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Review question / Objective: The main aim of this review is to analyse the impact factors of material physical activity in an ecological model and to analyse differences in influencing factors between pregnant women's PA and moderate-to-vigorous intensity physical activity (MVPA) , provide a reference for the research, intervention, and policy designation of maternal physical activity. Rationale: In combination with McLeroy et al. (1988)behavior is viewed as being determined by the following: (1) Personal level: the internal factors of the individual characteristics,(sociodemographic and biological, behavior, psychological ); (2) interpersonal level: interpersonal processes and primary groups-formal and informal social network and social support systems,(eg: family、public, etc.); (3)organization level: social institutions with organizational characteristics, such as health services, gyms and may also include influences from health care providers and Physical activity consultant, etc.; (4) community level: relationships among organizations, institutions, and informal networks within defined boundaries，（eg： appropriate facilities、living environment, etc.); and finally (5) public policy level: local, state, and national laws and policies.

A novel, engineered, liver-tropic adeno-associated virus vector expressing a hyperactive Padua factor IX (FIX) protein (BBM-H901) has been developed and is promising for haemophilia B gene therapy. We aimed to explore its safety and activity in increasing FIX concentrations and reducing bleeding frequency.We did a single-centre, single-arm, phase 1, pilot trial evaluating the safety and activity of a single intravenous infusion of BBM-H901 at the Institute of HematologyBlood Diseases Hospital, Chinese Academy of Medical SciencesPeking Union Medical College (Tianjin, China). We enrolled adult patients with haemophilia B (aged18 years) with baseline FIX coagulation activity (FIX:C) of less than 2 IU/dL, no FIX inhibitor, and low titre of neutralising antibodies (≤1:4) against vector capsid. Eligible participants were intravenously infused with a single dose of 5 × 10Between Oct 16, 2019, and Jan 13, 2021, 12 male participants were assessed, and ten Chinese participants were enrolled and infused with BBM-H901. After a median follow-up of 58 weeks (IQR 51·5-99·5), mean FIX:C reached mean 36·9 IU/dL (SD 20·5). No serious adverse events, no grade 3-4 adverse events were observed. Grade 1-2 adverse events related to BBM-H901 include pyrexia (1 [10%]) and elevation of aminotransferase(1 [10%]). No FIX inhibitors were observed. All participants developed antibodies against vector capsid after infusion. Eight (80%) participants had ALT and AST concentrations below the upper limit of normal throughout the follow-up period. Two (20%) participants had elevation of ALT and AST accompanied with decrease of FIX:C, which remained at 7 IU/dL and 11.8 IU/dL, respectively.This pilot study suggests that liver-tropic BBM-H901 is safe 1 year after infusion. Vector derived FIX:C concentration is sufficiently high to prevent bleeding events and minimise the need for replacement therapy in small populations with haemophilia B. These findings support further study.Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences, National Key Research and Development Program of China, National Natural Science Foundation of China, Tianjin Municipal Science and Technology Commission Grant, and Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences.

Subclinical bleeding is a haemorrhage event not clinically detected in haemophilia, and no reliable method is available for predicting subclinical bleeding. We investigated whether haemophilia mice have subclinical haemorrhage and evaluated potential biomarkers including multiple cytokine changes to predict subclinical haemorrhage. Plasma from naive FVIII-/- and FIX-/- mice and their wild-type counterparts (FVIII WT and FIX WT, respectively) were measured for prothrombin fragment 1 + 2 (F1 + 2) and multiple cytokines. Haemophilia mice with induced hemarthrosis were used as positive clinical bleeding controls. Naive haemophilia mice that displayed higher levels than positive bleeding control were counted. Univariate and multivariate analyses of cytokines were performed. Compared with wild-type mice (FVIII WT 1.1-6.2 vs. FIX WT 2.7-6.7 pmol/l), F1 + 2 widely varied in both haemophilia mouse strains (FVIII-/- 3.7-25.7 vs. FIX-/- 2.7-15.7 pmol/l). Each cytokine varied widely in both naive haemophilia A and B mice, but not significantly, for most cytokines. In comparison to haemophilia mice with hemarthrosis bleeding challenge, naive FVIII-/- mice had elevated pro-inflammatory cytokines and FIX-/- mice had elevated anti-inflammatory cytokines. In addition, interleukin (IL)-4, followed by IL-1, IL-6, TNF-α and MIP-1α in FVIII-/- mice and MIP-1α, followed by IL-1, IL-10 in FVIII-/- mice exhibited significant differences potentially associated with potential subclinical bleeding. Naive haemophilia mice showed elevated pro-inflammatory cytokines with different patterns, represented by pro-inflammatory cytokine elevation in more naive FVIII-/- mice and more anti-inflammatory cytokines in FIX-/- mice.

Neutralizing antibodies (NAbs) strongly limit adeno-associated virus (AAV) vector transduction and repeated administration. Previous studies have shown that NAbs induced by AAVs are associated with T and B cell activation and that the B7/CD28 and CD40/CD40L costimulation signaling pathways are involved. CTLA4 and CD40 are vital molecules that participate in the costimulatory pathway. In this study, we evaluated CTLA4-Ig and CD40-Ig immunosuppressive efficacies through AAV and investigated their effects on the feasibility for multiple systemic administrations of AAV vectors. The results showed that a single administration of AAV vector carrying either CTLA4-Ig alone or with CD40-Ig could greatly reduce the level of NAbs. An AAV serotype-specific immune tolerance could be successfully established, which enabled repeated, i.e., second and third, systemic administration of AAV vectors in the same mice. A combination of CTLA4-Ig and CD40-Ig delivered via AAV vectors significantly inhibited T and B cell activations without affecting immune response to the total immunoglobulin G (IgG) production and cytokines. Interestingly, exogenous gene expression significantly improved after multiple administrations of AAV vector in vivo. Our study generates a reliable and effective method for repeated dosing of AAV vectors that is needed on gene therapy.

Hemarthrosis is the primary cause of hemophiliac arthropathy (HA). Pro-inflammatory cytokines are thought to play an important role in the pathogenesis of HA, and thus, anti-cytokine approaches may be used as an adjuvant therapy. A novel series of enaminone compounds (JODI), that contain the N-aryl piperazino motif, have been shown in vitro to reduce pro-inflammatory cytokines and thus may be efficacious in vivo. In this report, we will assess whether JODI can suppress multiple cytokines which might be potentially responsible for joint inflammation in a mouse model of hemarthrosis. The results showed that JODI significantly improved the survival after LPS treatment, and most pro-inflammatory cytokines/chemokines were decreased significantly after JODI administration. In the hemophilia mouse model, hemarthrosis resulted in local cytokine/chemokine changes, represented by elevated pro-inflammatory (IL-6, MCP-1, MIP-1α, MIP-1β) and pro-angiogenic (VEGF and IL-33) cytokines, and decreased anti-pro-inflammatory cytokines IL-4 and IL-10. The changes were reversed by administration of JODI, which can be used as a novel approach to manage hemophilia arthropathy.

