IntroductionLiver-directed gene transfer using adeno-associated virus (AAV) vectors has the potential to serve as therapy for several inherited hematologic diseases. One such disease is the bleeding disorder hemophilia B, caused by a deficiency in coagulation factor IX (FIX). Currently, there are 2 clinical trials for hemophilia B that use liver-directed AAV-mediated gene transfer of the F9 gene (www. clinicaltrials.gov; identifiers NCT00515710 and NCT00979238). One of these trials reported transient efficacious circulating FIX levels (ϳ 10%) with the use of the vector AAV2-hFIX16. 1 Although AAV vectors are predominantly nonintegrating, with most of the transgene expression from stable episomes, 2 it has been shown through direct sequencing that integration can occur. 3,4 When integration takes place, there is a preference for integrating in regions where DNA breaks occur. These can be regions of endonuclease cleavage, 5 active transcription, 6-8 cytosine-phosphateguanosine (CpG) islands, 7,8 and palindromes. 9 All of these studies describing AAV vector genome integration identified vector integration sites through plasmid rescue of vectors containing bacterial origins of replication (ori). 4 Amplification of these plasmids in bacterial culture allows for sequencing of the integration junction between vector and host genome. Because of the bacterial selection involved in this method, bias may occur against recovering integrants whose size or sequence negatively affect bacterial growth, resulting in incomplete mapping of the full spectrum of integrants.Vector genome integration has been associated with adverse events; integrating ␥-retroviral vectors were implicated in the clonal expansion of transduced cells in 3 clinical studies, 2 for X-linked severe combined immunodeficiency 10,11 and the other for chronic granulomatous disease. 12,13 Although AAV vectors integrate at a much lower frequency than retroviral vectors, low-level AAV vector integration in transduced cells may still be a concern. A compelling argument supporting low genotoxic risk of AAV vectors comes from long-term follow-up of liver-directed AAVmediated gene transfer in canine and murine models. Of 77 dogs receiving AAV vector at doses up to 3.4 ϫ 10 12 vector genomes(vg)/kg and followed for Յ 10 years, none developed liver tumors as assayed by ultrasound, computed tomographic (CT) scan, and magnetic resonance imaging (MRI) 14,15 (K.A.H., V.R.A., and Timothy C. Nichols, unpublished data, October 15, 2010). Similarly, Ͼ 300 mice receiving AAV vectors with a therapeutic transgene at doses up to 4 ϫ 10 13 vg/kg and followed Յ 14 months have not shown a difference in tumor incidence compared with control mice. 16,17 However, a study by one group reported an increase in tumor incidence that was attributed to AAV vectors. 18 These investigators reported that administration of an AAV serotype 2 (AAV2) vector encoding ␤-glucuronidase in neonatal mice resulted in a significant increase in incidence of hepatocellular carcinoma (HCC), a tumor commonly fou...

Untreated cystathionine beta-synthase (CBS) deficiency in humans is characterized by extremely elevated plasma total homocysteine (tHcy>200 microM), with thrombosis as the major cause of morbidity. Treatment with vitamins and diet leads to a dramatic reduction in thrombotic events, even though patients often still have severe elevations in tHcy (>80 microM). To understand the difference between extreme and severe hyperhomocysteinemia, we have examined two mouse models of CBS deficiency: Tg-hCBS Cbs(-/-) mice, with a mean serum tHcy of 169 microM, and Tg-I278T Cbs(-/-) mice, with a mean tHcy of 296 microM. Only Tg-I278T Cbs(-/-) animals exhibited strong biological phenotypes, including facial alopecia, osteoporosis, endoplasmic reticulum (ER) stress in the liver and kidney, and a 20% reduction in mean survival time. Metabolic profiling of serum and liver reveals that Tg-I278T Cbs(-/-) mice have significantly elevated levels of free oxidized homocysteine but not protein-bound homocysteine in serum and elevation of all forms of homocysteine and S-adenosylhomocysteine in the liver compared to Tg-hCBS Cbs(-/-) mice. RNA profiling of livers indicate that Tg-I278T Cbs(-/-) and Tg-hCBS Cbs(-/-) mice have unique gene signatures, with minimal overlap. Our results indicate that there is a clear pathogenic threshold effect for tHcy and bring into question the idea that mild elevations in tHcy are directly pathogenic.

