Targeting the hepatitis B cccDNA with a sequence-specific ARCUS nuclease to eliminate hepatitis B virus in vivo.

Persistence of chronic hepatitis B (CHB) is attributed to maintenance of the intrahepatic pool of the viral covalently closed circular DNA (cccDNA), which serves as the transcriptional template for all viral gene products required for replication. Current nucleos(t)ide therapies for CHB prevent virus production and spread but have no direct impact on cccDNA or expression of viral genes. We describe a potential curative approach using a highly specific engineered ARCUS nuclease (ARCUS-POL) targeting the hepatitis B virus (HBV) genome. Transient ARCUS-POL expression in HBV-infected primary human hepatocytes produced substantial reductions in both cccDNA and hepatitis B surface antigen (HBsAg). To evaluate ARCUS-POL in vivo, we developed episomal adeno-associated virus (AAV) mouse and non-human primate (NHP) models containing a portion of the HBV genome serving as a surrogate for cccDNA. Clinically relevant delivery was achieved through systemic administration of lipid nanoparticles containing ARCUS-POL mRNA. In both mouse and NHP, we observed a significant decrease in total AAV copy number and high on-target indel frequency. In the case of the mouse model, which supports HBsAg expression, circulating surface antigen was durably reduced by 96%. Together, these data support a gene-editing approach for elimination of cccDNA toward an HBV cure.

Nanoparticle Delivery of RIG-I Agonist Enables Effective and Safe Adjuvant Therapy in Pancreatic Cancer.

Local immunomodulation can be a promising strategy to augment the efficacy and decrease off-target toxicities associated with cancer treatment. Pancreatic cancer is resistant to immunotherapies due to the immunosuppressive tumor microenvironment. Herein, we investigated a therapeutic approach involving delivery of a short interfering double-stranded RNA (dsRNA), specific to Bcl2, with 5' triphosphate ends, by lipid calcium phosphate nanoparticles, in an orthotopic allograft KPC model of pancreatic cancer. Retinoic acid-inducible gene I (RIG-I)-like receptors can bind to 5' triphosphate dsRNA (ppp dsRNA), a pathogen-associated molecular pattern, producing type I interferon, while Bcl2 silencing can drive apoptosis of cancer cells. Our approach demonstrated a robust enrichment of tumor tissue with therapeutic nanoparticles and enabled a significant tumor growth inhibition, prolonging median overall survival. Nanoparticles encapsulating dual-therapeutic ppp dsRNA allowed strong induction in levels of pro-inflammatory Th1 cytokines, further increasing proportions of CD8+ T cells over regulatory T cells, M1 over M2 macrophages, and decreased levels of immunosuppressive B regulatory and plasma cells in the tumor microenvironment. Thus, these results provide a new immunotherapy approach for pancreatic cancer.

Hepatoma-intrinsic CCRK inhibition diminishes myeloid-derived suppressor cell immunosuppression and enhances immune-checkpoint blockade efficacy.

OBJECTIVE: Myeloid-derived suppressor cells (MDSCs) contribute to tumour immunosuppressive microenvironment and immune-checkpoint blockade resistance. Emerging evidence highlights the pivotal functions of cyclin-dependent kinases (CDKs) in tumour immunity. Here we elucidated the role of tumour-intrinsic CDK20, or cell cycle-related kinase (CCRK) on immunosuppression in hepatocellular carcinoma (HCC). DESIGN: Immunosuppression of MDSCs derived from patients with HCC and relationship with CCRK were determined by flow cytometry, expression analyses and co-culture systems. Mechanistic studies were also conducted in liver-specific CCRK-inducible transgenic (TG) mice and Hepa1-6 orthotopic HCC models using CRISPR/Cas9-mediated Ccrk depletion and liver-targeted nanoparticles for interleukin (IL) 6 trapping. Tumorigenicity and immunophenotype were assessed on single or combined antiprogrammed death-1-ligand 1 (PD-L1) therapy. RESULTS: Tumour-infiltrating CD11b+CD33+HLA-DR- MDSCs from patients with HCC potently inhibited autologous CD8+T cell proliferation. Concordant overexpression of CCRK and MDSC markers (CD11b/CD33) positively correlated with poorer survival rates. Hepatocellular CCRK stimulated immunosuppressive CD11b+CD33+HLA-DR- MDSC expansion from human peripheral blood mononuclear cells through upregulating IL-6. Mechanistically, CCRK activated nuclear factor-κB (NF-κB) via enhancer of zeste homolog 2 (EZH2) and facilitated NF-κB-EZH2 co-binding to IL-6 promoter. Hepatic CCRK induction in TG mice activated the EZH2/NF-κB/IL-6 cascade, leading to accumulation of polymorphonuclear (PMN) MDSCs with potent T cell suppressive activity. In contrast, inhibiting tumorous Ccrk or hepatic IL-6 increased interferon γ+tumour necrosis factor-α+CD8+ T cell infiltration and impaired tumorigenicity, which was rescued by restoring PMN-MDSCs. Notably, tumorous Ccrk depletion upregulated PD-L1 expression and increased intratumorous CD8+ T cells, thus enhancing PD-L1 blockade efficacy to eradicate HCC. CONCLUSION: Our results delineate an immunosuppressive mechanism of the hepatoma-intrinsic CCRK signalling and highlight an overexpressed kinase target whose inhibition might empower HCC immunotherapy.

