Phase 1-2 Trial of AAVS3 Gene Therapy in Patients with Hemophilia B.

BACKGROUND: FLT180a (verbrinacogene setparvovec) is a liver-directed adeno-associated virus (AAV) gene therapy that uses a synthetic capsid and a gain-of-function protein to normalize factor IX levels in patients with hemophilia B. METHODS: In this multicenter, open-label, phase 1-2 trial, we assessed the safety and efficacy of varying doses of FLT180a in patients with severe or moderately severe hemophilia B (factor IX level, ≤2% of normal value). All the patients received glucocorticoids with or without tacrolimus for immunosuppression to decrease the risk of vector-related immune responses. After 26 weeks, patients were enrolled in a long-term follow-up study. The primary end points were safety and efficacy, as assessed by factor IX levels at week 26. RESULTS: Ten patients received one of four FLT180a doses of vector genomes (vg) per kilogram of body weight: 3.84×1011 vg, 6.40×1011 vg, 8.32×1011 vg, or 1.28×1012 vg. After receiving the infusion, all the patients had dose-dependent increases in factor IX levels. At a median follow-up of 27.2 months (range, 19.1 to 42.4), sustained factor IX activity was observed in all the patients except one, who resumed factor IX prophylaxis. As of the data-cutoff date (September 20, 2021), five patients had normal factor IX levels (range, 51 to 78%), three patients had levels from 23 to 43%, and one had a level of 260%. Of the reported adverse events, approximately 10% were related to FLT180a and 24% to immunosuppression. Increases in liver aminotransferase levels were the most common FLT180a-related adverse events. Late increases in aminotransferase levels occurred in patients who had received prolonged tacrolimus beyond the glucocorticoid taper. A serious adverse event of arteriovenous fistula thrombosis occurred in the patient with high factor IX levels. CONCLUSIONS: Sustained factor IX levels in the normal range were observed with low doses of FLT180a but necessitated immunosuppression with glucocorticoids with or without tacrolimus. (Funded by Freeline Therapeutics; ClinicalTrials.gov numbers, NCT03369444 and NCT03641703; EudraCT numbers, 2017-000852-24 and 2017-005080-40.).

Preclinical evaluation of FLT190, a liver-directed AAV gene therapy for Fabry disease.

Fabry disease is an X-linked lysosomal storage disorder caused by loss of alpha-galactosidase A (α-Gal A) activity and is characterized by progressive accumulation of glycosphingolipids in multiple cells and tissues. FLT190, an investigational gene therapy, is currently being evaluated in a Phase 1/2 clinical trial in patients with Fabry disease (NCT04040049). FLT190 consists of a potent, synthetic capsid (AAVS3) containing an expression cassette with a codon-optimized human GLA cDNA under the control of a liver-specific promoter FRE1 (AAV2/S3-FRE1-GLAco). For mouse studies FLT190 genome was pseudotyped with AAV8 for efficient transduction. Preclinical studies in a murine model of Fabry disease (Gla-deficient mice), and non-human primates (NHPs) showed dose-dependent increases in plasma α-Gal A with steady-state observed 2 weeks following a single intravenous dose. In Fabry mice, AAV8-FLT190 treatment resulted in clearance of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) in plasma, urine, kidney, and heart; electron microscopy analyses confirmed reductions in storage inclusion bodies in kidney and heart. In NHPs, α-Gal A expression was consistent with the levels of hGLA mRNA in liver, and no FLT190-related toxicities or adverse events were observed. Taken together, these studies demonstrate preclinical proof-of-concept of liver-directed gene therapy with FLT190 for the treatment of Fabry disease.

The cost-effectiveness of gene therapy for severe hemophilia B: a microsimulation study from the United States perspective.

