Complete substitution with modified nucleotides suppresses the early interferon response and increases the potency of self-amplifying RNA.

Self-amplifying RNA (saRNA) will revolutionize vaccines and in situ therapeutics by enabling protein expression for longer duration at lower doses. However, a major barrier to saRNA efficacy is the potent early interferon response triggered upon cellular entry, resulting in saRNA degradation and translational inhibition. Substitution of mRNA with modified nucleotides (modNTPs), such as N1-methylpseudouridine (N1mΨ), reduce the interferon response and enhance expression levels. Multiple attempts to use modNTPs in saRNA have been unsuccessful, leading to the conclusion that modNTPs are incompatible with saRNA, thus hindering further development. Here, contrary to the common dogma in the field, we identify multiple modNTPs that when incorporated into saRNA at 100% substitution confer immune evasion and enhance expression potency. Transfection efficiency enhances by roughly an order of magnitude in difficult to transfect cell types compared to unmodified saRNA, and interferon production reduces by >8 fold compared to unmodified saRNA in human peripheral blood mononuclear cells (PBMCs). Furthermore, we demonstrate expression of viral antigens in vitro and observe significant protection against lethal challenge with a mouse-adapted SARS-CoV-2 strain in vivo . A modified saRNA vaccine, at 100-fold lower dose than a modified mRNA vaccine, results in a statistically improved performance to unmodified saRNA and statistically equivalent performance to modified mRNA. This discovery considerably broadens the potential scope of self-amplifying RNA, enabling entry into previously impossible cell types, as well as the potential to apply saRNA technology to non-vaccine modalities such as cell therapy and protein replacement.

Targeting tumor extracellular matrix activates the tumor-draining lymph nodes.

Disruption of the tumor extracellular matrix (ECM) may alter immune cell infiltration into the tumor and antitumor T cell priming in the tumor-draining lymph nodes (tdLNs). Here, we explore how intratumoral enzyme treatment (ET) of B16 melanoma tumors with ECM-depleting enzyme hyaluronidase alters adaptive and innate immune populations, including T cells, DCs, and macrophages, in the tumors and tdLNs. ET increased CD103+ DC abundance in the tdLNs, as well as antigen presentation of a model tumor antigen ovalbumin (OVA), eliciting local OVA-specific CD8+ T cell responses. Delivered in combination with a distant cryogel-based cancer vaccine, ET increased the systemic antigen-specific CD8+ T cell response. By enhancing activity within the tdLN, ET may broadly support immunotherapies in generating tumor-specific immunity.

Cryogel vaccines effectively induce immune responses independent of proximity to the draining lymph nodes.

The delivery location of traditional vaccines can impact immune responses and resulting efficacy. Cryogel-based cancer vaccines, which are typically injected near the inguinal lymph nodes (iLNs), recruit and activate dendritic cells (DC) in situ, induce DC homing to the iLNs, and have generated potent anti-tumor immunity against several murine cancer models. However, whether cryogel vaccination distance to a draining LN affects the kinetics of DC homing and downstream antigen-specific immunity is unknown, given the heightened importance of the scaffold vaccine site. We hypothesized that vaccination near the iLNs would lead to more rapid DC trafficking to the iLNs, thereby inducing faster and stronger immune responses. Here, mice were injected with cryogel vaccines against ovalbumin either adjacent or distal to the iLNs, and the resultant DC trafficking kinetics, T cell phenotypes, antigen-specific T cell and humoral responses, and prophylactic efficacy in an ovalbumin-expressing tumor model were assessed. Cryogel vaccines induced potent, long-lasting antigen-specific immune responses independent of distance to the iLNs, with no significant differences in DC trafficking kinetics, ovalbumin-specific T cell and antibody responses, or prophylactic efficacy. Moreover, DC trafficking and activation state were not impacted when cryogels were injected near a tumor. These results demonstrate a flexibility in vaccination location for scaffold-based vaccines, independent of draining LN distance.

Ultrasound-triggered release reveals optimal timing of CpG-ODN delivery from a cryogel cancer vaccine.