Bleeding into the joints represents the major morbidity of severe hemophilia and predisposes it to hemophilic arthropathy (HA). In a reproducible hemarthrosis mouse model, we found distinct changes in thrombin activity in joint tissue homogenate following exposure of the joint to blood in wide type (WT) and hemophilic B mice. Specifically, at early time points (4 h and 24 h) after hemarthrosis, thrombin activity in WT mice quickly peaked at 4 h, and returned to baseline after 1 week. In hemophilia B mice, there was no/minimal thrombin activity in joint tissues at 4 h and 24 h, whereas at 72 h and thereafter, thrombin activity kept rising, and persisted at a higher level. Nevertheless, prothrombin had not decreased in both WT and hemophilia. The pattern was also confirmed by Western blotting and immunostaining. To optimize the protection against development of HA, we tested different treatment regimens by administration of clotting factor IX into hemophilia B mouse after hemarthrosis induction, including a total of 600 IU/kg FIX within the first 24 h or the whole 2-week period. We concluded that timely (in the first 24 h) and sufficient hemostasis correction is critical for a better protection against the development of hemophilic arthropathy.

Adeno-associated virus (AAV) vectors have been used successfully in clinical trials for patients with hemophilia or blindness, but pre-existing neutralizing antibodies (Nab) are common in the general population and exclude many patients from clinical trials. Exploration of effective strategies to enhance AAV transduction and escape from Nab activity is still imperative. Previous studies have shown the compatibility of capsids from AAV serotypes and homology of recognition sites of AAV Nab located on different capsid subunits from one virion. In this study, we co-transfected AAV2 and AAV8 helper plasmids at different ratios (3:1, 1:1 and 1:3) to assemble haploid capsids and study both their transduction efficiency and Nab escape activity. After muscular injection, all of the haploid viruses induced higher transduction than their parental AAV vectors (2- to 9-fold over AAV2), with the highest of these being the haploid vector AAV2/8 3:1. After systemic administration, a 4-fold higher transduction in the liver was observed with haploid AAV2/8 1:3 than that with AAV8 alone. We then packaged the therapeutic factor IX cassette into haploid AAV2/8 1:3 capsids and injected them into FIX knockout mice via the tail vein. Higher FIX expression and improved phenotypic correction were achieved with the haploid AAV2/8 1:3 virus vector when compared to that of AAV8. Additionally, the haploid virus AAV2/8 1:3 was able to escape AAV2 neutralization and did not increase capsid antigen presentation capacity when compared to AAV8. To improve the Nab evasion ability of the haploid virus, we produced the triploid vector AAV2/8/9 by co-transfecting AAV2, AAV8 and AAV9 helper plasmids at a ratio of 1:1:1. After systemic administration, a 2-fold higher transduction in the liver was observed with the triploid vector AAV2/8/9 than that with AAV8. Nab analysis demonstrated that the triploid AAV2/8/9 vector was able to escape Nab activity from mouse sera immunized with parental serotypes. These results indicate that polyploid viruses might potentially acquire advantages from parental serotypes for enhancement of AAV transduction and evasion of Nab recognition without increasing capsid antigen presentation in target cells. Polyploid AAV vectors can be generated from any AAV serotype, whether natural, rational, library derived or a combination thereof, providing a novel strategy that should be explored in future clinical trials in patients with neutralizing antibodies.

Hemophilia arthropathy (HA) represents the majority of morbidity in severe hemophilia patients, especially in resource-limited countries. Adeno-associated virus (AAV)-mediated gene therapy is showing promise for managing hemophilia. However, patients with neutralizing antibodies (NAbs) against AAV, and inhibitors to clotting factors, are excluded from such therapy. This study explored the feasibility of AAV-mediated local gene therapy for HA. Factor VIII knockout (FVIII

Repeated intra-articular hemorrhages lead to hemophilic arthropathy in severe hemophilia. Inflammation and pro-inflammatory cytokines (e.g., tumor necrosis factor alpha (TNF&alpha, )) might be involved in this pathogenesis. We hypothesized that anti-TNF&alpha, may provide adjuvant protection for hemophilic arthropathy management. We measured TNF&alpha, in synovial lavage from hemophilia mice subjected to hemarthrosis induction and synovial fluid from patients with hemophilic arthropathy (n = 5). In hemophilia mice, recurrent hemarthroses were induced, anti-TNF&alpha, was initiated either from day (D)7 after one hemarthrosis episode or D21 after three hemarthroses episodes (n &ge, 7/treatment group). In patients with hemophilic arthropathy (16 patients with 17 affected joints), a single dose of anti-TNF&alpha, was administered intra-articularly. Efficacy, characterized by synovial membrane thickness and vascularity, was determined. Elevated TNF&alpha, in synovial lavage was found in the hemophilia mice and patients with hemophilic arthropathy. Hemophilia mice subjected to three hemarthroses developed severe synovitis (Synovitis score of 6.0 &plusmn, 1.6). Factor IX (FIX) replacement alone partially improved the pathological changes (Synovitis score of 4.2 &plusmn, 0.8). However, anti-TNF&alpha, treatment initiated at D7, not D21, significantly provided protection (Synovitis score of 1.8 &plusmn, 0.9 vs. 3.9 &plusmn, 0.3). In patients with hemophilic arthropathy, intra-articular anti-TNF&alpha, significantly decreased synovial thickness and vascularity during the observed period from D7 to D30. Collectively, this preliminary study seems to indicate that TNF&alpha, may be associated with the pathogenicity of hemophilic arthropathy and anti-TNF&alpha, could provide adjuvant protection against hemophilic arthropathy. Further studies are required to confirm the preliminary results shown in this study.

While joint damage is the primary co-morbidity of hemophilia, osteoporosis and osteopenia are also observed. Coagulation factor VIII deficient (FVIII−/−) mice develop an osteoporotic phenotype in the absence of induced hemarthrosis that is exacerbated two weeks after an induced joint injury. Here we have compared comprehensively the bone health of clotting factor VIII, factor IX, and Von Willebrand Factor knockout (FVIII−/−, FIX−/−, and VWF−/− respectively) mice both in the absence of joint hemorrhage and following induced joint injury. We found FVIII−/− and FIX−/− mice, but not VWF−/− mice, developmentally have an osteoporotic phenotype. Unilateral induced hemarthrosis causes further bone damage in both FVIII−/− and FIX−/− mice, but has little effect on VWF−/− bone health, indicating that the FVIII.VWF complex is not required for normal bone remodeling in vivo. To further investigate the bone healing following hemarthrosis in hemophilia we examined a two week time course using microCT, serum chemistry, and histological analysis. Elevated ratio of osteoprotegerin (OPG)/receptor activator of nuclear factor-kappa B ligand (RANKL), increased osterix+ osteoblastic cells, and decreased smoothness of the cortical bone surface were evident within several days of injury, indicative of acute heterotopic mineralization along the cortical surface. This was closely followed by increased interleukin-6 (IL-6) levels, increased osteoclast numbers, and significant trabecular bone loss. Uncoupled and disorganized bone formation and resorption continued for the duration of the study resulting in significant deterioration of the joint. Further elucidation of the shared mechanisms underlying abnormal bone homeostasis in the absence of FVIII or FIX is needed to guide evidence-based approaches to the screening and treatment of the prevalent bone defects in hemophilia A and B.