Adeno-associated virus (AAV)-mediated gene therapy is a novel treatment promising to reduce morbidity associated with hemophilia. Although multiple clinical trials continue to evaluate efficacy and safety, limited cost-effectiveness data have been published. This study compared the potential cost-effectiveness of AAV-mediated factor IX (FIX)-Padua gene therapy for patients with severe hemophilia B in the United States vs on-demand FIX replacement and primary FIX prophylaxis, using either standard or extended half-life FIX products. A microsimulation Markov model was constructed, and transition probabilities between health states and utilities were informed by using published data. Costs were aggregated by using a microcosting approach. A time horizon from 18 years old until death, from the perspective of a third-party payer in the United States, was conducted. Gene therapy was more cost-effective than both alternatives considering a $150 000/quality-adjusted life-year threshold. The price for gene therapy was assumed to be $2 000 000 in the base case scenario; however, one of the 1-way sensitivity analyses was conducted by using observed manufacturing, administration, and 5-year follow-up costs of $87 198 for AAV-mediated gene therapy vector as derived from the manufacturing facility and clinical practice at St Jude Children's Research Hospital. One-way sensitivity analyses revealed 10 of 102 scenarios in which gene therapy was not cost-effective compared with alternative treatments. Notably, gene therapy remained cost-effective in a hypothetical scenario in which we estimated that the discounted factor concentrate price was 20% of the wholesale acquisition cost in the United States. Probabilistic sensitivity analysis estimated gene therapy to be cost-effective at 92% of simulations considering a $150 000/quality-adjusted life-year threshold. In conclusion, based on detailed simulation inputs and assumptions, gene therapy was more cost-effective than on-demand treatment and prophylaxis for patients with severe hemophilia B.

Inducible localized delivery of an anti-PD-1 scFv enhances anti-tumor activity of ROR1 CAR-T cells in TNBC.

BACKGROUND: Chimeric antigen receptor (CAR)-T cells can induce powerful immune responses in patients with hematological malignancies but have had limited success against solid tumors. This is in part due to the immunosuppressive tumor microenvironment (TME) which limits the activity of tumor-infiltrating lymphocytes (TILs) including CAR-T cells. We have developed a next-generation armored CAR (F i-CAR) targeting receptor tyrosine kinase-like orphan receptor 1 (ROR1), which is expressed at high levels in a range of aggressive tumors including poorly prognostic triple-negative breast cancer (TNBC). The F i-CAR-T is designed to release an anti-PD-1 checkpoint inhibitor upon CAR-T cell activation within the TME, facilitating activation of CAR-T cells and TILs while limiting toxicity. METHODS: To bolster potency, we developed a F i-CAR construct capable of IL-2-mediated, NFAT-induced secretion of anti-PD-1 single-chain variable fragments (scFv) within the tumor microenvironment, following ROR1-mediated activation. Cytotoxic responses against TNBC cell lines as well as levels and binding functionality of released payload were analyzed in vitro by ELISA and flow cytometry. In vivo assessment of potency of F i-CAR-T cells was performed in a TNBC NSG mouse model. RESULTS: F i-CAR-T cells released measurable levels of anti-PD-1 payload with 5 h of binding to ROR1 on tumor and enhanced the cytotoxic effects at challenging 1:10 E:T ratios. Treatment of established PDL1 + TNBC xenograft model with F i-CAR-T cells resulted in significant abrogation in tumor growth and improved survival of mice (71 days), compared to non-armored CAR cells targeting ROR1 (F CAR-T) alone (49 days) or in combination with systemically administered anti-PD-1 antibody (57 days). Crucially, a threefold increase in tumor-infiltrating T cells was observed with F i-CAR-T cells and was associated with increased expression of genes related to cytotoxicity, migration and proliferation. CONCLUSIONS: Our next-generation of ROR1-targeting inducible armored CAR platform enables the release of an immune stimulating payload only in the presence of target tumor cells, enhancing the therapeutic activity of the CAR-T cells. This technology provided a significant survival advantage in TNBC xenograft models. This coupled with its potential safety attributes merits further clinical evaluation of this approach in TNBC patients.

A hemophilia A mouse model for the in vivo assessment of emicizumab function.