Glycoconjugated Site-Selective DNA-Methylating Agent Targeting Glucose Transporters on Glioma Cells.

DNA-alkylating drugs continue to remain an important weapon in the arsenal against cancers. However, they typically suffer from several shortcomings because of the indiscriminate DNA damage that they cause and their inability to specifically target cancer cells. We have developed a strategy for overcoming the deficiencies in current DNA-alkylating chemotherapy drugs by designing a site-specific DNA-methylating agent that can target cancer cells because of its selective uptake via glucose transporters, which are overexpressed in most cancers. The design features of the molecule, its synthesis, its reactivity with DNA, and its toxicity in human glioblastoma cells are reported here. In this molecule, a glucosamine unit, which can facilitate uptake via glucose transporters, is conjugated to one end of a bispyrrole triamide unit, which is known to bind to the minor groove of DNA at A/T-rich regions. A methyl sulfonate moiety is tethered to the other end of the bispyrrole unit to serve as a DNA-methylating agent. This molecule produces exclusively N3-methyladenine adducts upon reaction with DNA and is an order of magnitude more toxic to treatment resistant human glioblastoma cells than streptozotocin is, a Food and Drug Administration-approved, glycoconjugated DNA-methylating drug. Cellular uptake studies using a fluorescent analogue of our molecule provide evidence of uptake via glucose transporters and localization within the nucleus of cells. These results demonstrate the feasibility of our strategy for developing more potent anticancer chemotherapeutics, while minimizing common side effects resulting from off-target damage.

Facile Fabrication of Tumor Redox-Sensitive Nanoassemblies of Small-Molecule Oleate Prodrug as Potent Chemotherapeutic Nanomedicine.

The conjugate of paclitaxel (PTX) and docosahexaenoic acid has entered into clinical trials. However, the most recent clinical outcomes fell short of expectations, due to the extremely slow drug release from the hydrophobic conjugates. Herein, a novel prodrug-based nanoplatform self-assembled by the disulfide bond linked conjugates of PTX and oleic acid for rapid and differential release of PTX in tumor cells is reported. This redox-responsive prodrug-nanosystem demonstrates multiple therapeutic advantages, including one-step facile fabrication, high drug-loading efficiency (56%, w/w), on-demand drug release responding to redox stimuli, as well as favorable cellular uptake and biodistribution. These advantages result in significantly enhanced antitumor efficacy in vivo, with the tumor almost completely disappearing in mice. Such a uniquely engineered prodrug-nanosystem has great potential to be used as potent chemotherapeutic nanomedicine in clinical cancer therapy.

Paralabral Cyst of the Hip Causing Deep Vein Thrombosis Treated with Arthroscopic Decompression and Labral Repair.