Recent hemophilia B clinical trials using adeno-associated virus (AAV) gene delivery have demonstrated much lower coagulation factor IX (FIX) production in patients compared with the high levels observed in animal models and AAV capsid-specific cytotoxic T lymphocyte response elicited at high doses of AAV vectors. These results emphasize the necessity to explore effective approaches for enhancement of AAV transduction. Initially, we found that incubation of all AAV vectors with human serum enhanced AAV transduction. Complementary analytical experiments demonstrated that human serum albumin (HSA) directly interacted with the AAV capsid and augmented AAV transduction. The enhanced transduction was observed with clinical grade HSA. Mechanistic studies suggest that HSA increases AAV binding to target cells, and that the interaction of HSA with AAV does not interfere with the AAV infection pathway. Importantly, HSA incubation during vector dialysis also increased transduction. Finally, HSA enhancement of AAV transduction in a model of hemophilia B displayed greater than a fivefold increase in vector-derived circulating FIX, which improved the bleeding phenotype correction. In conclusion, incubation of HSA with AAV vectors supports a universal augmentation of AAV transduction and, more importantly, this approach can be immediately transitioned to the clinic for the treatment of hemophilia and other diseases.

At present, the overall level of football in China is not high, but the school sports with "health first" as the guiding ideology, football is a good choice. Compared with professional training bases, ordinary colleges generally do not have expensive training equipment and sophisticated Physiological and biochemical monitoring system, therefore, football training in colleges and universities should also be distinguished from professional football training, but the mechanism of physical quality improvement is the same. All training should be done on the basis of scientific feasibility, and the improvement of physical quality is also required. This study aims to study the physical fitness training of college football in the actual environment of colleges and universities, and realize the soccer fitness training method with the purpose of scientific football training as the means and the improvement of students’ physical quality. At the same time, relying on the background of computer technology, the design concept of a health and fitness network teaching platform based on football projects is proposed.

Background Following induced joint hemorrhage, hemophilia B results in the abnormal persistence of iron deposition, inflammation, and neovascularity of the synovial tissue, as well as deterioration of the bone articular surface and strength. Previously, we demonstrated that a factor IX (FIX) replacement protein with extended circulating FIX activity, glycoPEGylated FIX nonacog beta pegol (N9-GP), could improve synovial and osteochondral parameters in F9 knockout mice when administered after joint injury. Objective We explored the use of N9-GP prior to unilateral joint hemorrhage and compared to unmodified recombinant FIX (rFIX). Methods Pharmacodynamics, histology, and microcomputed tomography were used to assess the effects of prophylactic administration of glycoPEGylated FIX. Results In comparison to rFIX, N9-GP significantly improved soft tissue histological parameters, as well as bone outcome at 2 weeks post injury, while performing equally in reduction of blood present in the joint space assessed 1 day after injury. Conclusions These results indicate that, in comparison to rFIX, the prophylactic use of extended half-life FIX provides superior protection from bleeding-induced joint damage, manifested by improved correction of histologic parameters.

Data from clinical trials for hemophilia B using adeno-associated virus (AAV) vectors have demonstrated decreased transgenic coagulation factor IX (hFIX) expression 6-10 weeks after administration of a high vector dose. While it is likely that capsid-specific cytotoxic T lymphocytes eliminate vector-transduced hepatocytes, thereby resulting in decreased hFIX, this observation is not intuitively consistent with restored hFIX levels following prednisone application. Although the innate immune response is immediately activated following AAV vector infection via TLR pathways, no studies exist regarding the role of the innate immune response at later time points after AAV vector transduction. Herein, activation of the innate immune response in cell lines, primary human hepatocytes, and hepatocytes in a human chimeric mouse model was observed at later time points following AAV vector transduction. Mechanistic analysis demonstrated that the double-stranded RNA (dsRNA) sensor MDA5 was necessary for innate immune response activation and that transient knockdown of MDA5, or MAVS, decreased IFN-�� expression while increasing transgene production in AAV-transduced cells. These results both highlight the role of the dsRNA-triggered innate immune response in therapeutic transgene expression at later time points following AAV transduction and facilitate the execution of effective strategies to block the dsRNA innate immune response in future clinical trials.

Vector capsid dose-dependent inflammation of transduced liver has limited the ability of adeno-associated virus (AAV) factor IX (FIX) gene therapy vectors to reliably convert severe to mild hemophilia B in human clinical trials. These trials also identified the need to understand AAV neutralizing antibodies and empty AAV capsids regarding their impact on clinical success. To address these safety concerns, we have used a scalable manufacturing process to produce GMP-grade AAV8 expressing the FIXR338L gain-of-function variant with minimal (

The development of inhibitory autoantibodies to the infused clotting factor VIII (FVIII) is a major complication for severe hemophilia A management. Novel therapy options for hemophilia have significantly progressed in the last decade, and a gene therapy cure for hemophilia is becoming a reality. However, mechanistic studies of FVIII autoantibodies (FVIII inhibitors) have lagged behind and remain a challenge for both protein replacement and gene therapy. FVIII inhibitor formation is assumed to be a classical T cell–dependent immune response in which cytokines/chemokines play an important role. The study of cytokine profile changes during FVIII inhibitor development may be helpful to understand the mechanism of inhibitor development and to explore potential novel approaches that will minimize the risk. After FVIII–/– mice were treated with intravenous administration of an adeno-associated virus 8 vector encoding human FVIII, FVIII expression peaked at week 2 (W2), and FVIII inhibitor was thoroughly develop...

While therapeutic expression of coagulation factors from adeno-associated virus (AAV) vectors has been successfully achieved in patients with hemophilia, neutralizing antibodies to the vector and inhibitory antibodies to the transgene severely limit efficacy. Indeed, approximately 40% of mice transduced with human factor VIII using the AAV8 serotype developed inhibitory antibodies to factor VIII (FVIII inhibitor), as well as extremely high titers (≥1:500) of neutralizing antibodies to AAV8. To correct hemophilia in these mice, AAV9, a serotype with low in vitro cross-reactivity (≤1:5) to anti-AAV8, was used to deliver mouse-activated factor VII (mFVIIa). It was found that within 6 weeks of systemic administration of 2 × 1013 particles/kg of AAV9/mFVIIa, hemophiliac mice with FVIII inhibitors and neutralizing antibodies (NAb) to AAV8 achieved hemostasis comparable to that in wild-type mice, as measured by rotational thromboelastometry. A level of 737 ng/mL mFVIIa was achieved after AAV9/mFVIIa adminstration compared to around 150 ng/mL without vector treatment, and concomitantly prothrombin time was shortened. Tissues collected after intra-articular hemorrhage from FVIII-deficient mice and mice with FVIII inhibitors were scored 4.7 and 5.5, respectively, on a scale of 0–10, indicating significant pathological damage. However, transduction with AAV9/mFVIIa decreased pathology scores to 3.6 and eliminated hemosiderin iron deposition in the synovium in most mice. Collectively, these results suggest that application of alternative serotypes of AAV vector to deliver bypassing reagents has the potential to correct hemophilia and prevent hemoarthrosis, even in the presence of FVIII inhibitor and neutralizing antibodies to AAV.

