Immune profiling of adeno-associated virus response identifies B cell-specific targets that enable vector re-administration in mice.

Adeno-associated virus (AAV) vector-based gene therapies can be applied to a wide range of diseases. AAV expression can last for months to years, but vector re-administration may be necessary to achieve life-long treatment. Unfortunately, immune responses against these vectors are potentiated after the first administration, preventing the clinical use of repeated administration of AAVs. Reducing the immune response against AAVs while minimizing broad immunosuppression would improve gene delivery efficiency and long-term safety. In this study, we quantified the contributions of multiple immune system components of the anti-AAV response in mice. We identified B-cell-mediated immunity as a critical component preventing vector re-administration. Additionally, we found that IgG depletion alone was insufficient to enable re-administration, suggesting IgM antibodies play an important role in the immune response against AAV. Further, we found that AAV-mediated transduction is improved in µMT mice that lack functional IgM heavy chains and cannot form mature B-cells relative to wild-type mice. Combined, our results suggest that B-cells, including non-class switched B-cells, are a potential target for therapeutics enabling AAV re-administration. Our results also suggest that the µMT mice are a potentially useful experimental model for gene delivery studies since they allow repeated dosing for more efficient gene delivery from AAVs.

Reducing Peri-implant Capsule Thickness in Submuscular Rodent Model of Breast Reconstruction With Delayed Radiotherapy.

INTRODUCTION: Capsular contracture remains the most common complication following device-based breast reconstruction, occurring in up to 50% of women who also undergo adjuvant radiotherapy either before or after device-based reconstruction. While certain risk factors for capsular contracture have been identified, there remains no clinically effective method of prevention. The purpose of the present study is to determine the effect of coating the implant with the novel small molecule Met-Z2-Y12, with and without delayed, targeted radiotherapy, on capsule thickness and morphologic change around smooth silicone implants placed under the latissimus dorsi in a rodent model. METHODS: Twenty-four female Sprague Dawley rats each had 2 mL smooth round silicone breast implants implanted bilaterally under the latissimus dorsi muscle. Twelve received uncoated implants and twelve received implants coated with Met-Z2-Y12. Half of the animals from each group received targeted radiotherapy (20 Gray) on postoperative day ten. At three and 6 months after implantation, the tissue surrounding the implants was harvested for analysis of capsular histology including capsule thickness. Additionally, microCT scans were qualitatively analyzed for morphologic change. RESULTS: Capsules surrounding Met-Z2-Y12-coated implants were significantly thinner (P = 0.006). The greatest difference in capsule thickness was seen in the irradiated 6-month groups, where mean capsule thickness was 79.1 ± 27.3 µm for uncoated versus 50.9 ± 9.6 µm for Met-Z2-Y12-coated implants (P = 0.038). At the time of explant, there were no capsular morphologic differences between the groups either grossly or per microCT. CONCLUSIONS: Met-Z2-Y12 coating of smooth silicone breast implants significantly reduces capsule thickness in a rodent model of submuscular breast reconstruction with delayed radiotherapy.

Colony stimulating factor-1 receptor is a central component of the foreign body response to biomaterial implants in rodents and non-human primates.

Host recognition and immune-mediated foreign body response to biomaterials can compromise the performance of implanted medical devices. To identify key cell and cytokine targets, here we perform in-depth systems analysis of innate and adaptive immune system responses to implanted biomaterials in rodents and non-human primates. While macrophages are indispensable to the fibrotic cascade, surprisingly neutrophils and complement are not. Macrophages, via CXCL13, lead to downstream B cell recruitment, which further potentiated fibrosis, as confirmed by B cell knockout and CXCL13 neutralization. Interestingly, colony stimulating factor-1 receptor (CSF1R) is significantly increased following implantation of multiple biomaterial classes: ceramic, polymer and hydrogel. Its inhibition, like macrophage depletion, leads to complete loss of fibrosis, but spares other macrophage functions such as wound healing, reactive oxygen species production and phagocytosis. Our results indicate that targeting CSF1R may allow for a more selective method of fibrosis inhibition, and improve biomaterial biocompatibility without the need for broad immunosuppression.

