Long-term control of diabetes by tofacitinib-based immunosuppressive regimen after allo islet transplantation in diabetic rhesus monkeys that rejected previously transplanted porcine islets.

Porcine islet xenotransplantation has been highlighted as an alternative to allo islet transplantation. Despite the remarkable progress that has been made in porcine-islet pre-clinical studies in nonhuman primates, immunological tolerance to porcine islets has not been achieved to date. Therefore, allo islet transplantation could be required after the failure of porcine islet xenotransplantation. Here, we report the long-term control of diabetes by allogeneic pancreatic islet transplantation in diabetic rhesus monkeys that rejected previously transplanted porcine islets. Four diabetic male rhesus monkeys received the porcine islets and then allo islets (5700-19 000 IEQ/kg) were re-transplanted for a short or long period after the first xeno islet rejection. The recipient monkeys were treated with an immunosuppressive regimen consisting of ATG, humira, and anakinra for induction, and sirolimus and tofacitinib for maintenance therapy. The graft survival days of allo islets in these monkeys were >440, 395, >273, and 127, respectively, similar to that in allo islet transplanted cynomolgus monkeys that received the same immunosuppressive regimen without xeno sensitization. Taken together, it is likely that prior islet xenotransplantation does not affect the survival of subsequent allo islets under clinically applicable immunosuppressants.

Long-term control of diabetes in a nonhuman primate by two separate transplantations of porcine adult islets under immunosuppression.

Porcine islet transplantation is an alternative to allo-islet transplantation. Retransplantation of islets is a routine clinical practice in islet allotransplantation in immunosuppressed recipients and will most likely be required in islet xenotransplantation in immunosuppressed recipients. We examined whether a second infusion of porcine islets could restore normoglycemia and further evaluated the efficacy of a clinically available immunosuppression regimen including anti-thymocyte globulin for induction; belimumab, sirolimus, and tofacitinib for maintenance and adalimumab, anakinra, IVIg, and tocilizumab for inflammation control in a pig to nonhuman primate transplantation setting. Of note, all nonhuman primates were normoglycemic after the retransplantation of porcine islets without induction therapy. Graft survival was >100 days for all 3 recipients, and 1 of the 3 monkeys showed insulin independence for >237 days. Serious lymphodepletion was not observed, and rhesus cytomegalovirus reactivation was controlled without any serious adverse effects throughout the observation period in all recipients. These results support the clinical applicability of additional infusions of porcine islets. The maintenance immunosuppression regimen we used could protect the reinfused islets from acute rejection.

The blockade of cytoplasmic HMGB1 modulates the autophagy/apoptosis checkpoint in stressed islet beta cells.

High mobility group (HMGB1) is an alarmin known to be harmful to pancreatic beta cells and associated with diabetes mellitus pathogenesis and pancreatic islet graft failure. It has been long thought that the suppression of HMGB1 molecule is beneficial to the beta cells. However, recent studies have indicated that cytoplasmic HMGB1 (cHMGB1) could function as a modulator to relieve cells from apoptotic stress by autophagy induction. Particularly, pancreatic beta cells have been known to utilize the autophagy-to-apoptosis switch when exposed to hypoxia or lipotoxicity. This study aimed to investigate the beta cells under hypoxic and lipotoxic stress while utilizing a small molecule inhibitor of HMGB1, inflachromene (ICM) which can suppress cHMGB1 accumulation. It was revealed that under cellular stress, blockade of cHMGB1 accumulation decreased the viability of islet grafts, primary islets and MIN6 cells. MIN6 cells under cHMGB1 blockade along with lipotoxic stress showed decreased autophagic flux and increased apoptosis. Moreover, cHMGB1 blockade in HFD-fed mice produced unfavorable outcomes on their glucose tolerance. In sum, these results suggested the role of cHMGB1 within beta cell autophagy/apoptosis checkpoint. Given the importance of autophagy in beta cells under apoptotic stresses, this study might provide further insights regarding HMGB1 and diabetes.

Long-term porcine islet graft survival in diabetic non-human primates treated with clinically available immunosuppressants.