While chronic degenerative arthropathy is the main morbidity of haemophilia, a very high prevalence of low bone density is also seen in men and boys with haemophilia. This study investigates bone degradation in the knee joint of haemophilic mice resulting from haemarthrosis and the efficacy of aggressive treatment with factor VIII in the period surrounding injury to prevent bone pathology. Skeletally mature factor VIII knock-out mice were subjected to knee joint haemorrhage induced by puncture of the left knee joint capsule. Mice received either intravenous factor VIII treatment or placebo immediately prior to injury and at hours 4, 24, 48, 72 and 96 after haemorrhage. Mice were killed 2-weeks after injury and the joint morphology and loss of bone in the proximal tibia was assessed using microCT imaging. Quantitative microCT imaging of the knee joint found acute bone loss at the proximal tibia following injury including loss of trabecular bone volumetric density and bone mineral density, as well as trabecular connectivity density, number and thickness. Unexpectedly, joint injury also resulted in calcification of the joint soft tissues including the tendons, ligaments, menisci and cartilage. Treatment with factor VIII prevented this bone and soft tissue degeneration. Knee joint haemorrhage resulted in acute changes in adjacent bone including loss of bone density and mineralization of joint soft tissues. The rapid calcification and loss of bone has implications for the initiation and progression of osteoarthritic degradation following joint bleeding.

Summary Background The most common morbidity that results from hemophilia is bleeding-induced hemophilic arthropathy (HA), which once established may not be interrupted completely even by prophylactic clotting factor replacement. Specific therapies to oppose inflammatory cytokines, including Interleukin 6 (IL-6) receptor antagonists, have become important in the management of inflammatory arthritides. Objectives We investigated combining therapy using MR16-1, a rat IgG antibody directed against mouse IL-6 receptor (anti-IL-6R), with factor VIII (FVIII) replacement to protect against bleeding-induced arthropathy in hemophilia A mice. Methods Three recurrent hemarthroses were induced in the knee joint capsule of FVIII knockout mice. Treatment at the time of each hemorrhage included either: no treatment; FVIII replacement given at the time of hemorrhage; FVIII replacement at hemorrhage plus anti-IL-6R as 4-weekly injections; FVIII replacement with non-specific control antibody (rat IgG); and anti-IL-6R alone without FVIII replacement. Six weeks following the first hemarthosis, joints were harvested and histopathology was scored for synovitis, for cartilage integrity and for macrophage infiltration. Results Animals that received anti-IL-6R as an adjunct to FVIII replacement demonstrated the best survival and the least acute joint swelling and pathology on histologic examination of the synovium and cartilage (P

Wound healing requires interactions between coagulation, inflammation, angiogenesis, cellular migration, and proliferation. Healing in dermal wounds of hemophilia B mice is delayed when compared with hemostatically normal wild-type (WT) mice, with abnormal persistence of iron deposition, inflammation, and neovascularity. We observed healing following induced joint hemorrhage in WT and factor IX (FIX) knockout (FIX-/-) mice, examining also parameters previously studied in an excisional skin wound model. Hemostatically normal mice tolerated this joint bleeding challenge, cleared blood from the joint, and healed with minimal pathology, even if additional autologous blood was injected intra-articularly at the time of wounding. Following hemarthrosis, joint wound healing in hemophilia B mice was impaired and demonstrated similar abnormal histologic features as previously described in hemophilic dermal wounds. Therefore, studies of pathophysiology and therapy of hemophilic joint bleeding performed in hemostatically normal animals are not likely to accurately reflect the healing defect of hemophilia. We additionally explored the hypothesis that the use of a FIX replacement protein with extended circulating FIX activity could improve synovial and osteochondral wound healing in hemophilic mice, when compared with treatment with unmodified recombinant FIX (rFIX) in the established joint bleeding model. Significantly improved synovial wound healing and preservation of normal osteochondral architecture are achieved by extending FIX activity after hemarthrosis using glycoPEGylated FIX when compared with an equivalent dose of rFIX. These results suggest that treating joint bleeding only until hemostasis is achieved may not result in optimal joint healing, which is improved by extending factor activity.

Delivery of genes that are larger than the wild-type adeno-associated virus (AAV) 4,681 nucleotide genome is inefficient using AAV vectors. We previously demonstrated in vitro that concurrent proteasome inhibitor (PI) treatment improves transduction by AAV vectors encoding oversized transgenes. In this study, an AAV vector with a 5.6 kilobase (kb) factor VIII expression cassette was used to test the effect of an US Food and Drug Administration–approved PI (bortezomib) treatment concurrent with vector delivery in vivo. Intrahepatic vector delivery resulted in factor VIII expression that persisted for >1 year in hemophilia mice. Single-dose bortezomib given with AAV2 or AAV8 factor VIII vector enhanced expression on average ~600 and ~300%, respectively. Moreover, coadministration of AAV8.canineFVIII (1 × 1013 vg/kg) and bortezomib in hemophilia A dogs (n = 4) resulted in normalization of the whole blood clotting time (WBCT) and 90% reduction in hemorrhages for >32 months compared to untreated hemophilia A dogs (n = 3) or dogs administered vector alone (n = 3). Demonstration of long-term phenotypic correction of hemophilia A dogs with combination adjuvant bortezomib and AAV vector expressing the oversized transgene establishes preclinical studies that support testing in humans and provides a working paradigm to facilitate a significant expansion of therapeutic targets for human gene therapy.

Data to characterize novel anti-inflammatory agent enaminone E121 have shown a marked decrease in the release of pro-inflammatory cytokines in macrophages stimulated with lipopolysaccharide (LPS) compared to the control (DMSO) group. Literature has described that when an N-alkylated piperazine motif is added to the terminal end of the lead compound, it is able to increase the CCR2 antagonistic activity. We hypothesized that incorporating the N-alkylated piperazine motif into the existing E-121 enaminone pharmacophore could lead to a ligand with dual functionality as a cytokine inhibitor and chemokine receptor antagonist. The resultant series (JODI) were studied in vitro, and data suggest they can suppress TNF-alpha and IL-6 in a dose-dependent manner, superior to that of dexamethasone. Additionally, macrophages stimulated with chemokine MCP-1 and treated with JODI compounds, (but not E121), are able to block CCR2 signaling as evidenced by decreased total ERK1/2. To determine if JODI-19 has activity in vivo, it was utilized in a mouse model of hemarthrosis to assess decreased inflammation as evidenced by local cytokine suppression. In a pilot study, FIX−/−mice were subjected to hemarthrosis induction with or without JODI administration. At different timepoints from four hours to four weeks after hemarthrosis, synovial fluid was collected by lavage for multiple cytokine analyses. Administration of JODI-19 significantly decreased TNFα, IL-1α, MCP-1 and MIP-1α levels in the synovial fluid. As such, JODI-19 may be a novel approach to better manage hemophilia arthropathy and its associated bone damage.