Size- and shape-dependent foreign body immune response to materials implanted in rodents and non-human primates.

The efficacy of implanted biomedical devices is often compromised by host recognition and subsequent foreign body responses. Here, we demonstrate the role of the geometry of implanted materials on their biocompatibility in vivo. In rodent and non-human primate animal models, implanted spheres 1.5 mm and above in diameter across a broad spectrum of materials, including hydrogels, ceramics, metals and plastics, significantly abrogated foreign body reactions and fibrosis when compared with smaller spheres. We also show that for encapsulated rat pancreatic islet cells transplanted into streptozotocin-treated diabetic C57BL/6 mice, islets prepared in 1.5-mm alginate capsules were able to restore blood-glucose control for up to 180 days, a period more than five times longer than for transplanted grafts encapsulated within conventionally sized 0.5-mm alginate capsules. Our findings suggest that the in vivo biocompatibility of biomedical devices can be significantly improved simply by tuning their spherical dimensions.

Injectable and Glucose-Responsive Hydrogels Based on Boronic Acid-Glucose Complexation.

Injectable hydrogels have been widely used for a number of biomedical applications. Here, we report a new strategy to form an injectable and glucose-responsive hydrogel using the boronic acid-glucose complexation. The ratio of boronic acid and glucose functional groups is critical for hydrogel formation. In our system, polymers with 10-60% boronic acid, with the balance being glucose-modified, are favorable to form hydrogels. These hydrogels are shear-thinning and self-healing, recovering from shear-induced flow to a gel state within seconds. More importantly, these polymers displayed glucose-responsive release of an encapsulated model drug. The hydrogel reported here is an injectable and glucose-responsive hydrogel constructed from the complexation of boronic acid and glucose within a single component polymeric material.

Uncovering the interleukin-12 pharmacokinetic desensitization mechanism and its consequences with mathematical modeling.

The cytokine interleukin-12 (IL-12) is a potential immunotherapy because of its ability to induce a Th1 immune response. However, success in the clinic has been limited due to a phenomenon called IL-12 desensitization - the trend where repeated exposure to IL-12 leads to reduced IL-12 concentrations (pharmacokinetics) and biological effects (pharmacodynamics). Here, we investigated IL-12 pharmacokinetic desensitization via a modeling approach to (i) validate proposed mechanisms in literature and (ii) develop a mathematical model capable of predicting IL-12 pharmacokinetic desensitization. Two potential causes of IL-12 pharmacokinetic desensitization were identified: increased clearance or reduced bioavailability of IL-12 following repeated doses. Increased IL-12 clearance was previously proposed to occur due to the upregulation of IL-12 receptor on T cells that causes increased receptor-mediated clearance in the serum. However, our model with this mechanism, the accelerated-clearance model, failed to capture trends in clinical trial data. Alternatively, our novel reduced-bioavailability model assumed that upregulation of IL-12 receptor on T cells in the lymphatic system leads to IL-12 sequestration, inhibiting the transport to the blood. This model accurately fits IL-12 pharmacokinetic data from three clinical trials, supporting its biological relevance. Using this model, we analyzed the model parameter space to illustrate that IL-12 desensitization occurs over a robust range of parameter values and to identify the conditions required for desensitization. We next simulated local, continuous IL-12 delivery and identified several methods to mitigate systemic IL-12 exposure. Ultimately, our results provide quantitative validation of our proposed mechanism and allow for accurate prediction of IL-12 pharmacokinetics over repeated doses.

Are implantable, living pharmacies within reach?

Cell-based drug factories could produce therapies on demand inside patients.

Rapid method to screen biomaterial angiogenesis in vivo using fluorescence imaging in mice.