BACKGROUND: Although pancreatic islet transplantation is becoming an effective therapeutic option for patients with type 1 diabetes (T1D) who suffer from a substantially impaired awareness of hypoglycemia, its application is limited due to the lack of donors. Thus, pig-to-human islet xenotransplantation has been regarded as a promising alternative due to the unlimited number of "donor organs." Long-term xenogeneic islet graft survival in pig-to-non-human primate (NHP) models has mainly been achieved by administering the anti-CD154 mAb-based immunosuppressant regimen. Since the anti-CD154 mAb treatment has been associated with unexpected fatal thromboembolic complications in clinical trials, the establishment of a new immunosuppressant regimen that is able to be directly applied in clinical trials is an urgent need. METHODS: We assessed an immunosuppressant regimen composed of clinically available agents at porcine islet transplantation in consecutive diabetic NHPs. RESULTS: Porcine islet graft survival in consecutive diabetic NHPs (n = 7; >222, >200, 181, 89, 62, 55, and 34 days) without severe adverse events. CONCLUSION: We believe that our study could contribute greatly to the initiation of islet xenotransplantation clinical trials.

CD4+ /CD8+ T-cell ratio correlates with the graft fate in pig-to-non-human primate islet xenotransplantation.

BACKGROUND: Xenogeneic islet transplantation using porcine pancreata has been a promising option for substituting human islet transplantation. Moreover, recent advances in pre-clinical results have put islet xenotransplantation closer to the possibility of clinical application. While preparing for the era of clinical xenotransplantation, developing non-invasive immune monitoring method which could predict the graft fate could benefit the patient. However, there are few reports showing predictive immune parameters associated with the fate of the graft in islet xenotransplantation. METHODS: The absolute number and ratio of T-cell subsets have been measured via flow cytometry from the peripheral blood of 16 rhesus monkeys before and after porcine islet xenotransplantation. The correlation between the graft survival and the absolute number or ratio of T cells was retrospectively analyzed. RESULTS: The ratio of CD4+ versus CD8+ T cells was significantly reduced due to CD8+ effector memory cells' increase. Correlation analyses revealed that CD4+ /CD8+ , CD4+ /CD8+ naïve, CD4+ naïve/CD8+ naïve, and CD4+ central memory/CD8+ naïve cell ratios negatively correlated with the duration of graft survival. Conversely, further analyses discovered strong, positive correlation of CD4+ /CD8+ cell ratios within the early graft-rejected monkeys (≤60 days). CONCLUSIONS: This retrospective study demonstrated that CD4+ /CD8+ ratios correlated with graft survival, especially in recipients which rejected the graft in early post-transplantation periods. CD4+ /CD8+ ratios could be used as a surrogate marker to predict the graft fate in pig-to-NHP islet xenotransplantation.

Absence of spontaneous regeneration of endogenous pancreatic ß-cells after chemical-induced diabetes and no effect of GABA on α-to-ß cell transdifferentiation in rhesus monkeys.

ß-cell deficiency is common feature of type 1 and late-stage of type 2 diabetes mellitus. Thus, ß-cell replacement therapy has been the focus of regenerative medicine past several decades. Particularly, evidences suggest that ß-cell regeneration via transdifferentiation from sources including α-cells is promising. However, data using higher mammals besides rodents are scarce. Here, we examined whether endogenous pancreatic ß-cells could regenerate spontaneously or under normoglycemia following porcine islet transplantation for varied periods up to 1197 days after streptozotocin-induced diabetes, and remaining α-cells transdifferentiate into ß-cells by GABA treatment in vivo and in vitro. The results showed that endogenous ß-cells rarely regenerate in both conditions as evidenced by stagnant serum C-peptide levels and ß-cell number in the pancreas, and the remaining α-cells did not transdifferentiate into ß-cells by GABA treatment. Collectively, we concluded that monkey ß-cells had relatively low regenerative potential compared with rodent counterpart and GABA treatment could not induce α-to-ß-cell transdifferentitation.

High mobility group box 1 secretion blockade results in the reduction of early pancreatic islet graft loss.

Pancreatic islet transplantation has been known as the best cure for patients suffering from severe type 1 diabetes mellitus (T1DM). Despite meaningful advances in human allogeneic islet transplantation field, significant amounts of islet loss in early post-transplantation periods is still a big concern for clinicians. One of the major factors determining the fate of the islets is the danger-associated molecular patterns (DAMPs) secreted by activated immune cells or islets themselves under hypoxic stress. High mobility group box 1 (HMGB1) protein is one of the best characterized DAMP molecules associated with islets. HMGB1 is known to be passively released by transplanted murine islet cells after taking damages from cytokines, reactive oxygen species, and other DAMPS, and the released HMGB1 harms neighboring islet cells by interacting with receptors expressed on murine islets such as toll-like receptor 2 (TLR2) and TLR4, thereby forming a vicious cycle. Here, we show that a small molecule inhibitor inflachromene (ICM) was capable of blocking the secretion of HMGB1 from murine islet cells during the normoxic and hypoxic post-isolation period. Notably, the treatment of ICM during the islet isolation process resulted in decreased HMGB1 levels during the subsequent cell culture. ICM's in vivo efficacy was evaluated in murine syngeneic islet transplantation model, and it significantly reduced the serum and graft level of HMGB1. Ultimately, the intraperitoneal administration of ICM prevented the loss of marginal-mass islet grafts and reversed the diabetes in mice.