The bispecific antibody emicizumab is increasingly used for hemophilia A treatment. However, its specificity for human factors IX and X (FIX and FX) has limited its in vivo functional analysis to primate models of acquired hemophilia. Here, we describe a novel mouse model that allows emicizumab function to be examined. Briefly, FVIII-deficient mice received IV emicizumab 24 hours before tail-clip bleeding was performed. A second infusion with human FIX and FX, administered 5 minutes before bleeding, generated consistent levels of emicizumab (0.7-19 mg/dL for 0.5-10 mg/kg doses) and of both FIX and FX (85 and 101 U/dL, respectively, after dosing at 100 U/kg). Plasma from these mice display FVIII-like activity in assays (diluted activated partial thromboplastin time and thrombin generation), similar to human samples containing emicizumab. Emicizumab doses of 1.5 mg/kg and higher significantly reduced blood loss in a tail-clip-bleeding model using FVIII-deficient mice. However, reduction was incomplete compared with mice treated with human FVIII concentrate, and no difference in efficacy between doses was observed. From this model, we deducted FVIII-like activity from emicizumab that corresponded to a dose of 4.5 U of FVIII per kilogram (ie, 9.0 U/dL). Interestingly, combined with a low FVIII dose (5 U/kg), emicizumab provided enough additive activity to allow complete bleeding arrest. This model could be useful for further in vivo analysis of emicizumab.

Haemophilia, the journey in search of a cure. 1960-2020.

The single most important step on the path to our modern understanding of blood coagulation and haemophilia in the 20th century was taken by British pathologist Robert Gwyn Macfarlane with his 1964 publication 'An enzyme cascade in the blood clotting mechanism, and its function as a biochemical amplifier'. In the same year, Ratnoff and Davie in the USA reached the same conclusion. Macfarlane and Rosemary Biggs had previously, in 1952, discovered factor IX as the factor deficient in haemophilia B. In 1973, Arthur Bloom defined the distinct role of Factor VIII and von Willebrand factor in haemophilia A and von Willebrand's disease respectively. This inspired the efforts of Tuddenham and his group towards the purification of Factor VIII which reached homogeneity in 1982, leading to the cloning of the Factor VIII gene in 1984 in collaboration with US scientists at Genentech, which in turn enabled development of safe recombinant factor concentrates for patients with haemophilia. Brownlee cloned the factor IX gene in 1982 at the Sir William Dunn Institute of Pathology in Oxford. This led eventually to the first successful trial of gene therapy for haemophilia B in 2011 by the Nathwani group at UCL, which built on pioneering work of US groups and was partnered with St Jude in Memphis where Nathwani started the project. This trial has fuelled the current quest for a functional cure of haemophilia A and B. The UK has, therefore, made a rich contribution to advances in haemostasis over the last 60 years, often in partnership with other groups across the world.

Therapeutic expression of human clotting factors IX and X following adeno-associated viral vector-mediated intrauterine gene transfer in early-gestation fetal macaques.