Effective vascularization is crucial for repairing and enhancing the longevity of engineered tissues and organs. As the field advances, there is a vital need for efficient and reliable methods for assessing vascularization in real-time. The integration and performance of constructed biomaterials in living organisms rely on angiogenesis and vascularization, making it essential to evaluate vascular development and networks within biomaterials. Current histology-based methods are limited and labor-intensive. On the other hand, fluorescence imaging offers promise for efficient, real-time evaluation of angiogenesis, reducing the time needed for screening many compounds and offering a high-throughput alternative to histology-based methods. Here, we investigated a novel, non-invasive method for quick and repeated analysis of the angiogenic and vascularization process in biomaterials via fluorescence IVIS imaging. Multi-domain peptides (MDPs), self-assembling peptide hydrogels that can possess pro-angiogenic properties depending on their primary sequence, were synthesized and utilized as angiogenic biomaterials and screened with a fluorescence IVIS probe to demonstrate real-time rapid angiogenesis in vivo. The fluorescence-based imaging showed the influence of the peptide chemistry, volume, and concentration on angiogenesis, with one particular MDP, SLanc, promoting robust angiogenesis after one week at 2 w/v%. Through this method, we were able to identify the optimal peptide for rapid and sustained angiogenesis. This approach enables real-time monitoring of angiogenic responses and vascularization processes in the same living subject. It promotes the development of new biomaterials that facilitate vascularization and validates an advanced in vivo screening technique for angiogenesis.

Development of a 3D printed perfusable in vitro blood-brain barrier model for use as a scalable screening tool.

Despite recent technological advances in drug discovery, the success rate for neurotherapeutics remains alarmingly low compared to treatments for other areas of the body. One of the biggest challenges for delivering therapeutics to the central nervous system (CNS) is the presence of the blood-brain barrier (BBB). In vitro blood-brain barrier models with high predictability are essential to aid in designing parameters for new therapeutics, assess their ability to cross the BBB, and investigate therapeutic strategies that can be employed to enhance transport. Here, we demonstrate the development of a 3D printable hydrogel blood-brain barrier model that mimics the cellular composition and structure of the blood-brain barrier with human brain endothelial cells lining the surface, pericytes in direct contact with the endothelial cells on the abluminal side of the endothelium, and astrocytes in the surrounding printed bulk matrix. We introduce a simple, static printed hemi-cylinder model to determine design parameters such as media selection, co-culture ratios, and cell incorporation timing in a resource-conservative and high-throughput manner. Presence of cellular adhesion junction, VE-Cadherin, efflux transporters, P-glycoprotein (P-gp) and Breast cancer resistance protein (BCRP), and receptor-mediated transporters, Transferrin receptor (TfR) and low-density lipoprotein receptor-related protein 1 (LRP1) were confirmed via immunostaining demonstrating the ability of this model for screening in therapeutic strategies that rely on these transport systems. Design parameters determined in the hemi-cylinder model were translated to a more complex, perfusable vessel model to demonstrate its utility for determining barrier function and assessing permeability to model therapeutic compounds. This 3D-printed blood-brain barrier model represents one of the first uses of projection stereolithography to fabricate a perfusable blood-brain barrier model, enabling the patterning of complex vessel geometries and precise arrangement of cell populations. This model demonstrates potential as a new platform to investigate the delivery of neurotherapeutic compounds and drug delivery strategies through the blood-brain barrier, providing a useful in vitro screening tool in central nervous system drug discovery and development.

Persistent tailoring of MSC activation through genetic priming.

Mesenchymal stem/stromal cells (MSCs) are an attractive platform for cell therapy due to their safety profile and unique ability to secrete broad arrays of immunomodulatory and regenerative molecules. Yet, MSCs are well known to require preconditioning or priming to boost their therapeutic efficacy. Current priming methods offer limited control over MSC activation, yield transient effects, and often induce the expression of pro-inflammatory effectors that can potentiate immunogenicity. Here, we describe a genetic priming method that can both selectively and sustainably boost MSC potency via the controlled expression of the inflammatory-stimulus-responsive transcription factor interferon response factor 1 (IRF1). MSCs engineered to hyper-express IRF1 recapitulate many core responses that are accessed by biochemical priming using the proinflammatory cytokine interferon-γ (IFN-γ). This includes the upregulation of anti-inflammatory effector molecules and the potentiation of MSC capacities to suppress T cell activation. However, we show that IRF1-mediated genetic priming is much more persistent than biochemical priming and can circumvent IFN-γ-dependent expression of immunogenic MHC class II molecules. Together, the ability to sustainably activate and selectively tailor MSC priming responses creates the possibility of programming MSC activation more comprehensively for therapeutic applications.