Peri-graft porcine-specific CD4+ FoxP3+ regulatory T cells by CD40-CD154 blockade prevented the rejection of porcine islet graft in diabetic mice.

BACKGROUND: Anti-CD154 monoclonal antibody (mAb) treatment has been known to have potential to induce immune tolerance in organ transplantation. Several studies have suggested the involvement of CD4+ regulatory T cells (Treg s) in xeno-immune tolerance. However, the characteristics of Treg s and the mechanisms of their regulatory functions in islet xenotransplantation have not been clearly defined. METHOD: Adult porcine islet cells were isolated and purified, and were transplanted under the kidney capsule of diabetic C57BL/6J mice with the administration of 0.5 mg/mouse of anti-CD154 mAb on 0, 1, 3, 5, and 7 days post-transplantation (DPT). The graft survival was monitored by blood glucose level. The islet graft and recipients' cells were analyzed by immunohistochemistry (IHC), flow cytometry, enzyme-linked immunosorbent spot (ELISPOT) assay, and mixed-lymphocyte reaction. RESULTS: Short-term anti-CD154 mAb monotherapy enabled the porcine islet graft to survive indefinitely in diabetic mice (n = 18). Immunohistochemical staining showed significantly higher ratio of CD4+ Foxp3+ Treg s in the peri-graft site, but not in the spleen and kidney-draining lymph node of the recipient mice. Depletion of Treg s evoked graft rejection, and adoptive transfer of Treg s from anti-CD154 mAb-treated recipients provided protection to the graft from rejection. These Treg s showed more potent suppressive capacity than those from the untreated control and were found to be porcine antigen-specific. CONCLUSIONS: In this study, we showed that anti-CD154 mAb monotherapy resulted in indefinite porcine islet graft survival in mice. The porcine-specific CD4+ Foxp3+ Treg s in the peri-graft site played the critical role in the protection of islet graft from rejection, which suggests a prospective immunosuppressive strategy for islet xenotransplantation.

Ascites formation accompanied by portal vein thrombosis after porcine islet xenotransplantation via the portal vein in Rhesus macaque (Macaca mulatta).

Pig-to-nonhuman primate (NHP) islet transplantation has been widely conducted as a preclinical xenotransplantation model prior to human clinical trial. Portal vein thrombosis is one of the complications associated with islet infusion through the portal vein into the liver. Here, we briefly report severe case of ascites formation accompanied by portal vein thrombi after pig-to-NHP islet xenotransplantation in a rhesus monkey. Meticulous prophylactic treatment such as continuous heparin infusion should be implemented to prevent portal vein thrombi in pig-to-NHP islet transplantation models.

Construction of EMSC-islet co-localizing composites for xenogeneic porcine islet transplantation.

Pancreatic islet transplantation is an ultimate solution for treating patients with type 1 diabetes (T1D). The pig is an ideal donor of islets for replacing scarce human islets. Besides immunological hurdles, non-immunological hurdles including fragmentation and delayed engraftment of porcine islets need solutions to succeed in porcine islet xenotransplantation. In this study, we suggest a simple but effective modality, a cell/islet co-localizing composite, to overcome these challenges. Endothelial-like mesenchymal stem cells (EMSCs), differentiated from bone-marrow derived mouse mesenchymal stem cells (MSCs), and MSCs evenly coated the surface of porcine islets (>85%) through optimized culture conditions. Both MSCs and EMSCs significantly reduced the fragmentation of porcine islets and increased the islet masses, designated as islet equivalents (IEQs). In fibrin in vitro and in vivo angiogenesis analysis, constructed EMSC-islet composites showed higher angiogenic potentials than naked islets, MSC-islet composites, or human endothelial cell-islet composites. This novel delivery method of porcine islets may have beneficial effects on the engraftment of transplanted islets by prevention of fragmentation and enhancement of revascularization.

Therapeutic effects of endoscopic ablation in patients with Hunner type interstitial cystitis.