In the setting of femoroacetabular impingement of the hip joint, paralabral cysts are well-documented sequelae. These cysts are typically associated with labral tears caused by CAM and/or pincer-type bony lesions. Synovial fluid extravasation through a tear in the labrum, similar to a popliteus cyst, leads to formation of a capsular-based cyst that is usually self-limiting. Few documented cases of these cysts causing compression of nearby neurovascular structures exist. There are several studies documenting arthroscopic decompression of these cysts, but none reporting compression of the femoral vein by a paralabral cyst resulting in deep vein thrombosis. We present the case of a large anterior paralabral cyst causing compression of the right femoral vein in a patient presenting with deep vein thrombosis and hip pain. Treatment consisted of arthroscopic decompression, followed by definitive aspiration by interventional radiology after labral repair and bipolar hip osteoplasty. The purpose of this case report was to document this rare presentation and offer learning points from our experience.

The Effect of Postoperative Sleeping Position on Knee Extension and Range of Motion Following Primary Total Knee Arthroplasty.

Total knee arthroplasty (TKA) is an orthopaedic operation that improves quality of life and reduces pain in patients with disabling arthritis of the knee. One commonly recognized complication is flexion contracture of the knee. Early physical therapy helps prevent flexion contracture and improve range of motion (ROM) postoperatively. This study evaluated postoperative sleeping position and its effect on terminal knee extension and ROM following primary TKA. We hypothesized that patients who slept in the supine position would achieve earlier knee extension and greater ROM when compared to those in the lateral recumbent position. A total of 150 consecutive primary TKAs were performed by a single surgeon (J.M.C.) from April 2014 to December 2014. The data were collected prospectively to determine preoperative ROM, postoperative ROM, and sleeping position. Mean postoperative terminal extension ROM at 1 month was 2.9 degrees in the supine group versus 6.0 degrees (p< .001) in the lateral group. No significant demographic differences between the two groups at baseline were found. Our results demonstrate that sleeping position affects initial postoperative terminal extension, however, not overall ROM. We found a statistically significant difference in extension when comparing patients in the supine versus lateral group. Patients who slept in the lateral position lacked 6 degrees of extension which is greater than the 5 degrees needed for normal gait mechanics. Those in the supine group lacked 2.9 degrees of extension, allowing for normal gait mechanics. This study identifies an easy, effective means of increasing patients initial ability to achieve knee extension and satisfaction following TKA.

A Case Report of Bilateral Lipoma Arborescens: An Unusual Consideration for Knee Pain.

Introduction: Lipoma arborescens (LAs) is a benign, intra-articular proliferation of fat cells in villous projections, creating a tree-like pattern on magnetic resonance imaging (MRI). The suprapatellar pouch is usually affected, and symptoms are typically gradual in nature, and patients may report painless swelling of the knee. Only ten cases of bilateral LA have been reported in the literature so far. Early recognition of this disease process and treatment may help to prevent prolonged symptoms and delays in care. Case Report: A 49-year-old female with bilateral knee pain and intermittent swelling for over 20 years presented to our clinic with complaints of bilateral knee pain and swelling. She had previous steroid injection but no relief. After MRI was obtained concerning for LA, a surgical discussion was had with the patient about arthroscopic removal. She elected to proceed with surgery and underwent arthroscopic debridement of both knees. At her follow-up at 6 months for the right knee and 2 months for the left knee, she had a significant improvement in pain and quality of life. Conclusion: LA of the knee is a rare condition, particularly bilateral, and in this patient, the diagnosis was missed for many years, and her definitive treatment was delayed. In her case, arthroscopic debridement of her bilateral LA proved to be a viable treatment option which significantly improved the patient's quality of life and function.

Workers' Compensation: The Burden on Healthcare Resource Utilization After Foot and Ankle Surgery.

INTRODUCTION: The purpose of this study is to investigate the amount of healthcare resources dedicated to patients with WC after common foot or ankle procedures compared with a procedure-matched control group. METHODS: A retrospective review was performed of patients with WC and without WC undergoing foot and ankle procedures. Measures of healthcare burden included clinical communications, documents, prescriptions, office visits, and days to return to work and clinic discharge. RESULTS: Collectively, 142 patients met the inclusion criteria. Patients with workers' compensation had increased office communication encounters (P < 0.001), processed documents (P < 0.001), medication prescriptions (P < 0.001), number of office visits (P < 0.001), number of days until return to work (P < 0.001), and days until final disposition from clinic (P < 0.001). Patients with workers' compensation were more likely to have postoperative complications (OR 2.1; 95% CI, 1.0 to 4.3; P = 0.04), secondary surgeries (OR 8.2; 95% CI, 2.3 to 29.4; P < 0.001), and new complaints during the perioperative period (OR 1.9; 95% CI, 0.9 to 4.0; P = 0.07) but were less likely to cancel appointments (OR 0.41; 95% CI, 0.19 to 0.86; P = 0.02). DISCUSSION: When undergoing common foot and ankle orthopaedic procedures, patients with WC demonstrated increased healthcare utilization of resources. This included more office staff work burden dedicated to patients with WC for longer amounts of time, effectively doubling the effort of a non-WC cohort.