Adeno-associated viral vectors (AAVs) achieve stable therapeutic expression without long-term toxicity in adults with hemophilia. To avert irreversible complications in congenital disorders producing early pathogenesis, safety and efficacy of AAV-intrauterine gene transfer (IUGT) requires assessment. We therefore performed IUGT of AAV5 or -8 with liver-specific promoter-1 encoding either human coagulation factors IX (hFIX) or X (hFX) into Macaca fascicularis fetuses at ∼0.4 gestation. The initial cohort received 1 × 1012 vector genomes (vgs) of AAV5-hFIX ( n = 5; 0.45 × 1013 vg/kg birth weight), resulting in ∼3.0% hFIX at birth and 0.6-6.8% over 19-51 mo. The next cohort received 0.2-1 × 1013 vg boluses. AAV5-hFX animals ( n = 3; 3.57 × 1013 vg/kg) expressed <1% at birth and 9.4-27.9% up to 42 mo. AAV8-hFIX recipients ( n = 3; 2.56 × 1013 vg/kg) established 4.2-41.3% expression perinatally and 9.8-25.3% over 46 mo. Expression with AAV8-hFX ( n = 6, 3.12 × 1013 vg/kg) increased from <1% perinatally to 9.8-13.4% >35 mo. Low expressers (<1%, n = 3) were postnatally challenged with 2 × 1011 vg/kg AAV5 resulting in 2.4-13.2% expression and demonstrating acquired tolerance. Linear amplification-mediated-PCR analysis demonstrated random integration of 57-88% of AAV sequences retrieved from hepatocytes with no events occurring in or near oncogenesis-associated genes. Thus, early-IUGT in macaques produces sustained curative expression related significantly to integrated AAV in the absence of clinical toxicity, supporting its therapeutic potential for early-onset monogenic disorders.-Chan, J. K. Y., Gil-Farina I., Johana, N., Rosales, C., Tan, Y. W., Ceiler, J., Mcintosh, J., Ogden, B., Waddington, S. N., Schmidt, M., Biswas, A., Choolani, M., Nathwani, A. C., Mattar, C. N. Z. Therapeutic expression of human clotting factors IX and X following adeno-associated viral vector-mediated intrauterine gene transfer in early-gestation fetal macaques.

Immunosuppression overcomes insulin- and vector-specific immune responses that limit efficacy of AAV2/8-mediated insulin gene therapy in NOD mice.

We report the restoration of euglycaemia in chemically induced diabetic C57BL/6 mice and spontaneously diabetic Non Obese Diabetic (NOD) mice by intravenous systemic administration of a single-stranded adeno-associated virus (ssAAV2/8) codon optimised (co) vector encoding furin cleavable human proinsulin under a liver-specific promoter. There were no immunological barriers to efficacy of insulin gene therapy in chemically induced C57BL/6 mice, which enjoyed long-lasting correction of hyperglycaemia after therapy, up to 250 days. Euglycaemia was also restored in spontaneously diabetic NOD mice, although these mice required a 7-10-fold higher dose of vector to achieve similar efficacy as the C57BL/6 mice and the immunodeficient NODscid mice. We detected CD8+ T cell reactivity to insulin and mild inflammatory infiltration in the livers of gene therapy recipient NOD mice, neither of which were observed in the treated C57BL/6 mice. Efficacy of the gene therapy in NOD mice was partially improved by targeting the immune system with anti-CD4 antibody treatment, while transfer of NOD mouse AAV2/8-reactive serum to recipients prevented successful restoration of euglycaemia in AAV2/8-HLP-hINSco-treated NODscid mice. Our data indicate that both immune cells and antibodies form a barrier to successful restoration of euglycaemia in autoimmune diabetic recipient mice with insulin gene therapy, but that this barrier can be overcome by increasing the dose of vector and by suppressing immune responses.

Potential limits of AAV-based gene therapy with the use of new transgenes expressing factor IX fusion proteins.

INTRODUCTION: The variety of treatment for haemophilia B (HB) has recently improved with the emergence of both AAV-based gene therapy and bioengineered human factor IX (hFIX) molecules with prolonged half-life due to fusion to either albumin (Alb) or immunoglobulin Fc fragment (Fc). AIM: Adeno-associated viral vectors (AAV) mediating expression of hFIX-Alb and hFIX-Fc fusion proteins was investigated for gene therapy of HB to explore if their extended half-life translates to higher plasma levels of FIX. METHODS: Single-stranded cross-packaged AAV2/8 vectors expressing hFIX-Alb, hFIX-Fc and hFIX were evaluated in vitro, and in mice. RESULTS: Both hFIX-Alb and hFIX-Fc fusion proteins were synthesized and expressed as single chains of expected size following AAV-mediated gene transfer in vitro and in vivo. The procoagulant properties of these hFIX-fusion proteins were comparable to wild-type hFIX. However, their expression levels were threefold lower than wild-type hFIX in vivo most likely due to inefficient secretion. CONCLUSION: This, the first, evaluation of hFIX-fusion proteins in the context of AAV gene transfer suggests that the hFIX-fusion proteins are secreted inefficiently from the liver, thus preventing their optimal use in gene therapy approaches.

