report the design and in vivo performance of systemically injected lipid-polymer nanoparticles encapsulating small interfering RNA (siRNA), for the silencing of genes in bone-marrow endothelial cells. In mice, nanoparticles encapsulating siRNA sequences targeting the proteins stromal-derived factor 1 (Sdf1) or monocyte chemotactic protein 1 (Mcp1) enhanced (when silencing Sdf1) or inhibited (when silencing Mcp1) the release of stem and progenitor cells and of leukocytes from the bone marrow. In a mouse model of myocardial infarction, nanoparticle-mediated inhibition of cell release from the haematopoietic niche via Mcp1 silencing reduced leukocytes in the diseased heart, improved healing after infarction and attenuated heart failure. Nanoparticle-mediated RNA interference in the haematopoietic niche could be used to investigate haematopoietic processes for therapeutic applications in cancer, infection and cardiovascular disease.

Vascularized Muscle Flap to Reduce Wound Breakdown During Flexible Electrode-Mediated Functional Electrical Stimulation After Peripheral Nerve Injury.

The success of devices delivering functional electrical stimulation (FES) has been hindered by complications related to implants including skin breakdown and subsequent wound dehiscence. Our hypothesis was that a vascularized muscle flap along the dorsal surface of an epimysial electrode would prevent skin breakdown during FES therapy to treat atrophy of the gastrocnemius muscle during peripheral nerve injury. Resection of a tibial nerve segment with subsequent electrode implantation on the dorsal surfaces of the gastrocnemius muscle was performed on ten Lewis rats. In five rats, the biceps femoris (BF) muscle was dissected and placed along the dorsal surface of the electrode (Flap group). The other five animals did not undergo flap placement (No Flap group). All animals were treated with daily FES therapy for 2 weeks and degree of immune response and skin breakdown were evaluated. The postoperative course of one animal in the No Flap group was complicated by complete wound dehiscence requiring euthanasia of the animal on postoperative day 4. The remaining 4 No Flap animals showed evidence of ulceration at the implant by postoperative day 7. The 5 animals in the Flap group did not have ulcerative lesions. Excised tissue at postoperative day 14 examined by histology and in vivo Imaging System (IVIS) showed decreased implant-induced inflammation in the Flap group. Expression of specific markers for local foreign body response were also decreased in the Flap group.

Flexible Multielectrode Array for Skeletal Muscle Conditioning, Acetylcholine Receptor Stabilization and Epimysial Recording After Critical Peripheral Nerve Injury.

Complete re-innervation after a traumatic injury severing a muscle's peripheral nerve may take years. During this time, the denervated muscle atrophies and loses acetylcholine receptors, a vital component of the neuromuscular junction, limiting functional recovery. One common clinical treatment for atrophy is electrical stimulation; however, epimysial electrodes currently used are bulky and often fail due to an excessive inflammatory response. Additionally, there remains a need for a device providing in vivo monitoring of neuromuscular regeneration and the maintenance of acetylcholine receptors. Here, an implantable, flexible microelectrode array (MEA) was developed that provides surface neuromuscular stimulation and recording during long-term denervation. Methods: The MEA uses a flexible polyimide elastomer and an array of gold-based microelectrodes featuring Peano curve motifs, which together maintain electrode flexibility. The devices were implanted along the denervated gastrocnemius muscles of 5 rats. These rats underwent therapeutic stimulation using the MEA daily beginning on post-operative day 2. Another 5 rats underwent tibial nerve resection without implantation of MEA. Tissues were harvested on post-operative day 14 and evaluated for quantification of acetylcholine receptors and muscle fiber area using immunofluorescence and histological staining. Results: The Young's modulus was 1.67 GPa, which is comparable to native tendon and muscle. The devices successfully recorded electromyogram data when implanted in rats. When compared to untreated denervated muscles, MEA therapy attenuated atrophy by maintaining larger muscle fiber cross-sectional areas (p < 0.05). Furthermore, the acetylcholine receptor areas were markedly larger with MEA treatment (p < 0.05). Conclusions: This proof-of-concept work successfully demonstrates the ability to combine conformability, tensile strength-enhancing metal micropatterning, electrical stimulation and recording into a functional implant for both epimysial stimulation and recording.