Erratum to 'Left Profunda Femoral Artery Pseudoaneurysm After Revision Total Hip Arthroplasty' [Arthroplasty Today 6 (2020) 906-910].

[This corrects the article DOI: 10.1016/j.artd.2020.09.004.].

A Survey Analysis of Patient Understanding and Preferences for Podiatrists Versus Foot and Ankle Orthopaedic Surgeons.

BACKGROUND: Recent studies suggest poorer outcomes and higher costs associated with patients treated by podiatrists, yet no studies have evaluated patient perception and preference for foot and ankle providers. This study aims to determine patient perception of training for podiatrists compared to orthopaedic surgeons and patient preference for type of provider seen. METHODS: A 20-question survey was administered to new patients seeing either a podiatrist or foot and ankle orthopaedic surgeon. Questions pertained to demographics, patient knowledge of their provider, perception of training requirements, number of years in professional training, and differences in surgical volume during training. Patients were asked their preference for a particular type of foot and ankle provider, and whether they perceived a difference in surgical skillset or a provider's ability to manage different pathology. RESULTS: In all, 147 podiatry and 115 orthopaedic patients were included in the study. Demographics were similar between groups. Both groups believed orthopedists required more years of education and surgical training. In all, 61.5% of orthopaedic patients believed that orthopedists performed more foot and ankle surgeries and were more skilled as compared to podiatrists, while only about a third of podiatry patients believed this to be true (68.7% vs 38.6%; P < .001). Most patients believed orthopedists were more skilled in treating fractures. In all, 48.7% of orthopaedic patients preferred seeing an orthopedist compared to only 3.5% of podiatry patients. CONCLUSIONS: Our study demonstrates that patients are knowledgeable about the type of foot and ankle provider they are seeing. Most patients believe orthopaedic surgeons require more years of education and surgical training compared to podiatrists and believe they are more skilled in fracture-related surgery. Fewer podiatry patients expressed a preference for an orthopaedic surgeon. Providers must play an active role in discussing their training background prior to treating foot and ankle patients, especially in the setting of fracture-related pathology. CLINICAL RELEVANCE: This study demonstrates that most patients seeking care from a podiatrist or foot and ankle orthopaedic surgeon are relatively knowledgeable about the type of provider they are seeing; however, there are some differences. Most patients understand that orthopaedic surgeons require more years of education and surgical training and also believe orthopaedic surgeons are more skilled in fracture work and taking care of arthritic conditions. In general, podiatry patients have less preference for seeing an orthopaedic surgeon; however, many of these patients are seeking care for wounds and infections. With expanding roles and scope of practice among podiatry providers, it is important that providers become more active in explaining their training background and qualifications when treating foot and ankle conditions. LEVELS OF EVIDENCE: Level II: Prospective.

Progression of Intracranial Hemorrhage After Chemical Prophylaxis Using Low-Molecular-Weight Heparin in Patients With Traumatic Brain Injury.

INTRODUCTION: Chemical prophylaxis using low-molecular-weight heparin (LMWH) is considered a standard of care for venous thromboembolism in trauma patients. Our center performs a head computed tomography (CT) scan 24 hours after initiation with prophylactic LMWH in the setting of a known traumatic brain injury (TBI). The purpose was to determine the overall incidence of ICH progression after chemoprophylaxis in patients with a TBI. METHODS: This retrospective study was performed at a Level I trauma center, from 1/1/2014 to 12/31/2017. Study patients were drawn from the institution's trauma registry based on Abbreviated Injury Score codes. RESULTS: 778 patients met all inclusion criteria after initial chart review. The proportion of patients with an observed radiographic progression of intracranial hemorrhage after LMWH was 5.8%. 3.1% of patients had a change in clinical management. Observed radiographic progression after LMWH prophylaxis and the presence of SDH on initial CT, the bilateral absence of pupillary response in the emergency department, and a diagnosis of dementia were found to have statistically significant correlation with bleed progression after LMWH was initiated. CONCLUSION: Over a 4-year period, the use of CT to evaluate for radiographic progression of traumatic intracranial hemorrhage 24 hours after receiving LMWH resulted in a change in clinical management for 3.1% of patients. The odds of intracranial hemorrhage progression were approximately 6.5× greater in patients with subdural hemorrhage on initial CT, 3.1× greater in patients with lack of bilateral pupillary response in ED, and 4.2× greater in patients who had been diagnosed with dementia.