OBJECTIVE: To investigate the efficacy of endoscopic ablation of Hunner lesions (HLs) in patients with interstitial cystitis (IC) and to find predictors of early recurrence of HLs. PATIENTS AND METHODS: A prospective study was performed of patients with Hunner type IC who underwent transurethral ablation. We repeated endoscopic ablation when symptoms and HLs recurred during the follow-up period. The primary endpoint was recurrence-free time. Secondary endpoints were a change in frequency, nocturia, and urgency episodes, and changes in visual analogue scale (VAS) pain scores and other symptom indices at follow-up visits. RESULTS: A total of 72 patients were analysed. The median (range) follow-up period was 29.5 (12.0-50.0) months. After primary ablation treatment, HLs recurred in 75.0% (54/72) of patients, and the median (sd) recurrence-free time was 12.0 (1.6) months. Amongst the 54 patients with recurrence, 50 underwent a second ablation treatment. HLs occurred in 44.0% (22/50) of individuals after the second operation, and the median (sd) recurrence-free time was 18.0 (5.1) months. Lower maximal cystometric capacity (odds ratio 1.01, 95% confidence interval 1.001-1.013) was the predictive factor for early recurrence. There were significant improvements in the VAS pain scores, O'Leary-Sant Interstitial Cystitis Symptom Index and Problem Index, Pelvic Pain and Urgency/Frequency Patient Symptom Scale after treatment (all, P < 0.05). CONCLUSIONS: Endoscopic ablation is an effective treatment option for HLs and significantly reduces pain and improves voiding symptoms. Repeat ablation upon recurrence could help symptom control and bladder preservation only if the bladder capacity is maintained.

Computed tomography or necropsy diagnosis of multiple bullae and the treatment of pneumothorax in rhesus macaques (Macaca mulatta).

Pulmonary bullae and pneumothorax have various etiologies in veterinary medicine. We diagnosed multiple pulmonary bullae combined with or without pneumothorax by computed tomography (CT) or necropsy in seven rhesus macaques (Macaca mulatta) imported from China. Two of seven rhesus macaques accompanied by pneumothorax were cured by fixation of ruptured lung through left or right 3rd intercostal thoracotomy. Pneumonyssus simicola, one of the etiologies of pulmonary bullae, was not detected from tracheobronchiolar lavage. To the best of our knowledge, this is the first case report on the CT-aided diagnosis of pulmonary bullae and the successful treatment of combined pneumothorax by thoracotomy in non-human primates (NHPs).

Cell enrichment-free massive ex-vivo expansion of peripheral CD20⁺ B cells via CD40-CD40L signals in non-human primates.

Non-human primates (NHPs) are valuable as preclinical resources that bridge the gap between basic science and clinical application. B cells from NHPs have been utilized for the development of B-cell targeted drugs and cell-based therapeutic modalities; however, few studies on the ex-vivo expansion of monkey B cells have been reported. In this study, we developed a highly efficient ex-vivo expansion protocol for monkey B cells resulting in 99% purity without the requirement for prior cell-enrichment procedures. To this end, monkey peripheral blood mononuclear cells (PBMCs) were stimulated for 12 days with cells constitutively expressing monkey CD40L in expansion medium optimized for specific and massive expansion of B cells. The B cells expansion rates obtained were 2-5 times higher than those previously reported in humans, with rates ranging from 7.9 to 16.6 fold increase. Moreover, expanded B cells sustained high expression of co-stimulatory molecules including CD83 and CD86 until day 12 of culture, and the simple application of a brief centrifugation resulted in a CD20(+) B cell purity rate of greater than 99%. Furthermore, small amounts of CD3(+)CD20(+)BT-like cells were generated and CD16 was expressed at moderate levels on expanded B cells. Thus, the establishment of this protocol provides a method to produce quantities of homogeneous, mature B cells in numbers sufficient for the in vitro study of B cell immunity as well as for the development of B cell-diagnostic tools and cell-based therapeutic modalities.

Optimal CXCR5 Expression during Tfh Maturation Involves the Bhlhe40-Pou2af1 Axis.