Hemophilic bleeding into joints causes synovial and microvascular proliferation and inflammation (hemophilic synovitis) that contribute to end-stage joint degeneration (hemophilic arthropathy), the major morbidity of hemophilia. New therapies are needed for joint deterioration that progresses despite standard intravenous (IV) clotting factor replacement. To test whether factor IX within the joint space can protect joints from hemophilic synovitis, we established a hemophilia B mouse model of synovitis. Factor IX knockout (FIX−/−) mice received a puncture of the knee joint capsule with a needle to induce hemarthrosis; human factor IX (hFIX) was either injected through the needle into the joint space (intraarticularly) or immediately delivered IV. FIX−/− mice receiving intraarticular FIX protein were protected from synovitis compared with mice receiving same or greater doses of hFIX IV. Next, adeno-associated virus (AAV) gene transfer vectors expressing hFIX were injected into knee joints of FIX−/− mice. Joints treated with 1010 vector genomes (vg)/joint AAV2-, AAV5-, or AAV8-hFIX or 2.5 × 109 vg/joint AAV5-hFIX developed significantly fewer pathologic changes 2 weeks after injury compared with the pathology of control injured contralateral hind limbs. Extravascular factor activity and joint-directed gene transfer may ameliorate hemophilic joint destruction, even in the absence of circulating FIX.

Self-complementary adeno-associated virus (scAAV) vectors can significantly minimize the vector load required to achieve sustained transgene expression. In this study, transcriptional regulatory elements were systematically screened to produce constitutive and liver-specific scAAV factor IX (FIX) expression cassettes. In addition, optimization of GC content, cis- regulatory elements, and codon usage in the human FIX (hFIX) transgene increased expression 4-20-fold. A vector was developed that was capable of expressing high FIX levels in comparison with the single-stranded (ss) AAV vector used in a recent clinical trial. The ssAAV and scAAV vectors display different transgene expression and genome stability patterns in the liver, as determined by immunohistochemical staining, in situ messenger RNA (mRNA) hybridization and vector genome quantitation. The ssAAV2 vector promoted strong FIX expression in only a subset of hepatocytes. The scAAV2-hFIX vector showed widespread ( approximately 80% of hepatocytes), moderate FIX expression levels similar to normal livers with correction of coagulation function in FIX-deficient mice. The ability of low dose scAAV-FIX vectors to achieve near-physiological expression may circumvent inflammatory responses in the liver. In addition to providing an improved scAAV vector for potential application in future hemophilia B clinical trials and liver-directed gene delivery, these studies underscore the need for rigorous analysis and optimization of vector genome cassettes.

Glycans are key determinants of host range and transmissibility in several pathogens. In the case of adeno-associated viruses (AAV), different carbohydrates serve as cellular receptors in vitro ; however, their contributions in vivo are less clear. A particularly interesting example is adeno-associated virus serotype 9 (AAV9), which displays systemic tropism in mice despite low endogenous levels of its primary receptor (galactose) in murine tissues. To understand this further, we studied the effect of modulating glycan binding avidity on the systemic fate of AAV9 in mice. Intravenous administration of recombinant sialidase increased tissue levels of terminally galactosylated glycans in several murine tissues. These conditions altered the systemic tropism of AAV9 into a hepatotropic phenotype, characterized by markedly increased sequestration within the liver sinusoidal endothelium and Kupffer cells. In contrast, an AAV9 mutant with decreased glycan binding avidity displayed a liver-detargeted phenotype. Altering glycan binding avidity also profoundly affected AAV9 persistence in blood circulation. Our results support the notion that high glycan receptor binding avidity appears to impart increased liver tropism, while decreased avidity favors systemic spread of AAV vectors. These findings may not only help predict species-specific differences in tropism for AAV9 on the basis of tissue glycosylation profiles, but also provide a general approach to tailor AAV vectors for systemic or hepatic gene transfer by reengineering capsid-glycan interactions.

The initiation of coagulation via tissue factor/factor VIIa is relatively weak in normal synovium and joint tissues, so that hemophilic patients with additional deficiency of the intrinsic pathway of coagulation are especially at risk for joint bleeding and the development of hemophilic synovitis. The inflamed joint that results from recurrent bleeding-induced injury, however, may be an environment in which pharmacologic doses of factor VIIa have potentially greater procoagulant action than in the uninjured joint. There is accumulating evidence suggesting that coagulation factors in the extravascular space, and not only circulating plasma factors, have the potential to contribute to hemostatic protection of joints. The potential role of extravascular factor VIIa is considered.

Von Hippel-Lindau (VHL) inactivation develops a tumor syndrome characterized by highly vascularized tumors as a result of hypoxia inducible factors (HIF) stabilization. The most common manifestation is the development of hemangioblastomas typically located in the central nervous system and other organs including the liver. PTEN (Phosphatase and tension homologue deleted on chromosome 10) inactivation also upregulates HIF-1alpha and may take part in promoting vascular lesions in tumors. The coordinate effect of loss of these tumor suppressors on HIF levels, and the subsequent effect on vascular lesion formation would elucidate the potential for mechanisms to modify HIF dosage supplementally and impact tumor phenotype. We therefore employed models of somatic conditional inactivation of Vhl, Pten, or both tumor suppressor genes in individual cells of the liver by Cre-loxP recombination to study the cooperativity of these two tumor suppressors in preventing tumor formation. Nine months after tumor suppressor inactivation, Vhl conditional deletion (Vhl (loxP/loxP)) mice showed no abnormalities, Pten conditional deletion (Pten (loxP/loxP)) mice developed liver steatosis and focal nodular expansion of hepatocytes containing lipid droplet and fat. Vhl and Pten conditional deletion (Vhl (loxP/loxP);Pten (loxP/loxP)) mice, however, developed multiple cavernous liver lesions reminiscent of hemangioblastoma. Liver hemangioblastomas in VHL disease may, therefore, require secondary mutation in addition to VHL loss of heterozygosity which is permissive for vascular lesion development or augments levels of HIF-1alpha.

Massive hepatectomy (MHX) leads to failure of remnant livers. Excessive metabolic burden in remnant livers may cause mitochondrial dysfunction. This study investigated whether blockade of the mitochondrial permeability transition (MPT) with N-methyl-4-isoleucine cyclosporine (NIM811) improves the outcome of MHX.Mice were gavaged with NIM811 (10 mg/kg before surgery and 5 mg/kg daily afterward) and underwent sham-operation or approximately 90% partial hepatectomy.Serum alanine aminotransferase, necrosis, and apoptosis increased, respectively, to approximately 1200 U/L, 6.1%, and 7% after MHX. NIM811 decreased peak alanine aminotransferase release, necrosis, and apoptosis by 70%, 100%, and 42%, respectively. 5-Bromo-2'-deoxyuridine incorporation, proliferating cell nuclear antigen expression, and the remnant liver weights were all increased significantly by NIM811 treatment, indicating improved liver regeneration. NIM811 also blunted hyperbilirubinemia by 54%, increased serum albumin by 51%, and improved survival from 6% to 40% after MHX. Hepatic mitochondrial depolarization, cell death, and MPT were detected by intravital confocal/multiphoton microscopy of rhodamine 123, propidium iodide, and calcein. Mitochondrial depolarization occurred in many viable hepatocytes (13 cells/high-power field), and nonviable hepatocytes increased slightly to approximately 1 cell/high-power field at 3 hr after MHX. Entry of calcein into mitochondria after MHX indicated MPT onset. Importantly, NIM811 decreased mitochondria depolarization by more than 60%, blocked MPT onset, and prevented cell death. Decreases of hepatic ATP, mitochondrial cytochrome c release, and caspase-3 activation after MHX were also partially blocked by NIM811.NIM811 minimized liver injury and improved liver regeneration after MHX, at least in part, by preventing MPT onset and subsequent compromised energy supply and proapoptotic cytochrome c release.