Impact of Cumulative Chemotherapy Dose on Survival With Adjuvant FEC-D Chemotherapy for Breast Cancer.

BACKGROUND: Reductions in adjuvant chemotherapy dose <85% for historical regimens (ie, cyclophosphamide/methotrexate/fluorouracil) are known to affect breast cancer survival. This threshold, in addition to early versus late dose reductions, are poorly defined for third-generation anthracycline/taxane-based chemotherapy. In patients with breast cancer receiving adjuvant 5-fluorouracil/epirubicin/cyclophosphamide followed by docetaxel (FEC-D), we evaluated the impact of chemotherapy total cumulative dose (TCD), and early (FEC) versus late (D only) dose reductions, on survival outcomes. PATIENTS AND METHODS: Women with stage I-III, hormone receptor-positive/negative, HER2-negative breast cancer treated with adjuvant FEC-D chemotherapy from 2007 through 2014 in Alberta, Canada, were included. TCD for cycles 1 to 6 of <85% or ≥85% was calculated. Average cumulative dose was also calculated for early (cycles 1-3) and late (cycles 4-6) chemotherapy. Survival outcomes (disease-free survival [DFS] and overall survival [OS]) were estimated using Kaplan-Meier and multivariate analysis. Cohorts were evaluated for uniformity. RESULTS: Characteristics were reasonably balanced for all cohorts. Overall, 1,302 patients were evaluated for dose reductions, with 16% being reduced <85% (n=202) relative to ≥85% (n=1,100; 84%). Patients who received TCD ≥85% relative to <85% had superior 5-year DFS (P=.025) and OS (P<.001) according to Kaplan-Meier analysis, which remained significant on univariate and multivariate analyses. In stratified late and early dose reduction cohorts, DFS and OS showed a significant inferior survival trend for dose reduction early in treatment administration in 5-year Kaplan-Meier (P=.002 and P<.001, respectively) and multivariate analyses (hazard ratio [HR], 1.46; P=.073, and HR, 1.77; P=.011, respectively). Dose delays of <14 or ≥14 days and granulocyte colony-stimulating factor use did not affect outcomes. CONCLUSIONS: Chemotherapy TCD <85% for adjuvant FEC-D affects breast cancer survival. Late reductions (D only) were not shown to adversely affect DFS or OS. Conversely, early reductions (FEC±D) negatively affected patient outcomes.

Polyimide Electrode-Based Electrical Stimulation Impedes Early Stage Muscle Graft Regeneration.

Given the increasing use of regenerative free muscle flaps for various reconstructive procedures and neuroprosthetic applications, there is great interest and value in their enhanced regeneration, revascularization, and reinnervation for improved functional recovery. Here, we implant polyimide-based mircroelectrodes on free flap grafts and perform electrical stimulation for 6 weeks in a murine model. Using electrophysiological and histological assessments, we compare outcomes of stimulated grafts with unstimulated control grafts. We find delayed reinnervation and abnormal electromyographic (EMG) signals, with significantly more polyphasia, lower compound muscle action potentials and higher fatigability in stimulated animals. These metrics are suggestive of myopathy in the free flap grafts stimulated with the electrode. Additionally, active inflammatory processes and partial necrosis are observed in grafts stimulated with the implanted electrode. The results suggest that under this treatment protocol, implanted epimysial electrodes and electrical stimulation to deinnervated, and devascularized flaps during the early recovery phase may be detrimental to regeneration. Future work should determine the optimal implantation and stimulation window for accelerating free muscle graft regeneration.

Endothelial miR-30c suppresses tumor growth via inhibition of TGF-ß-induced Serpine1.