Bioengineered AAV Capsids with Combined High Human Liver Transduction In Vivo and Unique Humoral Seroreactivity.

Existing recombinant adeno-associated virus (rAAV) serotypes for delivering in vivo gene therapy treatments for human liver diseases have not yielded combined high-level human hepatocyte transduction and favorable humoral neutralization properties in diverse patient groups. Yet, these combined properties are important for therapeutic efficacy. To bioengineer capsids that exhibit both unique seroreactivity profiles and functionally transduce human hepatocytes at therapeutically relevant levels, we performed multiplexed sequential directed evolution screens using diverse capsid libraries in both primary human hepatocytes in vivo and with pooled human sera from thousands of patients. AAV libraries were subjected to five rounds of in vivo selection in xenografted mice with human livers to isolate an enriched human-hepatotropic library that was then used as input for a sequential on-bead screen against pooled human immunoglobulins. Evolved variants were vectorized and validated against existing hepatotropic serotypes. Two of the evolved AAV serotypes, NP40 and NP59, exhibited dramatically improved functional human hepatocyte transduction in vivo in xenografted mice with human livers, along with favorable human seroreactivity profiles, compared with existing serotypes. These novel capsids represent enhanced vector delivery systems for future human liver gene therapy applications.

The murine lung as a factory to produce secreted intrapulmonary and circulatory proteins.

We have shown that a lentiviral vector (rSIV.F/HN) pseudotyped with the F and HN proteins from Sendai virus generates high levels of intracellular proteins after lung transduction. Here, we evaluate the use of rSIV.F/HN for production of secreted proteins. We assessed whether rSIV.F/HN transduction of the lung generates therapeutically relevant levels of secreted proteins in the lung and systemic circulation using human α1-anti-trypsin (hAAT) and factor VIII (hFVIII) as exemplars. Sedated mice were transduced with rSIV.F/HN carrying either the secreted reporter gene Gaussia luciferase or the hAAT or hFVIII cDNAs by nasal sniffing. rSIV.F/HN-hAAT transduction lead to therapeutically relevant hAAT levels (70 µg/ml) in epithelial lining fluid, with stable expression persisting for at least 19 months from a single application. Secreted proteins produced in the lung were released into the circulation and stable expression was detectable in blood. The levels of hFVIII in murine blood approached therapeutically relevant targets. rSIV.F/HN was also able to produce secreted hAAT and hFVIII in transduced human primary airway cells. rSIV.F/HN transduction of the murine lungs leads to long-lasting and therapeutically relevant levels of secreted proteins in the lung and systemic circulation. These data broaden the use of this vector platform for a large range of disease indications.

Recent advances in developing specific therapies for haemophilia.

Haemophilia therapy has undergone very rapid evolution in the last 10 years. The major limitation of current replacement therapy is the short half-life of factors VIII and IX. These half-lives have been extended by the addition of various moieties, allowing less frequent infusion regimens. Entirely novel approaches have also entered the clinic, including a bispecific antibody that mimics factor VIII and strategies that rebalance the haemostatic mechanism by reducing antithrombin through inhibition of synthesis. These two treatments are available by subcutaneous injection at infrequent intervals and both can be used in patients with neutralising antibodies (inhibitors). Finally, a cure may be on the horizon with preliminary evidence of success for gene therapy in haemophilia B and A.

Gene Therapy for Hemophilia.