18-Year Operative Incidence and Cost Analysis of the Treatment of Adult Acquired Flatfoot Deformity.

BACKGROUND: Adult acquired flatfoot (AAFD) is commonly treated by foot and ankle surgeons. Despite how commonly this disease presents, its incidence and economic impact have yet to be defined. We hypothesized that the operative incidence of AAFD and its economic burden would increase over the time period 1996 to 2014. METHODS: The South Carolina database was queried for data from acute care and ambulatory surgery centers. Bivariate descriptive statistics were used to analyze the data. Operative incidence was calculated and demographics and medical comorbidities of patients who progressed to operative intervention were analyzed. Costs associated with operative care episodes were calculated to determine the economic burden. RESULTS: A total of 1299 patients underwent AAFD corrective surgery between 1996 and 2014. Patients who underwent surgery for AAFD were most likely to be white, female, and in their fourth, fifth, and sixth decade of life. Operative incidence for AAFD rose from 0.26 per 100 000 covered lives in 1996 to 3.04 in 2014. The total health care costs associated with patients who underwent surgery for AAFD increased from $57 395.33 in 1996 to $6 859 723.60 in 2014. CONCLUSIONS: This data demonstrate that patients most commonly undergoing operative intervention for AAFD were white, female, and in their fourth, fifth, or sixth decade of life. There has been a significant increase in operative incidence, which may help direct attention to further exploration of outcome data in these patient populations, associated treatment costs, and preventative treatment options. LEVEL OF EVIDENCE: Level III, retrospective comparative study.

Evaluating Tourniquet Efficacy Comparing Exsanguination Techniques for the Upper Extremity.

Tourniquets have been used in the medical setting for centuries and have become the gold standard when preparing patients for surgery, particularly in orthopaedic surgery. Upper extremity tourniquet use improves intraoperative visibility and identification of anatomy. It also decreases blood loss intraoperatively and improves the safety of orthopaedic procedures. Despite the widespread use of tourniquets and differing methods of limb exsanguination, little research has been done quantifying its efficacy. The purpose of this study was to compare gravity exsanguination to Esmarch exsanguination of the upper extremity prior to tourniquet inflation in a large patient sample. A plethysmographic method based on water displacement served as a surrogate for the blood volume exsanguinated. Control measurements of water displacement were obtained from both upper extremities without tourniquet inflation. Water displacement was then measured with both gravity and Esmarch exsanguination techniques. Gender, handedness, height, weight, body mass index, and age were recorded for volunteers and used as covariates. Change in mean water displacement from control (un-exsanguinated) arm and gravity alone measurement was 37.2 ml. Change in mean water displacement between control arm and mean Esmarch measurement was 56.3 ml. Exsanguination using Esmarch compared to gravity alone resulted in a 51.2% increase in blood removal. Only age had a significant interaction effect for the Esmarch method. Analysis revealed that age accounted for 21.4% of all variance in blood exsanguinated using the Esmarch method when compared to the control group. The Esmarch technique was more efficacious for all demographics measured, but most efficacious in subjects who were older than 40 years. This data reaffirms that gravity exsanguination is more efficacious than no tourniquet use at all, and that the Esmarch technique is more efficacious than gravity. To our knowledge, this study is the most robust of its kind to critically and objectively compare upper extremity exsanguination methods and overall tourniquet use by age and supports the common practice of Esmarch exsanguination in orthopaedic extremity surgery.

A Case Report of Giant Cell Tumor in the Thumb Distal Phalanx.