Persistent tailoring of MSC activation through genetic priming.

Mesenchymal stem/stromal cells (MSCs) are an attractive platform for cell therapy due to their safety profile and unique ability to secrete broad arrays of immunomodulatory and regenerative molecules. Yet, MSCs are well known to require preconditioning or priming to boost their therapeutic efficacy. Current priming methods offer limited control over MSC activation, yield transient effects, and often induce expression of pro-inflammatory effectors that can potentiate immunogenicity. Here, we describe a 'genetic priming' method that can both selectively and sustainably boost MSC potency via the controlled expression of the inflammatory-stimulus-responsive transcription factor IRF1 (interferon response factor 1). MSCs engineered to hyper-express IRF1 recapitulate many core responses that are accessed by biochemical priming using the proinflammatory cytokine interferon-γ (IFNγ). This includes the upregulation of anti-inflammatory effector molecules and the potentiation of MSC capacities to suppress T cell activation. However, we show that IRF1-mediated genetic priming is much more persistent than biochemical priming and can circumvent IFNγ-dependent expression of immunogenic MHC class II molecules. Together, the ability to sustainably activate and selectively tailor MSC priming responses creates the possibility of programming MSC activation more comprehensively for therapeutic applications.

Ultrasonographic Identification of Shell Surface Types in Commercially Available Silicone Gel-Filled Breast Implants.

BACKGROUND: Breast implant safety issues have resulted in the need for global product recalls and medical device tracing. Conventional methods of breast implant tracing, have to date proven to be unsuccessful. This study aims to evaluate the effectiveness of high-resolution ultrasound (HRUS) screening in identifying implanted breast devices. METHODS: Data from 113 female patients undergoing preoperative ultrasound screening for secondary breast surgery between 2019 and 2022 was prospectively reviewed to evaluate the effectiveness of HRUS imaging with the aid of a sonographic surface catalog to identify the surface and brand type of implanted breast devices. To corroborate the findings and assess the reproducibility of the approach, further evaluations were replicated in New Zealand white rabbits and compared with the results found in humans. RESULTS: In the human recipients, implant surface and brand types were correctly identified by ultrasound imaging in 99% (112 of 113) and 96% (69 of 72) of the cases, either consultation-only or revision, respectively. This constituted an overall success rate of 98% (181 of 185). Furthermore, in a corroborating New Zealand white rabbit model where full-scale commercial implants were introduced and monitored over many months, from the total 28 analyzed, the surface was accurately identified in a total of 27 cases (the one failure being before generation of a sonograph surface catalogue), demonstrating an overall success rate of 96.4%. CONCLUSION: HRUS is, therefore, a valid and first-hand tool for breast implant imaging that can correctly evaluate both surface type and brand type alongside other variables such as implant placement, positioning, flipping, or rupture. CLINICAL RELEVANCE STATEMENT: HRUS is a valid and first-hand tool for the identification and traceability of breast implants that evaluates surface type and brand type. This low-cost, accessible, and reproducible practice provides patients with peace of mind and surgeons with a promising diagnostic tool.

Enhanced dynamic covalent chemistry for the controlled release of small molecules and biologics from a nanofibrous peptide hydrogel platform.