The X-linked bleeding disorder hemophilia causes frequent and exaggerated bleeding that can be life-threatening if untreated. Conventional therapy requires frequent intravenous infusions of the missing coagulation protein (factor VIII [FVIII] for hemophilia A and factor IX [FIX] for hemophilia B). However, a lasting cure through gene therapy has long been sought. After a series of successes in small and large animal models, this goal has finally been achieved in humans by in vivo gene transfer to the liver using adeno-associated viral (AAV) vectors. In fact, multiple recent clinical trials have shown therapeutic, and in some cases curative, expression. At the same time, cellular immune responses against the virus have emerged as an obstacle in humans, potentially resulting in loss of expression. Transient immune suppression protocols have been developed to blunt these responses. Here, we provide an overview of the clinical development of AAV gene transfer for hemophilia, as well as an outlook on future directions.

In Utero Transfer of Adeno-Associated Viral Vectors Produces Long-Term Factor IX Levels in a Cynomolgus Macaque Model.

The safe correction of an inherited bleeding disorder in utero prior to the onset of organ damage is highly desirable. Here, we report long-term transgene expression over more than 6 years without toxicity following a single intrauterine gene transfer (IUGT) at 0.9G using recombinant adeno-associated vector (AAV)-human factor IX (hFIX) in the non-human primate model we have previously described. Four of six treated animals monitored for around 74 months expressed hFIX at therapeutic levels (3.9%-120.0%). Long-term expression was 6-fold higher in males and with AAV8 compared to AAV5, mediated almost completely at this stage by random genome-wide hepatic proviral integrations, with no evidence of hotspots. Post-natal AAV challenge without immunosuppression was evaluated in two animals exhibiting chronic low transgene expression. The brief neutralizing immune reaction elicited had no adverse effect and, although expression was not improved at the dose administered, no clinical toxicity was observed. This long-term surveillance thus confirms the safety of late-gestation AAV-hFIX transfer and demonstrates that postnatal re-administration can be performed without immunosuppression, although it requires dose optimization for the desired expression. Nevertheless, eventual vector genotoxicity and the possibility of germline transmission will require lifelong monitoring and further evaluation of the reproductive function of treated animals.

Polonium-210 poisoning: a first-hand account.

BACKGROUND: Polonium-210 ((210)Po) gained widespread notoriety after the poisoning and subsequent death of Mr Alexander Litvinenko in London, UK, in 2006. Exposure to (210)Po resulted initially in a clinical course that was indistinguishable from infection or exposure to chemical toxins, such as thallium. METHODS: A 43-year-old man presented to his local hospital with acute abdominal pain, diarrhoea, and vomiting, and was admitted to the hospital because of dehydration and persistent gastrointestinal symptoms. He was initially diagnosed with gastroenteritis and treated with antibiotics. Clostridium difficile toxin was subsequently detected in his stools, which is when he first raised the possibility of being poisoned and revealed his background and former identity, having been admitted under a new identity with which he had been provided on being granted asylum in the UK. Within 6 days, the patient had developed thrombocytopenia and neutropenia, which was initially thought to be drug induced. By 2 weeks, in addition to bone marrow failure, he had evidence of alopecia and mucositis. Thallium poisoning was suspected and investigated but ultimately dismissed because blood levels of thallium, although raised, were lower than toxic concentrations. The patient continued to deteriorate and within 3 weeks had developed multiple organ failure requiring ventilation, haemofiltration, and cardiac support, associated with a drop in consciousness. On the 23rd day after he first became ill, he suffered a pulseless electrical activity cardiorespiratory arrest from which he could not be resuscitated and was pronounced dead. FINDINGS: Urine analysis using gamma-ray spectroscopy on day 22 showed a characteristic 803 keV photon emission, raising the possibility of (210)Po poisoning. Results of confirmatory analysis that became available after the patient's death established the presence of (210)Po at concentrations about 10(9)-times higher than normal background levels. Post-mortem tissue analyses showed autolysis and retention of (210)Po at lethal doses in several organs. On the basis of the measured amounts and tissue distribution of (210)Po, it was estimated that the patient had ingested several 1000 million becquerels (a few GBq), probably as a soluble salt (eg, chloride), which delivered very high and fatal radiation doses over a period of a few days. INTERPRETATION: Early symptoms of (210)Po poisoning are indistinguishable from those of a wide range of chemical toxins. Hence, the diagnosis can be delayed and even missed without a high degree of suspicion. Although body surface scanning with a standard Geiger counter was unable to detect the radiation emitted by (210)Po, an atypical clinical course prompted active consideration of poisoning with radioactive material, with the diagnosis ultimately being made with gamma-ray spectroscopy of a urine sample. FUNDING: UK NHS, Public Health England, and the UK Department of Health.