Recently, several injectable scaffold-based cancer vaccines have been developed that can recruit and activate host dendritic cells (DCs) and generate potent antitumor responses. However, the optimal timing of adjuvant delivery, particularly of the commonly used cytosine-phosphodiester-guanine-oligonucleotide (CpG-ODN), for scaffold-based cancer vaccines remains unknown. We hypothesized that optimally timed CpG-ODN delivery will lead to enhanced immune responses, and designed a cryogel vaccine system where CpG-ODN release can be triggered on-demand by ultrasound. CpG-ODN was first condensed with polyethylenimine and then adsorbed to cryogels. Little adsorbed CpG-ODN was released in vitro. Ultrasound stimulation triggered continuous CpG-ODN release, at an enhanced rate even after ultrasound was turned off, with minimal burst release. In vivo, ultrasound stimulation four days post-vaccination induced a significantly higher antigen-specific cytotoxic T-lymphocyte (CTL) response compared to control mice. Furthermore, ultrasound stimulation at this time point generated a significantly higher IgG2a/c antibody titer than all the groups except ultrasound stimulation eight days post-vaccination. This optimal timing of ultrasound-triggered release coincided with peak DC accumulation in the cryogels. By enabling temporal control of vaccine components through release on-demand, this system is a promising platform to study the optimal timing of delivery of immunomodulatory agents for cancer vaccination.

Correlation between Preoperative Serum Levels of Calcium, Phosphate, and Intact Parathyroid Hormone and Radiological Outcomes in Spinal Interbody Fusion among End-Stage Renal Disease Patients.

Spinal fusion surgery for end-stage renal disease (ESRD) patients is a clinical challenge. This study aimed to investigate whether postoperative radiological outcomes are related to preoperative serum calcium, phosphate, or intact parathyroid hormone (iPTH) levels in patients with ESRD who underwent spinal interbody fusion surgery. This study included 62-consecutive patients with ESRD who underwent anterior cervical discectomy and fusion (ACDF) or transforaminal lumbar interbody fusion (TLIF) surgery for symptomatic spinal disorder. The most recent preoperative serum calcium, phosphate, and iPTH levels were recorded, and the postoperative radiographic outcomes were assessed. A significant correlation was found between the occurrence of cage subsidence and higher blood phosphate, calcium-phosphate product (Ca × P), and iPTH levels in the TLIF group. The occurrence of pedicle screw loosening was related to higher blood phosphate and Ca × P product in the TLIF group. However, no correlation was found between the fusion grades and the serum levels in either the TLIF or ACDF groups. These results indicated that higher preoperative serum phosphate and Ca × P product are risk factors for both cage subsidence and screw loosening in patients with ESRD who underwent TLIF surgery. Higher iPTH levels are also a possible risk factor for cage subsidence.

Generation of the Compression-induced Dedifferentiated Adipocytes (CiDAs) Using Hypertonic Medium.

Current methods to obtain mesenchymal stem cells (MSCs) involve sampling, culturing, and expanding of primary MSCs from adipose, bone marrow, and umbilical cord tissues. However, the drawbacks are the limited numbers of total cells in MSC pools, and their decaying stemness during in vitro expansion. As an alternative resource, recent ceiling culture methods allow the generation of dedifferentiated fat cells (DFATs) from mature adipocytes. Nevertheless, this process of spontaneous dedifferentiation of mature adipocytes is laborious and time-consuming. This paper describes a modified protocol for in vitro dedifferentiation of adipocytes by employing an additional physical stimulation, which takes advantage of augmenting the stemness-related Wnt/ß-catenin signaling. Specifically, this protocol utilizes a polyethylene glycol (PEG)-containing hypertonic medium to introduce extracellular physical stimulation to obtain higher efficiency and introduce a simpler procedure for adipocyte dedifferentiation.

l-Arginine Grafted Poly(Glycerol Sebacate) Materials: An Antimicrobial Material for Wound Dressing.

