Background
Following protein replacement therapy, one‐third of severe hemophilia A patients develop antibodies to factor VIII (FVIII), which also hinders the efficacy of gene therapy. Regulatory T ...cells (Tregs) have a naturally suppressive function that potentially reduces the immune response to FVIII therapy. Furthermore, antigen‐specific Tregs are functionally much more potent than polyclonal cells. Adoptive transfer of antigen‐specific Tregs can effectively suppress anti‐FVIII antibody responses.
Objective
Develop a clinically feasible protocol to enrich and expand Tregs specific to FVIII for suppressing anti‐FVIII immune responses.
Methods
Regulatory T cells are isolated from FVIII‐sensitized mice, sorted on CD25high markers, and expanded specifically with FVIII, antigen‐presenting cells, and interleukin 2 (IL 2). Subsequently, Tregs are further cultured with anti‐CD3/anti‐CD28 beads, anti‐Crry antibodies, and IL 2 to achieve 10‐fold to 20‐fold expansion. Expanded Tregs are characterized and tested for their suppressive activity in vitro and in vivo.
Results
In vitro FVIII‐specific suppressive assays indicate that FVIII specifically expanded Tregs are more suppressive than non‐specifically expanded and naive Tregs. Adoptive transfer of expanded Tregs into HemA mice showed that FVIII‐specifically expanded Tregs are significantly more potent in suppressing anti‐FVIII immune responses in FVIII plasmid‐treated HemA mice. Moreover, the FVIII‐specific immune tolerance is maintained after a secondary challenge with FVIII plasmid.
Conclusions
Our results demonstrate that the FVIII‐specific sensitization and expansion protocol yields more potent Tregs to suppress anti‐FVIII antibody responses and induce long‐term tolerance to FVIII, increasing the potential for adoptive Treg cell therapy to modulate anti‐FVIII immune responses.
Hemophilia A (HemA) patients are currently treated with costly and inconvenient replacement therapy of short-lived factor VIII (FVIII) protein. Development of lipid nanoparticle (LNP)-encapsulated ...mRNA encoding FVIII can change this paradigm. LNP technology constitutes a biocompatible and scalable system to efficiently package and deliver mRNA to the target site. Mice intravenously infused with the luciferase mRNA LNPs showed luminescence signals predominantly in the liver 4 h after injection. Repeated injections of LNPs did not induce elevation of liver transaminases. We next injected LNPs carrying mRNAs encoding different variants of human FVIII (F8 LNPs) into HemA mice. A single injection of B domain-deleted F8 LNPs using different dosing regimens achieved a wide range of therapeutic activities rapidly, which can be beneficial for various usages in hemophilia treatment. The expression slowly declined yet remained above therapeutic levels up to 5–7 days post-injection. Furthermore, routine repeated injections of F8 LNPs in immunodeficient mice produced consistent expression of FVIII over time. In conclusion, F8 LNP treatment produced rapid and prolonged duration of FVIII expression that could be applied to prophylactic treatment and potentially various other treatment options. Our study showed potential for a safe and effective platform of new mRNA therapies for HemA.
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Messenger RNA (mRNA) encapsulated in lipid nanoparticles (LNPs) can be efficiently delivered into the liver. LNPs carrying FVIII mRNA (F8 LNPs) produced rapid and prolonged duration of FVIII expression in hemophilia A mice. This safe and effective platform of new mRNA therapies could be used for prophylactic treatment and potentially various other treatment options for hemophilia A patients.
•FVIII-specific Tregs were produced by transducing CD4+ T cells with F8CARmFoxp3-LV..•The gene-modified T cells suppressed anti-FVIII immune responses in vitro.•Adoptive FVIIICAR-Treg therapy ...prevented anti-FVIII antibody production in HemA mice.
Although protein replacement therapy provides effective treatment for hemophilia A patients, about a third of severe patients develop neutralizing inhibitor antibodies to factor VIII. Adoptive transfer of regulatory T cells (Tregs) has shown promise in treating unwanted immune responses. In previous studies, transferred polyclonal Tregs ameliorated the anti-factor VIII immune responses in hemophilia A mice. In addition, factor VIII-primed Tregs demonstrated increased suppressive function. However, antigen-specific Tregs are a small fraction of the total lymphocyte population. To generate large numbers of factor VIII-specific Tregs, the more abundant murine primary CD4+ T cells were lentivirally transduced ex vivo to express Foxp3 and a chimeric antigen receptor specific to factor VIII (F8CAR). Transduced cells significantly inhibited the proliferation of factor VIII-specific effector T cells in suppression assays. To monitor the suppressive function of the transduced chimeric antigen receptor expressing T cells in vivo, engineered CD4+CD25+Foxp3+F8CAR-Tregs were sorted and adoptively transferred into hemophilia A mice that are treated with hydrodynamically injected factor VIII plasmid. Mice receiving engineered F8CAR-Tregs showed maintenance of factor VIII clotting activity and did not develop anti-factor VIII inhibitors, while control CD4+T cell or PBS recipient mice developed inhibitors and had a sharp decrease in factor VIII activity. These results show that CD4+ cells lentivirally transduced to express Foxp3 and F8CAR can promote factor VIII tolerance in a murine model. With further development and testing, this approach could potentially be applied to human hemophilia patients.