INTRODUCTION: The following report describes a rare case of giant cell tumor (GCT) of the bone that presented in the distal phalanx of the thumb. GCT of the bone is a relatively rare, and typically benign condition that presents most frequently in the metaphysis of long bones in women age 30-50 years old. There are only three other instances in the literature describing GCT of the bone presenting in the distal phalanx of the thumb. Although rare, delayed or missed diagnosis can be very debilitating to the patient. CASE REPORT: A 28-year-old male laborer who is right hand dominant and works with his hands for a living presented to the emergency department (ED) with swelling and pain at the distal aspect of his left thumb with no known injury. The patient was seen 4 weeks previously and was treated for cellulitis of the hand with antibiotics. At that time, no radiographs were taken. Despite this treatment, the patient reported increased swelling and pain over the next 2 weeks. He then sought treatment in the ED where a hand surgeon was consulted and radiographs were obtained that displayed a lytic, disruptive, and mildly expansile lesion of the distal phalanx of the first finger concerning for sarcoma. The risks and benefits of surgery were discussed with the patient and surgical intervention was planned. CONCLUSION: Due to how rarely this condition presents clinically, the patient was initially misdiagnosed and definitive treatment was delayed. Although rare, this is an important diagnosis to consider in patients presenting similarly. The patient ultimately received adequate treatment, but the delay in diagnosis in combination with the locally aggressive nature of this tumor could have led to extensive surgical intervention with impairment in hand function. As a laborer whose income relies on daily use of his hands a delayed diagnosis; in this case could have had a catastrophic impact.

Left Profunda Femoral Artery Pseudoaneurysm After Revision Total Hip Arthroplasty.

We present a case report of an 84-year-old male who presented with a profunda femoris artery (PFA) pseudoaneurysm 8 years after the index revision total hip arthroplasty procedure. Failure of revision hardware and subsequent migration of implants led to damage of the PFA and pseudoaneurysm formation. The patient was hemodynamically unstable on presentation and required emergent endovascular intervention. Once medically stabilized, the patient underwent extensive debridement of the aneurysm and hematoma bed and broken hardware was removed to prevent further complications. At 6-month follow-up, the patient was able to mobilize independently and had returned to all prior levels of activities of daily living. We discuss the vascular anatomy of the hip, the paucity of literature on PFA pseudoaneurysm, as well as the likely etiology of total hip arthroplasty failures.

A Circle RNA Regulatory Axis Promotes Lung Squamous Metastasis via CDR1-Mediated Regulation of Golgi Trafficking.

Lung squamous carcinoma (LUSC) is a highly metastatic disease with a poor prognosis. Using an integrated screening approach, we found that miR-671-5p reduces LUSC metastasis by inhibiting a circular RNA (circRNA), CDR1as. Although the putative function of circRNA is through miRNA sponging, we found that miR-671-5p more potently silenced an axis of CDR1as and its antisense transcript, cerebellar degeneration related protein 1 (CDR1). Silencing of CDR1as or CDR1 significantly inhibited LUSC metastases and CDR1 was sufficient to promote migration and metastases. CDR1, which directly interacted with adaptor protein 1 (AP1) complex subunits and coatomer protein I (COPI) proteins, no longer promoted migration upon blockade of Golgi trafficking. Therapeutic inhibition of the CDR1as/CDR1 axis with miR-671-5p mimics reduced metastasis in vivo. This report demonstrates a novel role for CDR1 in promoting metastasis and Golgi trafficking. These findings reveal an miRNA/circRNA axis that regulates LUSC metastases through a previously unstudied protein, CDR1. SIGNIFICANCE: This study shows that circRNA, CDR1as, promotes lung squamous migration, metastasis, and Golgi trafficking through its complimentary transcript, CDR1.

Synergistic and low adverse effect cancer immunotherapy by immunogenic chemotherapy and locally expressed PD-L1 trap.