Maintaining safe and potent pharmaceutical drug levels is often challenging. Multidomain peptides (MDPs) assemble into supramolecular hydrogels with a well-defined, highly porous nanostructure that makes them attractive for drug delivery, yet their ability to extend release is typically limited by rapid drug diffusion. To overcome this challenge, we developed self-assembling boronate ester release (SABER) MDPs capable of engaging in dynamic covalent bonding with payloads containing boronic acids (BAs). As examples, we demonstrate that SABER hydrogels can prolong the release of five BA-containing small-molecule drugs as well as BA-modified insulin and antibodies. Pharmacokinetic studies revealed that SABER hydrogels extended the therapeutic effect of ganfeborole from days to weeks, preventing Mycobacterium tuberculosis growth better than repeated oral administration in an infection model. Similarly, SABER hydrogels extended insulin activity, maintaining normoglycemia for six days in diabetic mice after a single injection. These results suggest that SABER hydrogels present broad potential for clinical translation.

Harnessing cellular therapeutics for type 1 diabetes mellitus: progress, challenges, and the road ahead.

Type 1 diabetes mellitus (T1DM) is a growing global health concern that affects approximately 8.5 million individuals worldwide. T1DM is characterized by an autoimmune destruction of pancreatic ß cells, leading to a disruption in glucose homeostasis. Therapeutic intervention for T1DM requires a complex regimen of glycaemic monitoring and the administration of exogenous insulin to regulate blood glucose levels. Advances in continuous glucose monitoring and algorithm-driven insulin delivery devices have improved the quality of life of patients. Despite this, mimicking islet function and complex physiological feedback remains challenging. Pancreatic islet transplantation represents a potential functional cure for T1DM but is hindered by donor scarcity, variability in harvested cells, aggressive immunosuppressive regimens and suboptimal clinical outcomes. Current research is directed towards generating alternative cell sources, improving transplantation methods, and enhancing cell survival without chronic immunosuppression. This Review maps the progress in cell replacement therapies for T1DM and outlines the remaining challenges and future directions. We explore the state-of-the-art strategies for generating replenishable ß cells, cell delivery technologies and local targeted immune modulation. Finally, we highlight relevant animal models and the regulatory aspects for advancing these technologies towards clinical deployment.

In vivo safety evaluation of polyarginine coated magnetic nanovectors.

Safety and efficacy are of critical importance to any nanomaterial-based diagnostic and therapy. The innocuity and functionality of a nanomaterial in vivo is largely dependent on the physicochemical properties of the material, particularly its surface coating. Here, we evaluated the influence of polycationic coating on the efficacy, clearance organ uptake, and safety of magnetic nanovectors designed for siRNA delivery. Polyethylene glycol (PEG) coated superparamagnetic iron oxide nanoparticles (NPs) of 12 nm in core diameter were modified with a polycationic coating of either poly-l-arginine (pArg) or polyethylenimine (PEI) and further covalently functionalized with siRNA oligonucleotides. The produced NP-pArg-siRNA and NP-PEI-siRNA nanovectors were similar in hydrodynamic size (21 and 22 nm, respectively) but significantly differed in zeta potentials (+2.1 mV and +29.8 mV, respectively). Fluorescence quantification assays revealed that the NP-pArg-siRNA nanovector was 3-fold more potent than NP-PEI-siRNA in delivering siRNA and 1.8-fold more effective in gene silencing when tested in rat C6 glioblastoma cells. In vivo, both nanovector formulations were similarly taken up by the spleen and liver as determined by histopathological and hemopathological assays. However, PEI coated nanovectors elicited severe hemoincompatibility and damage to the liver and spleen, while pArg coated nanovectors were found to be safe and tolerable. Combined, our findings suggest that polycationic coatings of pArg were more effective and safer than commonly used PEI coatings for preparation of nanovectors. The NP-pArg-siRNA nanovector formulation developed here shows great potential for in vivo based biomedical applications.

Localized immunomodulation technologies to enable cellular and organoid transplantation.

Localized immunomodulation technologies are rapidly emerging as a new modality with the potential to revolutionize transplantation of cells and organs. In the past decade, cell-based immunomodulation therapies saw clinical success in the treatment of cancer and autoimmune diseases. In this review, we describe recent advances in engineering solutions for the development of localized immunomodulation techniques focusing on cellular and organoid transplantation. We begin by describing cell transplantation and highlighting notable clinical successes, particularly in the areas of stem cell therapy, chimeric antigen receptor (CAR)-T cell therapy, and islet transplantation. Next, we detail recent preclinical studies centered on genome editing and biomaterials to enhance localized immunomodulation. We close by discussing future opportunities to improve clinical and commercial success using these approaches to facilitate long-term immunomodulation technologies.