This study focuses on the development and evaluation of a novel wound dressing material. l-arginine grafted poly(glycerol sebacate) materials (PGS-g-Arg) are developed by chemical conjugation of l-arginine on poly(glycerol sebacate) chains and the mechanical property, water vapor transmission rate, antimicrobial functions and biocompatibility are investigated. At various l-arginine grafting ratio, the mechanical properties are tunable. It was found that between 7-13% l-arginine grafting ratios, the tensile strengths of PGS-g-Arg were similar to that of natural skin. These materials are shown with a low water vapor transmission rate, 6.1 to 10.3 g/m2/h, which may form a barrier and assist in the closure of wounds. Inhibition in the growth of Pseudomonas aeruginosa and Staphylococcus aureus was observed on PGS-g-Arg, and a series of experiments were conducted to confirm its biocompatibility. In summary, l -arginine grafted poly(glycerol sebacate) may offer a novel option for wound dressing.

Compression-induced dedifferentiation of adipocytes promotes tumor progression.

Dysregulated physical stresses are generated during tumorigenesis that affect the surrounding compliant tissues including adipocytes. However, the effect of physical stressors on the behavior of adipocytes and their cross-talk with tumor cells remain elusive. Here, we demonstrate that compression of cells, resulting from various types of physical stresses, can induce dedifferentiation of adipocytes via mechanically activating Wnt/ß-catenin signaling. The compression-induced dedifferentiated adipocytes (CiDAs) have a distinct transcriptome profile, long-term self-renewal, and serial clonogenicity, but do not form teratomas. We then show that CiDAs notably enhance human mammary adenocarcinoma proliferation both in vitro and in a xenograft model, owing to myofibrogenesis of CiDAs in the tumor-conditioned environment. Collectively, our results highlight unique physical interplay in the tumor ecosystem; tumor-induced physical stresses stimulate de novo generation of CiDAs, which feedback to tumor growth.

A biomaterial-based vaccine eliciting durable tumour-specific responses against acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is a malignancy of haematopoietic origin that has limited therapeutic options. The standard-of-care cytoreductive chemotherapy depletes AML cells to induce remission, but is infrequently curative. An immunosuppressive AML microenvironment in the bone marrow and the paucity of suitable immunotherapy targets limit the induction of effective immune responses. Here, in mouse models of AML, we show that a macroporous-biomaterial vaccine that delivers the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), the Toll-like-receptor-9 agonist cytosine-guanosine oligodeoxynucleotide and one or multiple leukaemia antigens (in the form of a defined peptide antigen, cell lysates or antigens sourced from AML cells recruited in vivo) induces local immune-cell infiltration and activated dendritic cells, evoking a potent anti-AML response. The biomaterial-based vaccine prevented the engraftment of AML cells when administered as a prophylactic and when combined with chemotherapy, and eradicated established AML even in the absence of a defined vaccine antigen. Biomaterial-based AML vaccination can induce potent immune responses, deplete AML cells and prevent disease relapse.

Topical Application of a Mast Cell Stabilizer Improves Impaired Diabetic Wound Healing.

Impaired wound healing in the diabetic foot is a major problem often leading to amputation. Mast cells have been shown to regulate wound healing in diabetes. We developed an indole-carboxamide type mast cell stabilizer, MCS-01, which proved to be an effective mast cell degranulation inhibitor in vitro and can be delivered topically for prolonged periods through controlled release by specifically designed alginate bandages. In diabetic mice, both pre- and post-wounding, topical MCS-01 application accelerated wound healing comparable to that achieved with systemic mast cell stabilization. Moreover, MCS-01 altered the macrophage phenotype, promoting classically activated polarization. Bulk transcriptome analysis from wounds treated with MCS-01 or placebo showed that MCS-01 significantly modulated the mRNA and microRNA profile of diabetic wounds, stimulated upregulation of pathways linked to acute inflammation and immune cell migration, and activated the NF-κB complex along with other master regulators of inflammation. Single-cell RNA sequencing analysis of 6,154 cells from wounded and unwounded mouse skin revealed that MCS-01 primarily altered the gene expression of mast cells, monocytes, and keratinocytes. Taken together, these findings offer insights into the process of diabetic wound healing and suggest topical mast cell stabilization as a potentially successful treatment for diabetic foot ulceration.

Delivery of targeted gene therapies using a hybrid cryogel-coated prosthetic vascular graft.