Gene therapy offers great promises for a cure of hemophilia A resulting from factor VIII (FVIII) gene deficiency. We have developed and optimized a non-viral ultrasound-mediated gene delivery (UMGD) ...strategy. UMGD of reporter plasmids targeting mice livers achieved high levels of transgene expression predominantly in hepatocytes. Following UMGD of a plasmid encoding human FVIII driven by a hepatocyte-specific promoter/enhancer (pHP-hF8/N6) into the livers of hemophilia A mice, a partial phenotypic correction was achieved in treated mice. In order to achieve persistent and therapeutic FVIII gene expression, we adopted a plasmid (pHP-hF8-X10) encoding an FVIII variant with significantly increased FVIII secretion. By employing an optimized pulse-train ultrasound condition and immunomodulation, the treated hemophilia A mice achieved 25%–150% of FVIII gene expression on days 1–7 with very mild transient liver damage, as indicated by a small increase of transaminase levels that returned to normal within 3 days. Therapeutic levels of FVIII can be maintained persistently without the generation of inhibitors in mice. These results indicate that UMGD can significantly enhance the efficiency of plasmid DNA transfer into the liver. They also demonstrate the potential of this novel technology to safely and effectively treat hemophilia A.
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Ultrasound-mediated gene delivery (UMGD) was demonstrated to efficiently transfer factor VIII plasmids driven by a hepatocyte-specific promoter/enhancer into the livers of hemophilia A mice. Persistent and therapeutic levels of factor VIII expression can be achieved, demonstrating the potential for UMGD to safely and effectively treat hemophilia A.
•AMD3100+G-CSF effectively reduced FVIII-specific LLPCs and inhibitor titers.•Reduction of LLPCs and inhibitors was paramount for the recovery of FVIII activity.•Regimens targeting both T and B cells ...reduced and maintained low inhibitor titers.
Long-lived plasma cells (LLPCs) can persistently produce anti-factor VIII (FVIII) antibodies which disrupt therapeutic effect of FVIII in hemophilia A patients with inhibitors. The migration of plasma cells to BM where they become LLPCs is largely controlled by an interaction between the chemokine ligand CXCL12 and its receptor CXCR4. AMD3100 combined with G-CSF inhibit their interactions, thus facilitating the mobilization of CD34+ cells and blocking the homing of LLPCs. These reagents were combined with anti-CD20 to reduce B-cells and the specific IL-2/IL-2mAb (JES6-1) complexes to induce Treg expansion for targeting anti-FVIII immune responses. Groups of mice primed with FVIII plasmid and protein respectively were treated with the combined regimen for six weeks, and a significant reduction of anti-FVIII inhibitor titers was observed, associated with the dramatic decrease of circulating and bone marrow CXCR4+ plasma cells. The combination regimens are highly promising in modulating pre-existing anti-FVIII antibodies in FVIII primed subjects.
Hemophilia A mice with pre-existing inhibitory antibodies against factor VIII (FVIII) were treated with single agents, AMD3100 and GCS-F, respectively. Inhibitor titers in treated mice and control ...HemA inhibitors mice were followed over time. Total B cells and plasma cells (PCs) were characterized by flow cytometry. HemA inhibitor mice were then treated with a combination regimen of IL-2/IL-2mAb complexes plus rapamycin and AMD3100. Finally, HemA inhibitor mice were treated with a new combination therapy using include IL-2/IL-2mAb complexes + Anti-CD20+AMD3100+G-CSF. The timeline of combination therapy was illustrated. Inhibitor titers following treatment in FVIII plasmid or protein induced inhibitor mice were evaluated overtime. A representative figure and gating strategies to characterize the subsets of Treg cells and B cells are presented. Please see http://dx.doi.org/10.1016/j.cellimm.2016.01.005 1 for interpretation and discussion of these data and results.