The pair of transcription factors Bcl6-Blimp1 is well-known for follicular T helper (Tfh) cell fate determination, however, the mechanism(s) for Bcl6-independent regulation of CXCR5 during Tfh migration into germinal center (GC) is still unclear. In this study, we uncovered another pair of transcription factors, Bhlhe40-Pou2af1, that regulates CXCR5 expression. Pou2af1 was specifically expressed in Tfh cells whereas Bhlhe40 expression was found high in non-Tfh cells. Pou2af1 promoted Tfh formation and migration into GC by upregulating CXCR5 but not Bcl6, while Bhlhe40 repressed this process by inhibiting Pou2af1 expression. RNA-Seq analysis of antigen-specific Tfh cells generated in vivo confirmed the role of Bhlhe40-Pou2af1 axis in regulating optimal CXCR5 expression. Thus, the regulation of CXCR5 expression and migration of Tfh cells into GC involves a transcriptional regulatory circuit consisting of Bhlhe40 and Pou2af1, which operates independent of the Bcl6-Blimp1 circuit that determines the Tfh cell fate.

Gracilimonas rosea sp. nov., isolated from tropical seawater, and emended description of the genus Gracilimonas.

A Gram-staining-negative, non-motile, spore-forming, rod-shaped, marine bacterial strain, CL-KR2(T), was isolated from tropical seawater near Kosrae, an island in the Federated States of Micronesia. Analysis of the 16S rRNA gene sequence of strain CL-KR2(T) revealed a clear affiliation with the genus Gracilimonas. Based on phylogenetic analysis, strain CL-KR2(T) showed the closest phylogenetic relationship to Gracilimonas tropica CL-CB462(T), with 16S rRNA gene sequence similarity of 96.6 %. DNA-DNA relatedness between strain CL-KR2(T) and G. tropica CL-CB462(T) was 6.7 % (reciprocal 9.5 %). Strain CL-KR2(T) grew in the presence of 1-20 % sea salts and the optimal salt concentration was 3.5-5 %. The temperature and pH optima for growth were 35 °C and pH 7.5. The major cellular fatty acids (≥10.0 %) of strain CL-KR2(T) were iso-C15 : 0, summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c) and iso-C17 : 1ω9c and the only isoprenoid quinone was MK-7. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, an unidentified phospholipid, two unidentified glycolipids and two unidentified lipids. The genomic DNA G+C content of strain CL-KR2(T) was 43.2 mol%. The combined phenotypic, chemotaxonomic and phylogenetic data showed that strain CL-KR2(T) could be distinguished from the only member of the genus Gracilimonas with a validly published name. Thus, strain CL-KR2(T) should be assigned to a novel species in the genus Gracilimonas, for which the name Gracilimonas rosea sp. nov. is proposed. The type strain is CL-KR2(T) ( = KCCM 90206(T) = JCM 18898(T)).

Mesenchymal stem cell-derived extracellular vesicles subvert Th17 cells by destabilizing RORγt through posttranslational modification.

Mesenchymal stem cell (MSC)-derived small extracellular vesicles (MSC-sEVs) are known to exert immunosuppressive functions. This study showed that MSC-sEVs specifically convert T helper 17 (Th17) cells into IL-17 low-producer (ex-Th17) cells by degrading RAR-related orphan receptor γt (RORγt) at the protein level. In experimental autoimmune encephalomyelitis (EAE)-induced mice, treatment with MSC-sEVs was found to not only ameliorate clinical symptoms but also to reduce the number of Th17 cells in draining lymph nodes and the central nervous system. MSC-sEVs were found to destabilize RORγt by K63 deubiquitination and deacetylation, which was attributed to the EP300-interacting inhibitor of differentiation 3 (Eid3) contained in the MSC-sEVs. Small extracellular vesicles isolated from the Eid3 knockdown MSCs by Eid3-shRNA failed to downregulate RORγt. Moreover, forced expression of Eid3 by gene transfection was found to significantly decrease the protein level of RORγt in Th17 cells. Altogether, this study reveals the novel immunosuppressive mechanisms of MSC-sEVs, which suggests the feasibility of MSC-sEVs as an attractive therapeutic tool for curing Th17-mediated inflammatory diseases.

Galectin-4 increases the ability of M2 macrophages to enhance antiviral CD4+ T-cell responses.