The immune response has been implicated as a critical factor in determining the success or failure of clinical gene therapy trials. Generally, such a response is elicited by the desired transgene product or, in some cases, the delivery system. In the current study, we report the previously uncharacterized finding that a therapeutic cassette currently being used for human investigation displays alternative reading frames (ARFs) that generate unwanted protein products to induce a cytotoxic T lymphocyte (CTL) response. In particular, we tested the hypothesis that antigenic epitopes derived from an ARF in coagulation factor IX (F9) cDNA can induce CTL reactivity, subsequently killing F9-expressing hepatocytes. One peptide (p18) of 3 candidates from an ARF of the F9 transgene induced CD8 + T cell reactivity in mice expressing the human MHC class I molecule B0702. Subsequently, upon systemic administration of adeno-associated virus (AAV) serotype 2 vectors packaged with the F9 transgene (AAV2/F9), a robust CD8 + CTL response was elicited against peptide p18. Of particular importance is that the ARF epitope-specific CTLs eliminated AAV2/F9-transduced hepatocytes but not AAV2/F9 codon-optimized (AAV2/F9-opt)-transduced liver cells in which p18 epitope was deleted. These results demonstrate a previously undiscovered mechanism by which CTL responses can be elicited by cryptic epitopes generated from a therapeutic transgene and have significant implications for all gene therapy modalities. Such unforeseen epitope generation warrants careful analysis of transgene sequences for ARFs to reduce the potential for adverse events arising from immune responses during clinical gene therapy protocols.

Background Recurrent bleeding into joints results in a crippling arthritis, the consequence of intra-articular (IA) pathologic lysed red blood cell iron and inflammatory signals from macrophages/monocytes. Hemophilic mice that experience joint bleeding develop synovial and osteochondral changes that mimic the changes seen in human hemophilia. We previously examined in hemophilia B mice the efficacy of conventional recombinant factor IX (rFIX) compared to a half-life extended polyethyleneglycol-conjugated factor IX (N9-GP) to support wound healing when administered "on demand" after the initiation of induced joint hemorrhage. In the "on-demand" scenario, N9-GP improved wound healing when compared to rFIX, although neither treatment group demonstrated completely normal joint healing (as observed in hemostatically normal mice that experienced the same injury). We test the hypothesis that the prophylactic use of N9-GP may support wound healing in synovium, cartilage, and bone and may do so more effectively than rFIX. Method: FIX deficient mice were treated prophylactically with either rFIX or N9-GP at the dose of 250 IUnits human factor IX/kg 30 minutes, 24hr or 72hr prior to the induction of unilateral hemarthosis. Injured hemostatically normal (iWT) mice were treated with I.V. normal saline as the controls. Joint diameter was measured daily to quantify joint swelling. Plasma and tissues were harvested from cohorts of mice (n = 8-10/group) at two weeks after hemarthrosis. Pathologic synovial hyperplasia, neovascularization, iron deposition and macrophage infiltration of the synovium were quantified by histologic examination. Integrity of trabecular bone neighboring the hemarthrosis was quantified by microComputed Tomography (mCT). Result: WT mice developed no detectable joint swelling at days 1-14 after the joint bleeding challenge, whereas mice treated prophylactically with rFIX were protected from joint swelling only in the cohort treated 24 hours prior to hemarthrosis. Joint swelling did not develop in groups that received prophylactic N9-GP using any of the 3 schedules. No mouse treated with rFIX (n = 28 total) or N9-GP (n=27 total) developed FIX inhibitors or FIX-binding IgG. Synovitis was graded 0 to 10 by Valentino mouse hemophilic synovitis score, with a mean score in the iWT mice of 1.0±0.5 and in the injured untreated FIX-/- mice of 4.9±1.4. Synovitis was greatly diminished although not eliminated by prophylactic N9-GP given 30 minutes prior to injury (1.7±1.2), and this result contrasted with the relatively less good healing provided by rFIX given 30 minutes prior to injury (3.9±1.1). Synovitis, iron staining, and persistence of macrophages within the joint were each diminished by N9-GP and rFIX prophylaxis. The degree of protection diminished as the interval between dosing and injury increased. Better protection was seen using N9-GP when compared to rFIX at each timepoint. Strikingly, rFIX at this dose provided minimal or no prophylactic protection from synovial neoangiogenesis (vessels/hpf) at two weeks after hemarthrosis even when given at the shortest interval preceding the injury. Injured, untreated FIX-/- mice developed 17.6±5.1 vessels/hpf compared to rFIX given 30 minutes prior to injury (16.1±4.8), 24 hours prior (17.8±5.4) or 72 hours prior (17.1±6.3). In contrast, iWT mice displayed 7.1±3.1 at two weeks after injury, which was comparable to N9-GP given 30 minutes prior to injury (9.4±2.4) and better than N9-GP given 24 hours prior (12.6±3.3) or 72 hours prior (13.1±5.6). Consistent with previous reports, hemophilic mice when compared to iWT mice demonstrated acute losses of bone mineral density in the bone adjacent to the joint experiencing bleeding, as well as structural defects in the number, thickness, and spacing of the bone trabeculae. Prophylactic rFIX or N9-GP given at 30 minutes or 24 hours prior to injury resulted in protection that did not differ statistically from findings in iWT mice. When prophylaxis was given at 72 hours prior to injury, significant volumetric bone mineral density loss of at least 20% was evident in the rFIX group (vBMD 98 mgHA) compared to iWT (131 mgHA) and N9-GP given 72 hours prior (125 mgHA). Conclusion: The support of normal wound healing in joint and bone following hemarthrosis was significantly improved by N9-GP prophylaxis when compared to rFIX in hemophilia B mice, with neoangiogenesis particularly improved. Disclosures Knudsen: Novo Nordisk: Employment. Monroe:Novo Nordisk: Honoraria, Research Funding. Ezban:Novo Nordisk: Employment. Bateman:Novo Nordisk: Honoraria, Research Funding; Baxter: Honoraria, Research Funding. Monahan:CSL Behring: Consultancy, Honoraria; Pfizer: Honoraria; Chatham LLC: Consultancy; Baxter/Baxalta: Consultancy, Honoraria, Research Funding; Prolor: Research Funding; Bayer: Consultancy; Novo Nordisk: Consultancy, Honoraria, Research Funding; Asklepios BioPharmaceutical: Consultancy, Patents & Royalties: Author I.P. licensed by UNC to AskBio, Research Funding.