It is currently unknown what factor(s) may promote entry into a weight loss plateau. Given intensive lifestyle interventions (ILI) for weight loss include changes in diet, we evaluated how diet ...quality impacts characteristics of a weight loss plateau. Daily weights were obtained remotely via electronic scale from 62 adults with obesity (73% female, mean age 42±11 y and BMI 37±5 kg/m2) undergoing a 24-week ILI. Periods (≥14 d) of active weight loss or plateau were identified by threshold regression modeling. Active weight loss was defined as a per day % weight change from baseline equivalent to ≥0.5 lb loss/wk and a weight loss plateau as ±0.25lbs/wk after a period of active weight loss (in which ≥3.5% weight loss was achieved). Three unannounced self-reported ASA24 dietary recalls were obtained at baseline and 3 mo. Diet quality was assessed by the healthy eating index (HEI)-2015. 53% reached a plateau after active weight loss (27% did not achieve >3.5% weight loss, 19% re-gained directly after loss). Weight loss (i.e., plateau depth) was associated with longer time to plateau (β=–10d, P<0.01). Higher baseline diet quality (total HEI) was associated with shorter time to plateau (β=–2.6d, P=0.03), but not overall depth of plateau (β=0.1%, P=0.28). Specifically, shorter time to plateau was related to lower baseline consumption of saturated fats (β=–15d, P=0.01) and greater plateau depth was related to lower baseline consumption of added sugars (β=1.3%, P=0.04). Mean diet quality improved minimally during ILI (ΔHEI 1.9±2.2). Lower baseline HEI correlated with greater improvement in diet quality at 3 mo (β=–0.5, P<0.001, N=46), but ΔHEI did not associate with any plateau characteristics (time: β=1.1d, P=0.15; depth: β=–0.1%, P=0.16, N=25). Higher diet quality upon entry into an ILI predicted a shorter duration of weight loss prior to reaching a plateau, suggesting that individuals with healthy eating patterns may derive less sustained weight loss benefit from participation in an ILI.
Disclosure
S.J.Melhorn: None. L.E.Sewaybricker: None. H.Gao: None. M.De leon: None. M.Webb: None. M.Lyle: None. S.J.Beatty: None. M.Kratz: Other Relationship; Nourished by Science LLC. E.Schur: None.
Funding
National Institutes of Health (DK089036, DK1176223, K24HL144917); University of Washington (NORC, DK035816)
Abstract
Hemophilia A patients lack or possess low levels of functional FVIII protein, which results in an inability for the blood to clot when injury occurs. The current treatment for hemophilia A ...patients is FVIII protein replacement, but this treatment is expensive and often results in anti-FVIII immune responses, neutralizing the clotting effect.
Regulatory T (Treg) cells help balance T effector (Teff) cells through their suppressive function during an immune response and keep autoimmunity in check. Treg cells possess a high affinity epitope of the IL2 receptor. With a low dose of IL2, the Treg population outcompetes the Teff cells, leading to an increase in activation and number. The increased suppressive activity may induce tolerance in hemophilia A mice treated with FVIII. Hemophilia A mice were hydrodynamically injected with IL2 and FVIII plasmids. Cell staining data showed a marked increase in Treg cell population and activation. In addition, the Treg/Teff ratio was significantly increased in the first 3 weeks and maintained at increased levels over several weeks afterwards. On Day 28-post FVIII injection, control mice had started to develop inhibitors associated with significant decrease of FVIII expression, while mice treated with IL2 showed no inhibitors with persistent FVIII expression. The treated mice are monitored to determine the potential long term tolerance induced by low dose IL2. Combined gene transfer of FVIII and IL2 plasmids can produce therapeutic FVIII and simultaneously prevent inhibitory antibody formation, thus providing a potentially effective treatment of hemophilia A.
Abstract
The immune response to factor VIII protein (FVIII; F8 in constructs) limits the effectiveness of treatments for hemophilia A (HemA) patients. Previously we demonstrated that regulatory T ...cells (Tregs) play a pivotal role in modulating anti-FVIII immune responses. For application of adoptive Treg therapy, we successfully expanded highly suppressive murine polyclonal Tregs antigen specifically in vitro. However, the FVIII-specific Tregs in the polyclonal population are still in very small numbers. Thus, we explored the strategy to generate FVIII-specific Tregs using the chimeric antigen receptor (CAR) approach. Lentiviral vector (LV) incorporating a high-binding anti-FVIII scFv linked to the CAR signaling domains and fused with a Foxp3 cDNA (F8CAR-Foxp3-LV) was prepared and used to transduce murine CD4+T cells. Flow cytometry analysis confirmed extracellular scFv and intracellular Foxp3 expression in transduced cells (F8CAR-Tregs). In vitro FVIII-specific suppressive assay showed that transduced cells had significantly higher suppressive activity than untransduced cells towards murine effector T cells. In addition, 1×106 transduced cells and untransduced cells were adoptively transferred into HemA mice and the treated mice were subsequently challenged with FVIII plasmid injected hydrodynamically. The anti-FVIII antibody titers are evaluated overtime. It is expected that F8CAR-Foxp3-LV transduced cells will prevent or decrease the production of anti-FVIII antibodies in HemA mice. We anticipate that compared with nonspecific or polyclonally expanded Tregs, FVIII-specific CAR Tregs will exert superior suppressive activity towards anti-FVIII immune responses without triggering systemic immune suppression.
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