Long-term safety and efficacy of factor IX gene therapy in hemophilia B.

BACKGROUND: In patients with severe hemophilia B, gene therapy that is mediated by a novel self-complementary adeno-associated virus serotype 8 (AAV8) vector has been shown to raise factor IX levels for periods of up to 16 months. We wanted to determine the durability of transgene expression, the vector dose-response relationship, and the level of persistent or late toxicity. METHODS: We evaluated the stability of transgene expression and long-term safety in 10 patients with severe hemophilia B: 6 patients who had been enrolled in an initial phase 1 dose-escalation trial, with 2 patients each receiving a low, intermediate, or high dose, and 4 additional patients who received the high dose (2×10(12) vector genomes per kilogram of body weight). The patients subsequently underwent extensive clinical and laboratory monitoring. RESULTS: A single intravenous infusion of vector in all 10 patients with severe hemophilia B resulted in a dose-dependent increase in circulating factor IX to a level that was 1 to 6% of the normal value over a median period of 3.2 years, with observation ongoing. In the high-dose group, a consistent increase in the factor IX level to a mean (±SD) of 5.1±1.7% was observed in all 6 patients, which resulted in a reduction of more than 90% in both bleeding episodes and the use of prophylactic factor IX concentrate. A transient increase in the mean alanine aminotransferase level to 86 IU per liter (range, 36 to 202) occurred between week 7 and week 10 in 4 of the 6 patients in the high-dose group but resolved over a median of 5 days (range, 2 to 35) after prednisolone treatment. CONCLUSIONS: In 10 patients with severe hemophilia B, the infusion of a single dose of AAV8 vector resulted in long-term therapeutic factor IX expression associated with clinical improvement. With a follow-up period of up to 3 years, no late toxic effects from the therapy were reported. (Funded by the National Heart, Lung, and Blood Institute and others; ClinicalTrials.gov number, NCT00979238.).

Rewiring of sIgM-Mediated Intracellular Signaling through the CD180 Toll-like Receptor.

Chronic lymphocytic leukemia (CLL) development and progression are thought to be driven by unknown antigens/autoantigens through the B cell receptor (BCR) and environmental signals for survival and expansion including toll-like receptor (TLR) ligands. CD180/RP105, a membrane-associated orphan receptor of the TLR family, induces normal B cell activation and proliferation and is expressed by approximately 60% of CLL samples. Half of these respond to ligation with anti-CD180 antibody by increased activation/phosphorylation of protein kinases associated with BCR signaling. Hence CLL cells expressing both CD180 and the BCR could receive signals via both receptors. Here we investigated cross-talk between BCR and CD180-mediated signaling on CLL cell survival and apoptosis. Our data indicate that ligation of CD180 on responsive CLL cells leads to activation of either prosurvival Bruton tyrosine kinase (BTK)/phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT-mediated, or proapoptotic p38 mitogen-activated protein kinase (p38MAPK)-mediated signaling pathways, while selective immunoglobulin M (sIgM) ligation predominantly engages the BTK/PI3K/AKT pathway. Furthermore, pretreatment of CLL cells with anti-CD180 redirects IgM-mediated signaling from the prosurvival BTK/PI3K/AKT toward the proapoptotic p38MAPK pathway. Thus preengaging CD180 could prevent further prosurvival signaling mediated via the BCR and, instead, induce CLL cell apoptosis, opening the door to therapeutic profiling and new strategies for the treatment of a substantial cohort of CLL patients.