OBJECTIVES: The success of prosthetic vascular grafts in the management of peripheral arterial disease is frequently limited by the development of anastomotic neointimal hyperplasia (ANIH), with the host response to prosthetic grafts beginning soon after implantation. To address this, we combine a platform of polyethylene terephthalate (PET) fabric with an applied cryogel layer containing biologic agents to create a bioactive prosthetic graft system, with the ability to deliver therapeutics targeting modulators of the ANIH-associated transcriptome response, along with antithrombotic agents. METHODS: Hybrid graft materials were synthesized by cryopolymerization of methacrylated alginate and heparin onto electrospun (ePET), knitted PET (kPET), or woven PET (wPET). Arg-Gly-Asp (RGD) peptides were added to increase cell adhesion. Scanning electron microscopy (SEM) was used to study the microstructure at 1 day, and 2, 4, and 8 weeks. Physical properties such as swelling ratio, pore connectivity, shape recovery, and stiffness were evaluated. Human aortic endothelial cell (HAoEC) adherence was visualized using confocal microscopy after 24 hours and proliferation was evaluated with a resazurin-based assay for 7 days. Confocal microscopy was used to assess delivery of adeno-associated virus (AAV-GFP) after incubation of hybrid grafts with HAoECs. Heparin activity of the materials was measured using an anti-Xa assay. RESULTS: SEM demonstrated large interconnected pores throughout the entire structure for all graft types, with minimal degradation of the cryogel after 8 weeks. Hybrid materials showed a trend towards increased shape recovery, increased stiffness, decreased swelling ratio, and no difference in pore connectivity. HAoECs incorporated, adhered, and proliferated over 7 days on all materials. HAoECs were successfully transduced with AAV-GFP from the hybrid graft materials. Anti-Xa assay confirmed continued activity of heparin from all materials for over 7 days. CONCLUSIONS: We have developed a bioactive prosthetic graft system with a cryogel coating capable of delivering biologic agents with antithrombotic activity. By applying the cryogel and selected agents onto PET prior to graft implantation, this study sets the stage for the system to be individualized and tailored to the patient, with bioengineering and targeted gene therapy strategies dovetailing to create an improved prosthetic graft adaptable to emerging knowledge and technologies.

Treating ischemia via recruitment of antigen-specific T cells.

Ischemic diseases are a leading cause of mortality and can result in autoamputation of lower limbs. We explored the hypothesis that implantation of an antigen-releasing scaffold, in animals previously vaccinated with the same antigen, can concentrate TH2 T cells and enhance vascularization of ischemic tissue. This approach may be clinically relevant, as all persons receiving childhood vaccines recommended by the Centers for Disease Control and Prevention have vaccines that contain aluminum, a TH2 adjuvant. To test the hypothesis, mice with hindlimb ischemia, previously vaccinated with ovalbumin (OVA) and aluminum, received OVA-releasing scaffolds. Vaccinated mice receiving OVA-releasing scaffolds locally concentrated antigen-specific TH2 T cells in the surrounding ischemic tissue. This resulted in local angiogenesis, increased perfusion in ischemic limbs, and reduced necrosis and enhanced regenerating myofibers in the muscle. These findings support the premise that antigen depots may provide a treatment for ischemic diseases in patients previously vaccinated with aluminum-containing adjuvants.

An injectable bone marrow-like scaffold enhances T cell immunity after hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for multiple disorders, but deficiency and dysregulation of T cells limit its utility. Here we report a biomaterial-based scaffold that mimics features of T cell lymphopoiesis in the bone marrow. The bone marrow cryogel (BMC) releases bone morphogenetic protein-2 to recruit stromal cells and presents the Notch ligand Delta-like ligand-4 to facilitate T cell lineage specification of mouse and human hematopoietic progenitor cells. BMCs subcutaneously injected in mice at the time of HSCT enhanced T cell progenitor seeding of the thymus, T cell neogenesis and diversification of the T cell receptor repertoire. Peripheral T cell reconstitution increased ~6-fold in mouse HSCT and ~2-fold in human xenogeneic HSCT. Furthermore, BMCs promoted donor CD4+ regulatory T cell generation and improved survival after allogeneic HSCT. In comparison to adoptive transfer of T cell progenitors, BMCs increased donor chimerism, T cell generation and antigen-specific T cell responses to vaccination. BMCs may provide an off-the-shelf approach for enhancing T cell regeneration and mitigating graft-versus-host disease in HSCT.