In tumors, extravascular fibrin forms provisional scaffolds for endothelial cell (EC) growth and motility during angiogenesis. We report that fibrin-mediated angiogenesis was inhibited and tumor growth delayed following postnatal deletion of Tgfbr2 in the endothelium of Cdh5-CreERT2 Tgfbr2fl/fl mice (Tgfbr2iECKO mice). ECs from Tgfbr2iECKO mice failed to upregulate the fibrinolysis inhibitor plasminogen activator inhibitor 1 (Serpine1, also known as PAI-1), due in part to uncoupled TGF-ß-mediated suppression of miR-30c. Bypassing TGF-ß signaling with vascular tropic nanoparticles that deliver miR-30c antagomiRs promoted PAI-1-dependent tumor growth and increased fibrin abundance, whereas miR-30c mimics inhibited tumor growth and promoted vascular-directed fibrinolysis in vivo. Using single-cell RNA-Seq and a NanoString miRNA array, we also found that subtypes of ECs in tumors showed spectrums of Serpine1 and miR-30c expression levels, suggesting functional diversity in ECs at the level of individual cells; indeed, fresh EC isolates from lung and mammary tumor models had differential abilities to degrade fibrin and launch new vessel sprouts, a finding that was linked to their inverse expression patterns of miR-30c and Serpine1 (i.e., miR-30chi Serpine1lo ECs were poorly angiogenic and miR-30clo Serpine1hi ECs were highly angiogenic). Thus, by balancing Serpine1 expression in ECs downstream of TGF-ß, miR-30c functions as a tumor suppressor in the tumor microenvironment through its ability to promote fibrin degradation and inhibit blood vessel formation.

BOLA (BolA Family Member 3) Deficiency Controls Endothelial Metabolism and Glycine Homeostasis in Pulmonary Hypertension.

BACKGROUND: Deficiencies of iron-sulfur (Fe-S) clusters, metal complexes that control redox state and mitochondrial metabolism, have been linked to pulmonary hypertension (PH), a deadly vascular disease with poorly defined molecular origins. BOLA3 (BolA Family Member 3) regulates Fe-S biogenesis, and mutations in BOLA3 result in multiple mitochondrial dysfunction syndrome, a fatal disorder associated with PH. The mechanistic role of BOLA3 in PH remains undefined. METHODS: In vitro assessment of BOLA3 regulation and gain- and loss-of-function assays were performed in human pulmonary artery endothelial cells using siRNA and lentiviral vectors expressing the mitochondrial isoform of BOLA3. Polymeric nanoparticle 7C1 was used for lung endothelium-specific delivery of BOLA3 siRNA oligonucleotides in mice. Overexpression of pulmonary vascular BOLA3 was performed by orotracheal transgene delivery of adeno-associated virus in mouse models of PH. RESULTS: In cultured hypoxic pulmonary artery endothelial cells, lung from human patients with Group 1 and 3 PH, and multiple rodent models of PH, endothelial BOLA3 expression was downregulated, which involved hypoxia inducible factor-2α-dependent transcriptional repression via histone deacetylase 1-mediated histone deacetylation. In vitro gain- and loss-of-function studies demonstrated that BOLA3 regulated Fe-S integrity, thus modulating lipoate-containing 2-oxoacid dehydrogenases with consequent control over glycolysis and mitochondrial respiration. In contexts of siRNA knockdown and naturally occurring human genetic mutation, cellular BOLA3 deficiency downregulated the glycine cleavage system protein H, thus bolstering intracellular glycine content. In the setting of these alterations of oxidative metabolism and glycine levels, BOLA3 deficiency increased endothelial proliferation, survival, and vasoconstriction while decreasing angiogenic potential. In vivo, pharmacological knockdown of endothelial BOLA3 and targeted overexpression of BOLA3 in mice demonstrated that BOLA3 deficiency promotes histological and hemodynamic manifestations of PH. Notably, the therapeutic effects of BOLA3 expression were reversed by exogenous glycine supplementation. CONCLUSIONS: BOLA3 acts as a crucial lynchpin connecting Fe-S-dependent oxidative respiration and glycine homeostasis with endothelial metabolic reprogramming critical to PH pathogenesis. These results provide a molecular explanation for the clinical associations linking PH with hyperglycinemic syndromes and mitochondrial disorders. These findings also identify novel metabolic targets, including those involved in epigenetics, Fe-S biogenesis, and glycine biology, for diagnostic and therapeutic development.

Immediate-term cognitive impairment following intravenous (IV) chemotherapy: a prospective pre-post design study.