Although great success has been obtained in the clinic, the current immune checkpoint inhibitors still face two challenging problems: low response rate and immune-related adverse effects (irAEs). Here we report the combination of immunogenic chemotherapy and locally expressed PD-L1 trap fusion protein for efficacious and safe cancer immunotherapy. We demonstrate that oxaliplatin (OxP) boosts anti-PD-L1 mAb therapy against murine colorectal cancer. By design of a PD-L1 trap and loading its coding plasmid DNA into a lipid-protamine-DNA nanoparticle, PD-L1 trap is produced transiently and locally in the tumor microenvironment, and synergizes with OxP for tumor inhibition. Significantly, unlike the combination of OxP and anti-PD-L1 mAb, the combination of OxP and PD-L1 trap does not induce obvious Th17 cells accumulation in the spleen, indicating better tolerance and lower tendency to irAEs. The reports here may highlight the potential of applying PD-L1 inhibitor, especially locally expressed PD-L1 trap, in cancer therapy following OxP-based chemotherapy.

Local Blockade of Interleukin 10 and C-X-C Motif Chemokine Ligand 12 with Nano-Delivery Promotes Antitumor Response in Murine Cancers.

In many cancers, the tumor microenvironment (TME) is largely immune suppressive, blocking the antitumor immunity and resulting in immunotherapy resistance. Interleukin 10 (IL-10) is a major player controlling the immunosuppressive TME in different murine tumor models. Increased IL-10 production suppresses intratumoral dendritic cell production of interleukin 12, thereby limiting antitumor cytotoxic T-cell responses and activation of NK cells during therapy. We engineered, formulated, and delivered genes encoding an IL-10 protein trap to change immunosuppressive TME, which could enhance antitumor immunity. Additionally, to achieve stronger and long-term therapeutic efficacy in a pancreatic cancer model, we targeted C-X-C motif chemokine ligand 12 (CXCL12), a key factor for inhibiting T-cell tumor infiltration, and simultaneously delivered an IL-10 trap. Following three injections of the lipid-protamine-DNA (LPD) nanoparticles loaded with trap genes (IL-10 trap and CXCL12 trap), we found tumor growth reduction and significantly prolonged survival of the host compared to control groups. Furthermore, the combination trap gene treatment significantly reduced immunosuppressive cells, such as M2 macrophages, MDSCs, and PD-L1+ cells, and activated immunosuppressive tolerogenic dendritic cells, NK cells, and macrophages intratumorally. We have also shown that, when effectively delivered to the tumor, the IL-10 trap gene alone can inhibit triple-negative breast cancer growth. This strategy may allow clinicians and researchers to change the immunosuppressive microenvironment in the tumor with either a single therapeutic agent or in combination with other immunotherapies to prime the immune system, preventing cancer invasion and prolonging patient survival.

Investigation of phosphorylated adjuvants co-encapsulated with a model cancer peptide antigen for the treatment of colorectal cancer and liver metastasis.

The lipid calcium phosphate nanoparticle is a versatile platform capable of encapsulating a wide range of phosphorylated molecules from single nucleotides to pDNA. The use of this platform has shown great success as an immunotherapeutic vaccine carrier, capable of delivering co-encapsulated phosphorylated adjuvants and peptides. Three potent vaccine formulations were investigated for anti-cancer efficacy. The phosphorylated adjuvants, CpG, 2'3'cGAMP, and 5'pppdsRNA were co-encapsulated with a model phosphorylated tumor specific peptide antigen (p-AH1-A5). The anti-cancer efficacy of these adjuvants was assessed using an orthotopic colorectal liver metastasis model based on highly aggressive and metastatic CT-26 FL3 cells implanted into the cecum wall. The results clearly indicate that the RIG-1 ligand, 5'pppdsRNA, co-encapsulated with the p-AH1-A5 peptide antigen greatly reduced the growth rate of the primary colon cancer as well as arrested the establishment of liver metastasis in comparison to the other adjuvant formulations and unvaccinated controls. Further evaluation of the immune cell populations within the primary tumor confirms the ability of the 5'pppdsRNA adjuvant to boost the adaptive CD8+ T-cell population, while not inciting increased populations of immune suppressive cell types such as T-regulatory cells or myeloid derived suppressor cells. Furthermore, to our knowledge this is the first study to investigate the anti-cancer efficacy of a specific RIG-1 receptor ligand, 5'pppdsRNA, alongside more established TLR 9 (CpG) and STING (2'3'cGAMP) adjuvants in a cancer vaccine. The 5'pppdsRNA vaccine formulation can be a potent immunotherapy, especially when combined with agents that remodel the immune suppressive microenvironment of the tumor.