Perfusable cell-laden matrices to guide patterning of vascularization in vivo.

The survival and function of transplanted tissue engineered constructs and organs require a functional vascular network. In the body, blood vessels are organized into distinct patterns that enable optimal nutrient delivery and oxygen exchange. Mimicking these same patterns in engineered tissue matrices is a critical challenge for cell and tissue transplantation. Here, we leverage bioprinting to assemble endothelial cells in to organized networks of large (>100 µm) diameter blood vessel grafts to enable spatial control of vessel formation in vivo. Acellular PEG/GelMA matrices with perfusable channels were bioprinted and laminar flow was confirmed within patterned channels, beneficial for channel endothelialization and consistent wall shear stress for endothelial maturation. Next, human umbilical vein endothelial cells (HUVECs) were seeded within the patterned channel and maintained under perfusion culture for multiple days, leading to cell-cell coordination within the construct in vitro. HUVEC and human mesenchymal stromal cells (hMSCs) were additionally added to bulk matrix to further stimulate anastomosis of our bioprinted vascular grafts in vivo. Among multiple candidate matrix designs, the greatest degree of biomaterial vascularization in vivo was seen within matrices fabricated with HUVECs and hMSCs encapsulated within the bulk matrix and HUVECs lining the walls of the patterned channels, dubbed design M-C_E. For this lead design, vasculature was detected within the endothelialized, perfusable matrix channels as early as two weeks and αSMA+ CD31+ vessels greater than 100 µm in diameter had formed by eight weeks, resulting in durable and mature vasculature. Notably, vascularization occurred within the endothelialized, bioprinted channels of the matrix, demonstrating the ability of bioprinted perfusable structures to guide vascularization patterns in vivo. The ability to influence vascular patterning in vivo can contribute to the future development of vascularized tissues and organs.

An Antifibrotic Breast Implant Surface Coating Significantly Reduces Periprosthetic Capsule Formation.

BACKGROUND: The body responds to prosthetic materials with an inflammatory foreign body response and deposition of a fibrous capsule, which may be deleterious to the function of the device and cause significant discomfort for the patient. Capsular contracture (CC) is the most common complication of aesthetic and reconstructive breast surgery. The source of significant patient morbidity, it can result in pain, suboptimal aesthetic outcomes, implant failure, and increased costs. The underlying mechanism remains unknown. Treatment is limited to reoperation and capsule excision, but recurrence rates remain high. In this study, the authors altered the surface chemistry of silicone implants with a proprietary anti-inflammatory coating to reduce capsule formation. METHODS: Silicone implants were coated with Met-Z2-Y12, a biocompatible, anti-inflammatory surface modification. Uncoated and Met-Z2-Y12-coated implants were implanted in C57BL/6 mice. After 21, 90, or 180 days, periprosthetic tissue was removed for histologic analysis. RESULTS: The authors compared mean capsule thickness at three time points. At 21, 90, and 180 days, there was a statistically significant reduction in capsule thickness of Met-Z2-Y12-coated implants compared with uncoated implants ( P < 0.05). CONCLUSIONS: Coating the surface of silicone implants with Met-Z2-Y12 significantly reduced acute and chronic capsule formation in a mouse model for implant-based breast augmentation and reconstruction. As capsule formation obligatorily precedes CC, these results suggest contracture itself may be significantly attenuated. Furthermore, as periprosthetic capsule formation is a complication without anatomical boundaries, this chemistry may have additional applications beyond breast implants, to a myriad of other implantable medical devices. CLINICAL RELEVANCE STATEMENT: Coating of the silicone implant surface with Met-Z2-Y12 alters the periprosthetic capsule architecture and significantly reduces capsule thickness for at least 6 months postoperatively in a murine model. This is a promising step forward in the development of a therapy to prevent capsular contracture.