BACKGROUND: Cognitive impairment is commonly reported in patients receiving chemotherapy, but the acuity of onset is not known. This study utilized the psychomotor vigilance test (PVT) and trail-making test B (TMT-B) to assess cognitive impairment immediately post-chemotherapy. METHODS: Patients aged 18-80 years receiving first-line intravenous chemotherapy for any stage of breast or colorectal cancer were eligible. Patient symptoms, peripheral neuropathy and Stanford Sleepiness Scale were assessed. A five-minute PVT and TMT-B were completed on a tablet computer pre-chemotherapy and immediately post-chemotherapy. Using a mixed linear regression model, changes in reciprocal transformed PVT reaction time (mean 1/RT) were assessed. A priori, an increase in median PVT reaction times by > 20 ms (approximating PVT changes with blood alcohol concentrations of 0.04-0.05 g%) was considered clinically relevant. RESULTS: One hundred forty-two cancer patients (73 breast, 69 colorectal, median age 55.5 years) were tested. Post-chemotherapy, mean 1/RT values were significantly slowed compared to pre-chemotherapy baseline (p = 0.01). This corresponded to a median PVT reaction time slowed by an average of 12.4 ms. Changes in PVT reaction times were not correlated with age, sex, cancer type, treatment setting, or use of supportive medications. Median post-chemotherapy PVT reaction time slowed by an average of 22.5 ms in breast cancer patients and by 1.6 ms in colorectal cancer patients. Post-chemotherapy median PVT times slowed by > 20 ms in 57 patients (40.1%). Exploratory analyses found no statistically significant association between the primary outcome and self-reported anxiety, fatigue or depression. TMT-B completion speed improved significantly post-chemotherapy (p = 0.03), likely due to test-retest phenomenon. CONCLUSIONS: PVT reaction time slowed significantly immediately post-chemotherapy compared to a pre-chemotherapy baseline, and levels of impairment similar to effects of alcohol consumption in other studies was seen in 40% of patients. Further studies assessing functional impact of cognitive impairment on patients immediately after chemotherapy are warranted.

Real-World Outcomes of Adjuvant Chemotherapy for Node-Negative and Node-Positive HER2-Positive Breast Cancer.

Background: Comparative real-world outcomes for patients with HER2-positive (HER2+) breast cancer receiving adjuvant trastuzumab outside of clinical trials are lacking. This study sought to retrospectively characterize outcomes for patients with node-negative and node-positive breast cancer receiving adjuvant trastuzumab in combination with docetaxel/cyclophosphamide (DCH), docetaxel/carboplatin/trastuzumab (TCH), or fluorouracil/epirubicin/cyclophosphamide followed by docetaxel/trastuzumab (FEC-DH) chemotherapy in Alberta, Canada, from 2007 through 2014. Methods: Disease-free survival and overall survival (OS) analyses for node-negative cohorts receiving DCH (n=111) or TCH (n=371) and node-positive cohorts receiving FEC-DH (n=146) or TCH (n=315) were compared using chi-square, Kaplan-Meier, or Cox multivariable analysis where appropriate. Results: Median follow-up was similar in node-negative (63.9 months) and node-positive (69.0 months) cohorts. The 5-year OS rates in patients with node-negative disease receiving DCH or TCH were similar (95.2% vs 96.9%; P=.268), whereas 5-year OS rates were higher but nonsignificant for patients with node-positive disease treated with FEC-DH compared with TCH (95.2% vs 91.4%; P=.160). Subgroup analysis of node-positive cohorts showed significantly improved OS with FEC-DH versus TCH in patients with estrogen receptor (ER)/progesterone receptor (PR)-positive breast cancer (98.3% vs 91.6%, respectively; P=.014). Conversely, patients with ER/PR-negative disease showed a nonsignificant trend toward higher OS rates with TCH versus FEC-DH (91.6% vs 83.3%, respectively; P=.298). Given the retrospective design, we were unable to capture all potential covariates that may have impacted treatment assignment and/or outcomes. Furthermore, cardiac toxicity data were unavailable. Conclusions: Survival rates of patients with HER2+ breast cancer in our study are comparable to those seen in clinical trials. Our findings support chemotherapy de-escalation in patients with node-negative disease and validate the efficacy of FEC-DH in those with node-positive disease.

Endothelial siRNA delivery in nonhuman primates using ionizable low-molecular weight polymeric nanoparticles.