A facile approach to enhance antigen response for personalized cancer vaccination.

Existing strategies to enhance peptide immunogenicity for cancer vaccination generally require direct peptide alteration, which, beyond practical issues, may impact peptide presentation and result in vaccine variability. Here, we report a simple adsorption approach using polyethyleneimine (PEI) in a mesoporous silica microrod (MSR) vaccine to enhance antigen immunogenicity. The MSR-PEI vaccine significantly enhanced host dendritic cell activation and T-cell response over the existing MSR vaccine and bolus vaccine formulations. Impressively, a single injection of the MSR-PEI vaccine using an E7 peptide completely eradicated large, established TC-1 tumours in about 80% of mice and generated immunological memory. When immunized with a pool of B16F10 or CT26 neoantigens, the MSR-PEI vaccine eradicated established lung metastases, controlled tumour growth and synergized with anti-CTLA4 therapy. Our findings from three independent tumour models suggest that the MSR-PEI vaccine approach may serve as a facile and powerful multi-antigen platform to enable robust personalized cancer vaccination.

Injectable, Tough Alginate Cryogels as Cancer Vaccines.

A covalently crosslinked methacrylated (MA)-alginate cryogel vaccine has been previously shown to generate a potent response against murine melanoma, but is not mechanically robust and requires a large 16G needle for delivery. Here, covalent and ionic crosslinking of cryogels are combined with the hypothesis that this will result in a tough MA-alginate cryogel with improved injectability. All tough cryogels can be injected through a smaller, 18G needle without sustaining any damage, while covalently crosslinked-only cryogels break after injection. Cytosine-phosphodiester-guanine (CpG)-delivering tough cryogels effectively activate dendritic cells (DCs). Granulocyte macrophage colony-stimulating factor releasing tough cryogels recruit four times more DCs than blank gels by day 7 in vivo. The tough cryogel vaccine induces strong antigen-specific cytotoxic T-lymphocyte and humoral responses. These vaccines prevent tumor formation in 80% of mice inoculated with HER2/neu-overexpressing DD breast cancer cells. The MA-alginate tough cryogels provide a promising minimally invasive delivery platform for cancer vaccinations.

Injectable Shape-Memorizing Three-Dimensional Hyaluronic Acid Cryogels for Skin Sculpting and Soft Tissue Reconstruction.

INTRODUCTION: Hyaluronic acid (HA)-based fillers are used for various cosmetic procedures. However, due to filler migration and degradation, reinjections of the fillers are often required. Methacrylated HA (MA-HA) can be made into injectable shape-memorizing fillers (three-dimensional [3D] MA-HA) aimed to address these issues. In this study, shape retention, firmness, and biocompatibility of 3D MA-HA injected subcutaneously in mice were evaluated. MATERIALS AND METHODS: Fifteen mice, each receiving two subcutaneous injections in their back, were divided into four groups receiving HA, MA-HA, 3D MA-HA, or saline, respectively. Digital imaging, scanning electron microscope (SEM) and in vivo imaging system (IVIS), durometry, and histology were utilized to evaluate in vitro/vivo degradation and migration, material firmness, and the angiogenic (CD31) and immunogenic (CD45) response of the host tissue toward the injected materials. RESULTS: Digital imaging, SEM, and IVIS revealed that 3D MA-HA fillers maintained their predetermined shape for at least 30 days in vitro and in vivo. Little volume effects were noted in the saline and other control groups. There were no differences in skin firmness between the groups or over time. Histology showed intact skin architecture in all groups. Three-dimensional MA-HA maintained its macroporous structure with significant angiogenesis at the 3D MA-HA/skin interfaces and throughout the 3D MA-HA. There was no significant inflammatory response to any of the injected materials. CONCLUSION: 3D MA-HA showed remarkable tissue compatibility, compliance, and shape predictability, as well as retention, and thus might be suitable for various skin sculpting and soft tissue reconstruction purposes.