Abstract 200 The development of neutralizing alloantibodies (inhibitors) against factor IX (FIX) in humans with hemophilia B is a potentially devastating complication; clinical investigation and evidence-based intervention for FIX inhibitors is complicated by their rarity. Current hemophilia B mouse models are not ideal for investigating the potential immunogenicity of hemophilia B therapies due to differences in mouse and human MHC class II structure and FIX epitope recognition. This recognition depends on CD4+ T cells and their direct interaction with surface-expressed MHC class II molecules complexed with antigen. We have developed a series of mouse lines that do not express mouse FIX or mouse MHC II, but instead express human FIX genes and humanized MHC II. The potential immunogenicity of human FIX and specific hFIX epitopes for CD4+ T cell recognition have been characterized in each of these “double humanized” (2H) mouse lines. Our derivation of the 2H mouse lines, all having first been bred through a complete knockout of mouse MHCII (mMHC II−/−), resulted in four lines of human HLA-DRB1*1501 mice. These four lines include 1) mouse FIX−/− (complete deletion; Cross-reacting material negative (CRM-)); 2) human R29X (expresses human FIX gene with early nonsense mutation; CRM-); 3) human R333Q (expresses human factor IX defective circulating protein with missense mutation; CRM+), and 4) human WT FIX (WT2 expresses hemostatic human FIX; CRM+). All lines were first shown to have similar numbers of MHCII and CD4+ expressing T cells in peripheral blood based on flow cytometry when compared with conventional (mouse MHC H2b) hemophilia B mice. To determine whether (hu-) mice have the same incidence and degree of immune response to hFIX compared to conventional hemophilia B mice, we immunized subcutaneously with hFIX emulsified in CFA. Importantly, control mMHC II−/− mice did not develop anti-FIX antibodies. Both strains of CRM- 2H hu-mice developed inhibitors and anti-hFIX IgG antibodies with similar intensity. CD4+-enriched splenocytes proliferated in vitro in response to hFIX challenge and secreted high amount of Th1-derived IFN-γ and moderate Th2 (IL-4 and IL-10) cytokines. However, when compared to the inhibitor-prone strains, conventional (mouse MHC H2b) and hu-DRB1*1501 CRM+ mice (both R333Q and WT2) tolerated hFIX, had larger total Treg populations, increased TGF-β1 cytokine, and demonstrated regulatory apoptosis of CD4+ Teffector cells. Additional lymphocyte response studies mapped four peptides derived from FIX catalytic domain as responsible for anti-FIX antibodies in the CRM- lines. (CD4+ T cells from CRM+ animals (hu-R333Q or hu-WT2 mice) did not proliferate to these four or any other hFIX peptides). The peptides identified in 2H hu- mice differed from immunostimulatory peptides identified in reported epitope mapping studies using FIX−/− mice having mouse MHC H2b, H2d, and H2kbackgrounds. These differences may reflect the fundamental structural and epitope recognition differences between murine and human MHC class II complexes. Finally, we have examined systemic and muscle-directed AAV-mediated gene therapy expressing either WT hFIX or a hFIX with a single amino acid gain of function variant (FIX Padua, R338LFIX). We have confirmed that expression of this single amino acid change does not increase inhibitor risk, as modeled in this double humanized hemophilia B model. This observation appears consistent with the extreme rarity of clinical inhibitor development in patients having underlying FIX missense mutations. In summary, the series of double humanized hemophilia B mouse models have hFIX recognition profiles that make them important tools for preclinical evaluation of novel hemophilia B therapeutics and to advance understanding of FIX inhibitors. Disclosures: No relevant conflicts of interest to declare.

Abstract 3124 Persistent factor IX expression and phenotypic improvement have been achieved in a human clinical trial for hemophilia B using liver-directed adeno-associated virus (AAV) gene therapy vectors. An ongoing clinical trial uses a vector incorporating self-complementing AAV (scAAV) genome form, factor IX codon optimization (FIXopt) and AAV serotype 8 capsid. As was seen in a previous single-strand AAV serotype 2 trial, dose escalation has been associated with apparent immune-mediated transient inflammation of vector-transduced liver, although in contrast to the previous trial persistent FIX expression has been maintained for the first time. Taken together, these important trials define a consistent threshold load of AAV capsid that has stimulated capsid-specific cytotoxic lymphocyte recognition and potential transaminitis. To advance the successes achieved in these trials while providing a clear margin of safety so that this immunogenic threshold need not be approached, we have pursued steps to limit further the AAV capsid load. Single amino acid substitutions at arginine 338 in the FIX catalytic domain generate FIX variants with increased specific activity. We separately substituted either R338A, R338Q, or R338L (FIX Padua) into a codon optimized human factor IX cDNA and evaluated F.IX expression in tissue culture following plasmid DNA transfection of HEK 293t cells. Each R338 substitution improved FIX specific activity, up to 10 times increased over wild type using the R338LFIXopt cDNA. We next generated scAAV8 vectors incorporating a liver-specific transthyretin (TTR) promoter to express optimized codon F.IX cDNA with or without the R338L substitution. FIX−/− mice receiving portal vein injection of 1 × 1010 vg/animal (4 ×1011 vg/kg) expressed 86.5% of normal FIX activity at 2 months post-transduction from the WTopt vector and 330% normal from the R338LFIXopt. Incorporation of R338Lopt variant resulted in at least 6 to 10 fold increase in FIX specific activity over a follow-up of > 40 weeks. At ten months following FIX gene delivery, mice underwent a tail transection bleeding challenge. FIX vector mice demonstrated therapeutic protection from this major bleeding challenge and furthermore all survived with no late rebleeding (a hallmark of hemophilic phenotype). Greater than 100% normal human FIX activity was maintained for >40 weeks following treatment with the R338LFIX vector (v. 26.3% at euthanasia in WTopt vector group). The prolonged follow-up permitted extended safety evaluation. Factor IX inhibitor antibodies were not detected in any mice throughout the follow-up; FIX-binding IgG1 and IgG2 were negative also. Thrombin/antithrombin III complexes (TAT) examined at 12 weeks and at >30 weeks of age in R338LFIXopt vector mice did not differ from levels in WTFIXopt vector-treated or age-matched C57Bl/6 hemostatically normal mice. Necropsy at 40–44 weeks after vector (1 year of age) showed only age-related changes with no microvascular or macrovascular thrombosis on H&E staining or specific immunostaining for fibrin/fibrinogen deposition; specific staining for fibrosis within myocardium or other sites was negative. We next synthesized a R338LFIXopt expression cassette containing the LP1 promoter/enhancer/intron sequence being used in the ongoing clinical trial and demonstrated equivalent FIX activity from either promoter construct. We then established that the R338LFIXopt vector gives a predictable dose-response across a range of doses as low as 1x 1010 vg/kg I.V. and as high as 4 × 1012 vg/kg I.V. Hemarthrosis is the most common bleeding complication in hemophilia and leads to chronic joint destruction. Bleeding was induced in the joint of FIX−/− mice that had been transduced 4 weeks earlier with the R338LFIX vector. Joints were collected at 2 weeks after induced bleed and the bleeding-induced joint damage was graded using an established histologic score. I.V. R338LFIXopt vector pretreatment resulted in protection against joint degeneration in a dose-dependent fashion in this most relevant clinical scenario. These preclinical studies demonstrate a safety :efficacy profile to advance hemophilia gene therapy using the scAAV8.R338LFIXopt vector. Disclosures: Monahan: Baxter: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asklepios BioPharmaceutical: Patents & Royalties, Research Funding; CSL Behring: Honoraria; NovoNordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees; PharmaIN: Research Funding; Prolor-Biotech: Research Funding. McPhee:Asklepios Biopharmaceutical: Employment. Samulski:Asklepios Biopharmaceutical: Employment, Patents & Royalties.