Galectin-4 (Gal-4) is a ß-galactoside-binding protein belonging to the galectin family. Although Gal-4 is known to be involved in several physiologic processes of the gastrointestinal tract, its immunomodulatory roles remain unclear. In this study, we investigated whether Gal-4 influences the function of M1 and M2 macrophages. Gal-4 treatment drove more robust changes in the gene expression of M2 macrophages compared to M1 macrophages. Antiviral immune response-related genes were significantly upregulated in Gal-4-treated M2 macrophages. Gal-4 significantly enhanced the immunostimulatory activity of M2 macrophages upon Toll-like receptor 7 stimulation or infection with lymphocytic choriomeningitis virus (LCMV). Moreover, the antibody production against LCMV infection and the antiviral CD4+ T-cell responses, but not the antiviral CD8+ T-cell responses, were greatly increased by Gal-4-treated M2 macrophages in vivo. The present results indicate that Gal-4 enhances the ability of M2 macrophages to promote antiviral CD4+ T-cell responses. Thus, Gal-4 could be used to boost antiviral immune responses.

Transcription factor TCF-1 regulates the functions, but not the development, of lymphoid tissue inducer subsets in different tissues.

Lymphoid tissue inducer (LTi) cells, a subset of innate lymphoid cells (ILCs), play an essential role in the formation of secondary lymphoid tissues. However, the regulation of the development and functions of this ILC subset is still elusive. In this study, we report that the transcription factor T cell factor 1 (TCF-1), just as GATA3, is indispensable for the development of non-LTi ILC subsets. While LTi cells are still present in TCF-1-deficient mice, the organogenesis of Peyer's patches (PPs), but not of lymph nodes, is impaired in these mice. LTi cells from different tissues have distinct gene expression patterns, and TCF-1 regulates the expression of lymphotoxin specifically in PP LTi cells. Mechanistically, TCF-1 may directly and/or indirectly regulate Lta, including through promoting the expression of GATA3. Thus, the TCF-1-GATA3 axis, which plays an important role during T cell development, also critically regulates the development of non-LTi cells and tissue-specific functions of LTi cells.

Acetyl-CoA carboxylase-1/2 blockade locks dendritic cells in the semimature state associated with FA deprivation by favoring FAO.

Immunometabolism is rising as an intriguing topic that reveals the connection between immune cell function and metabolic processes. Especially, fatty acid metabolism plays an essential role in the dendritic cells (DCs) during the differentiation and maturation period. We questioned whether regulation of acetyl-CoA carboxylases 1 and 2-(ACC1/2), the core enzymes of fatty acid synthesis (FAS), would control DC function. Here, we report that blocking ACC1/2 to prevent FAS during DC maturation switched their cellular metabolism into fatty acid oxidation to fuel oxidative phosphorylation. This action turned DCs to utilize exogenous fatty acids to sustain their basal energy demand and maintain a stable cellular respiration rate. Coincidentally, under the ACC1/2 inhibitor treatment, LPS-treated DCs exhibited a semimaturation phenotype with a maturation-resistance feature, with decreased expression of costimulatory molecules including CD86 and CD40, along with the reduction of IL-12 and IL-6. The migratory capability of DCs has been known to relate to the glycolysis pathway, and here we showed that the ACC1/2 blockade did not affect the expression of CCR7 and DC migration. Furthermore, we found that under the ACC1/2 blocking condition, DCs pulsed with OVA failed to activate OVA-specific CD4+ T cell proliferation even though their antigen uptake capacity was intact. Together, our data suggest ACC1/2 as a promising target to control DC fate.

Six-month sustained delivery of anti-VEGF from in-situ forming hydrogel in the suprachoroidal space.

Patients with wet age-related macular degeneration (AMD) require intravitreal injections of bevacizumab (Bev) or other drugs, often on a monthly basis, which is a burden on the healthcare system. Here, we developed an in-situ forming hydrogel comprised of Bev and hyaluronic acid (HA) crosslinked with poly(ethylene glycol) diacrylate for slow release of Bev after injection into the suprachoroidal space (SCS) of the eye using a microneedle. Liquid Bev formulations were cleared from SCS within 5 days, even when formulated with high viscosity, unless Bev was conjugated to a high molecular-weight HA (2.6 MDa), which delayed clearance until 1 month. To extend release to 6 months, we synthesized in-situ forming Bev-HA hydrogel initially as a low-viscosity mixture suitable for injection and flow in the SCS to cover a large area extending to the posterior pole of the eye where the macula is located in humans. Within 1 h after injection, Bev and HA were crosslinked, which retained Bev for slow release as the hydrogel biodegraded. In vivo studies in the rabbit eye reported Bev release for >6 months, depending on gel formulation and Bev assay. The in-situ forming Bev-HA hydrogel was well tolerated, as assessed by clinical exam, fundus imaging, histological analysis, and intraocular pressure measurement. We conclude that Bev released from an in-situ forming hydrogel may enable long-acting treatments of AMD and other posterior ocular indications.