Injectable cryogel-based whole-cell cancer vaccines.

A biomaterial-based vaccination system that uses minimal extracorporeal manipulation could provide in situ enhancement of dendritic cell (DC) numbers, a physical space where DCs interface with transplanted tumour cells, and an immunogenic context. Here we encapsulate GM-CSF, serving as a DC enhancement factor, and CpG ODN, serving as a DC activating factor, into sponge-like macroporous cryogels. These cryogels are injected subcutaneously into mice to localize transplanted tumour cells and deliver immunomodulatory factors in a controlled spatio-temporal manner. These vaccines elicit local infiltrates composed of conventional and plasmacytoid DCs, with the subsequent induction of potent, durable and specific anti-tumour T-cell responses in a melanoma model. These cryogels can be delivered in a minimally invasive manner, bypass the need for genetic modification of transplanted cancer cells and provide sustained release of immunomodulators. Altogether, these findings indicate the potential for cryogels to serve as a platform for cancer cell vaccinations.

Brain-penetrating nanoparticles improve paclitaxel efficacy in malignant glioma following local administration.

Poor drug distribution and short drug half-life within tumors strongly limit efficacy of chemotherapies in most cancers, including primary brain tumors. Local or targeted drug delivery via controlled-release polymers is a promising strategy to treat infiltrative brain tumors, which cannot be completely removed surgically. However, drug penetration is limited with conventional local therapies since small-molecule drugs often enter the first cell they encounter and travel only short distances from the site of administration. Nanoparticles that avoid adhesive interactions with the tumor extracellular matrix may improve drug distribution and sustain drug release when applied to the tumor area. We have previously shown model polystyrene nanoparticles up to 114 nm in diameter were able to rapidly diffuse in normal brain tissue, but only if coated with an exceptionally dense layer of poly(ethylene glycol) (PEG) to reduce adhesive interactions. Here, we demonstrate that paclitaxel (PTX)-loaded, poly(lactic-co-glycolic acid) (PLGA)-co-PEG block copolymer nanoparticles with an average diameter of 70 nm were able to diffuse 100-fold faster than similarly sized PTX-loaded PLGA particles (without PEG coatings). Densely PEGylated PTX-loaded nanoparticles significantly delayed tumor growth following local administration to established brain tumors, as compared to PTX-loaded PLGA nanoparticles or unencapsulated PTX. Delayed tumor growth combined with enhanced distribution of drug-loaded PLGA-PEG nanoparticles to the tumor infiltrative front demonstrates that particle penetration within the brain tumor parenchyma improves therapeutic efficacy. The use of drug-loaded brain-penetrating nanoparticles is a promising approach to achieve sustained and more uniform drug delivery to treat aggressive gliomas and potentially other brain disorders.

Non-invasive delivery of stealth, brain-penetrating nanoparticles across the blood-brain barrier using MRI-guided focused ultrasound.

The blood-brain barrier (BBB) presents a significant obstacle for the treatment of many central nervous system (CNS) disorders, including invasive brain tumors, Alzheimer's, Parkinson's and stroke. Therapeutics must be capable of bypassing the BBB and also penetrate the brain parenchyma to achieve a desired effect within the brain. In this study, we test the unique combination of a non-invasive approach to BBB permeabilization with a therapeutically relevant polymeric nanoparticle platform capable of rapidly penetrating within the brain microenvironment. MR-guided focused ultrasound (FUS) with intravascular microbubbles (MBs) is able to locally and reversibly disrupt the BBB with submillimeter spatial accuracy. Densely poly(ethylene-co-glycol) (PEG) coated, brain-penetrating nanoparticles (BPNs) are long-circulating and diffuse 10-fold slower in normal rat brain tissue compared to diffusion in water. Following intravenous administration of model and biodegradable BPNs in normal healthy rats, we demonstrate safe, pressure-dependent delivery of 60nm BPNs to the brain parenchyma in regions where the BBB is disrupted by FUS and MBs. Delivery of BPNs with MR-guided FUS has the potential to improve efficacy of treatments for many CNS diseases, while reducing systemic side effects by providing sustained, well-dispersed drug delivery into select regions of the brain.