Objective: Progressive destruction of joints resulting from recurrent intra-articular haemorrhage represents the major morbidity resulting from haemophilia A or B. In addition to systemic clotting factor replacement, therapies localized to haemophilic joints may provide adjunctive protection. In a factor VIII-/- mice model, we investigated if extra-vascular delivery of recombinant human clotting factor VIII (rhFVIII) via intra-articular (IA) injection can prevent bleeding-induced joint damage, and also examined the possibility that IA delivery of FVIII carries greater risk of developing anti-rhFVIII inhibitor antibody. Methods: FVIII-/- mice received rhFVIII by inserting a 30.5 G needle into the left knee joint, along with a range doses of FVIII(100, 25 and 5 IU kg−1) in 5 μL, normal saline as the control. Comparison group received the same needle injury and intravenous (IV) rhFVIII (100, 25 and 5 IU kg−1). 14 days after injury, both knee joints were collected for histological examination. To exclude the possibility that IA clotting factor was entering into circulation, mice received 100 IU kg−1 rhFVIII IA, and FVIII activity was measured by aPTT. To see if IA rhFVIII delivery can carry greater risk of developing anti-FVIII antibody, mice were treated with a total dose of 300 IU kg−1 rhFVIII over 10 days, either by IA or IV. 14 days after exposure, anti-FVIII was detected. After induction of anti-FVIII antibody by IV rhFVIII, mice were subjected either to needle puncture under coverage of bypassing agent (FEIBA) 100 IU kg−1 or 100 IU kg−1 IV rhFVIII, or needle puncture with 25 IU kg−1 rhFVIII. Control mice received needle puncture with normal saline. Two weeks later, knee joints were collected for histological examination. Summary: Mice receiving only saline at the time of needle puncture developed synovitis (mean score 5.0 ± 0.5). Mice treated with 25 IU kg−1 IA rhFVIII developed better protection than mice treated with 100 IU kg−1 IV rhFVIII (lower pathology score for IA, 0.733 ± 0.278 vs. IV 2.57 ± 1.70) and even better protection was achieved by the dose of 100IU IU kg−1 IA (Pathology score of 0.25 ± 0.31). IA injection of 100 IU kg−1 rhFVIII did not lead to increased circulating FVIII activity at any time point up to 48 h. In IV-treated mice, 100% of mice developed anti-FVIII antibody (8.06BU), while only 50% of mice developed anti-FVIII inhibitor at the lowest detection limit (0.61BU). In the presence of inhibitory antibody, only 46% of mice receiving IV FVIII survived the needle injury, 58% with FEIBA and 100% of mice survived with 25 IU kg−1 FVIII IA injection. In the saline-injected control mice, needle injury led to a mean pathology score of 6.8. Neither IV FVIII nor FEIBA provided effective protection, with pathology scores of 6.3 and 5.4, respectively. Surprisingly, 25 IU kg−1 IA rhFVIII produced a pathology score of only 1.7. Conclusion: Extravascular rhFVIII in the joint space can contribute protection against bleeding-induced joint damage. Intra-articular rhFVIII delivery did not induce greater risk of inhibitory antibody formation in FVIII knockout mice than circulating factor VIIII challenge; in fact, a lower incidence was observed. In the presence of anti-FVIII inhibitory antibodies, IA delivery of FVIII still can offer protection from bleeding-induced joint damage.

Hemophilic arthropathy is the major morbidity of congenital factor VIII and IX deficiency. Therapies localized to hemophilic joints could provide adjunctive protection, in addition to that provided by systemic factor replacement. However, the ability of extravascular clotting factors to contribute to hemostatic protection within joint tissue is unknown. We hypothesized that replacing deficient factor VIII or IX within the injured joint capsule of mice with hemophilia A (FVIII −/ −) or hemophilia B (FIX −/ −), respectively, would decrease the progression of synovitis. We developed a bleeding model consisting of a unilateral knee joint capsule needle puncture to induce hemorrhage in hemophilic mice. Pathology of the joint at two weeks after the injury is graded 0 to 10 using a murine hemophilic synovitis grading system (Valentino, Hakobyan. Haemophilia, 2006). Hemostatically normal mice do not develop synovitis following this injury, but > 95% of FIX −/ − mice develop bleeding and synovitis with a mean grade of 3–4 or greater. Coincident with needle puncture, recombinant human coagulation factor doses ranging from 0 to 20 IU/kg body weight of factor IX or 0 to 25 IU/kg of factor VIII were instilled intraarticularly (I.A.). Comparison groups received the same injury and intravenous (I.V.) factor IX or VIII doses of 25 IU/kg to 100 IU/kg (n= 4–7 mice per study group). Joint bleeding phenotype of the two strains of mice was similar. Mice receiving only saline injection at the time of needle puncture developed mean synovitis scores of 5 ±0.5 in the FVIII −/ − mice and 6 ±0.5 in the FIX −/ − mice. Protection by human clotting factor in the mouse coagulation system was incomplete; mice receiving 100 IU/kg I.V. of factor VIII or factor IX developed synovitis scores of 2.6 ± 1.7 and 2.1 ± 0.2, respectively. In contrast, pathology grade of FVIII −/ − mice dosed with 25 IU/kg I.A. was 0.67 ± 0.3 (p = 0.05 for comparison of 25 IU/kg I.A. with 100 IU/kg IV); FIX−/ − mice receiving 20 IU/kg I.A. had synovitis scores of 0.45 ± 0.58 (p < 0.01 for comparison of 25 IU/kg I.A. with 100 IU/kg I.V.). We next ruled out the possibility that I.A. factor was entering the circulation, and via that route resulting in joint protection, either through technical error at the time of injection, or from a depot effect in the joint with late equilibration into the circulation. Additional groups of mice received factor VIII or IX intravenously at 100 IU/kg, or intraarticularly at 4 times the doses used in the hemarthrosis challenge (80 IU/kg FIX or 100 IU/kg FVIII), and factor activity assays were performed at 1, 4, 12, 24, and 48 hours. Expected circulation kinetics were seen following I.V. dosing; no increase in circulating factor VIII or IX activity were seen in the intraarticular dosing groups at any timepoint. In considering the potential immunogenicity of an intraarticular therapy approach for hemophilic joint therapy, factor VIII −/ − mice were treated with three doses of human factor VIII 100 IU/kg at five day intervals either I.V. or I.A. At two weeks after exposure, 5/5 I.V.-treated mice developed inhibitor antibodies with titers ranging 0.8–7.2 BU; 2/5 I.A.-treated mice had detectable low-titer antibodies (1.3 BU), indicating no greater immunogenicity in the I.A. model. Extravascular factor VIII and factor IX can contribute to protection against blood-induced joint deterioration; enhancing local tissue hemostasis with protein or gene therapy may prove a useful adjunct to systemic replacement.