Dysfunctional endothelial cells contribute to the pathophysiology of many diseases, including vascular disease, stroke, hypertension, atherosclerosis, organ failure, diabetes, retinopathy, and cancer. Toward the goal of creating a new RNA-based therapy to correct aberrant endothelial cell gene expression in humans, efficient gene silencing in the endothelium of nonhuman primates was achieved by delivering small interfering RNA (siRNA) with 7C1, a low-molecular weight, ionizable polymer that forms nanoparticles. After a single intravenous administration of 1 mg of siRNA per kilogram of animal, 7C1 nanoparticles delivering Tie2 siRNA caused Tie2 mRNA levels to decrease by approximately 80% in the endothelium of the lung. Significant decreases in Tie2 mRNA were also found in the heart, retina, kidney, pancreas, and bone. Blood chemistry and liver function analysis before and after treatment all showed protein and enzyme concentrations within the normal reference ranges. Furthermore, after controlling for siRNA-specific effects, no significant increases in inflammatory cytokine concentrations were found in the serum. Similarly, no gross lesions or significant underlying pathologies were observed after histological examination of nonhuman primate tissues. This study is the first demonstration of endothelial gene silencing in multiple nonhuman primate organs using systemically administered siRNA nanoparticles and highlights the potential of this approach for the treatment of disease in humans.

MicroRNA regulation of the MRN complex impacts DNA damage, cellular senescence, and angiogenic signaling.

MicroRNAs (miRs) contribute to biological robustness by buffering cellular processes from external perturbations. Here we report an unexpected link between DNA damage response and angiogenic signaling that is buffered by a miR. We demonstrate that genotoxic stress-induced miR-494 inhibits the DNA repair machinery by targeting the MRE11a-RAD50-NBN (MRN) complex. Gain- and loss-of-function experiments show that miR-494 exacerbates DNA damage and drives endothelial senescence. Increase of miR-494 affects telomerase activity, activates p21, decreases pRb pathways, and diminishes angiogenic sprouting. Genetic and pharmacological disruption of the MRN pathway decreases VEGF signaling, phenocopies miR-494-induced senescence, and disrupts angiogenic sprouting. Vascular-targeted delivery of miR-494 decreases both growth factor-induced and tumor angiogenesis in mouse models. Our work identifies a putative miR-facilitated mechanism by which endothelial cells can be insulated against VEGF signaling to facilitate the onset of senescence and highlight the potential of targeting DNA repair to disrupt pathological angiogenesis.

Drug development for breast, colorectal, and non-small cell lung cancers from 1979 to 2014.

BACKGROUND: Understanding the drug development pathway is critical for streamlining the development of effective cancer treatments. The objective of the current study was to delineate the drug development timeline and attrition rate of different drug classes for common cancer disease sites. METHODS: Drugs entering clinical trials for breast, colorectal, and non-small cell lung cancer were identified using a pharmaceutical business intelligence database. Data regarding drug characteristics, clinical trials, and approval dates were obtained from the database, clinical trial registries, PubMed, and regulatory Web sites. RESULTS: A total of 411 drugs met the inclusion criteria for breast cancer, 246 drugs met the inclusion criteria for colorectal cancer, and 315 drugs met the inclusion criteria for non-small cell lung cancer. Attrition rates were 83.9% for breast cancer, 87.0% for colorectal cancer, and 92.0% for non-small cell lung cancer drugs. In the case of non-small cell lung cancer, there was a trend toward higher attrition rates for targeted monoclonal antibodies compared with other agents. No tumor site-specific differences were noted with regard to cytotoxic chemotherapy, immunomodulatory, or small molecule kinase inhibitor drugs. Drugs classified as "others" in breast cancer had lower attrition rates, primarily due to the higher success of hormonal medications. Mean drug development times were 8.9 years for breast cancer, 6.7 years for colorectal cancer, and 6.6 years for non-small cell lung cancer. CONCLUSIONS: Overall oncologic drug attrition rates remain high, and drugs are more likely to fail in later-stage clinical trials. The refinement of early-phase trial design may permit the selection of drugs that are more likely to succeed in the phase 3 setting. Cancer 2017;123:4672-4679. © 2017 American Cancer Society.