Hepatocellular carcinoma (HCC) represents a unique challenge for physicians and patients. There is no definitively curative treatment. Rather, many treatment and management modalities exist with differing advantages and disadvantages. Both current guidelines and individual patient concerns must be taken into account in order to properly manage HCC. In addition, quality of life issues are particularly complex in patients with HCC and these concerns must also be factored into treatment strategies. Thus, considering all the options and their various pros and cons can quickly become complex for both clinicians and patients. In this review, we systematically discuss the current treatment modalities available for HCC, detailing relevant clinical data, risks and rewards and overall outcomes for each approach. Surgical options discussed include resection, transplantation and ablation. We also discuss the radiation modalities: conformal radiotherapy, yttrium 90 microspheres and proton and heavy ion radiotherapy. The biologic agent Sorafenib is discussed as a promising new approach, and recent clinical trials are reviewed. We then detail currently described molecular pathways implicated in the initiation and progression of HCC, and we explore the potential of each pathway as an avenue for drug exploitation. We hope this comprehensive and forward-looking review enables both clinicians and patients to understand various options and thereby make more informed decisions regarding this disease.

Grape polyphenols can act as antioxidants, antiangiogenics, and selective estrogen receptor (ER) modifiers and are therefore especially relevant for gynecological cancers such as breast cancer. The major polyphenols of red wine (resveratrol, quercetin, and catechin) have been individually shown to have anticancer properties. However, their combinatorial effect on metastatic breast cancers has not been investigated in vivo. We tested the effect of low dietary concentrations of resveratrol, quercetin, and catechin on breast cancer progression in vitro by analyzing cell proliferation and cell cycle progression. The effects of these compounds on fluorescently tagged breast tumor growth in nude mice were assessed using in situ fluorescence image analysis. Individual polyphenols at 0.5 microM neither decreased breast cancer cell proliferation nor affected cell cycle progression in vitro. However, a combination of resveratrol, quercetin, and catechin at 0.5, 5, or 20 microM each significantly reduced cell proliferation and blocked cell cycle progression in vitro. Furthermore, using in situ image analysis, we determined that combined dietary polyphenols at 0.5, 5, or 25 mg/kg reduced primary tumor growth of breast cancer xenografts in a nude mouse model. Peak inhibition was observed at 5 mg/kg. These results indicate that grape polyphenols may inhibit breast cancer progression.

Intramuscular injection of adeno-associated viral (AAV) vector to skeletal muscle of humans with hemophilia B is safe, but higher doses are required to achieve therapeutic factor IX (F.IX) levels. The efficacy of this approach is hampered by the retention of F.IX in muscle extracellular spaces and by the limiting capacity of muscle to synthesize fully active F.IX at high expression rates. To overcome these limitations, we constructed AAV vectors encoding F.IX variants for muscle- or liver-directed expression in hemophilia B mice. Circulating F.IX levels following intramuscular injection of AAV-F.IX-K5A/V10K, a variant with low-affinity to extracellular matrix, were 2-5 fold higher compared with wild-type (WT) F.IX, while the protein-specific activities remained similar. Expression of F.IX-R338A generated a protein with 2- or 6-fold higher specific activity than F.IX-WT following vector delivery to skeletal muscle or liver, respectively. F.IX-WT and variant forms provide effective hemostasis in vivo upon challenge by tail-clipping assay. Importantly, intramuscular injection of AAV-F.IX variants did not trigger antibody formation to F.IX in mice tolerant to F.IX-WT. These studies demonstrate that F.IX variants provide a promising strategy to improve the efficacy for a variety of gene-based therapies for hemophilia B.

Effective therapies are needed to control excessive bleeding in a range of clinical conditions. We describe a surprisingly useful approach to improve hemostasis in vivo using a variant of coagulation factor Xa (FXaI16L). This conformationally pliant derivative is partially inactive due to a defect in transitioning from zymogen to protease 1,2. Using mouse models of hemophilia, we show that FXaI16L has a prolonged half-life, relative to wild-type FXa and does not cause excessive activation of coagulation. Once clotting mechanisms are activated to produce its cofactor FVa, FXaI16L is driven to the protease state and restores hemostasis in hemophilic animals upon vascular injury. Moreover, using human or murine analogs, we show that FXaI16L is more efficacious than FVIIa which is used to treat bleeding in hemophilia inhibitor patients3. Because of its underlying mechanism of action, FXaI16L may provide an effective strategy to enhance blood clot formation and act as a rapid pan-hemostatic agent for the treatment of bleeding conditions.

IntroductionGene replacement therapy attempts to restore the function of the defective gene product through sustained therapeutic transgene expression. However, depending on the nature of the mutation in the defective gene (eg, a deletion or nonsense mutation), the therapeutic gene product may represent a nonself protein to the host and thus may be subject to an immune response, which can eliminate transgene expression through antibody-mediated or cellular mechanisms. Therefore, understanding mechanisms of immune activation is of great clinical significance.Adeno-associated viral (AAV) vectors have been extensively tested for therapeutic gene delivery by means of in vivo gene transfer to nondividing target cells such as muscle fibers and hepatocytes. 1 AAV serotype 2 vectors are derived from a nonpathogenic, replication-deficient parvovirus with an approximate 4.7-kb single-stranded DNA genome. 2 These vectors do not contain viral-coding sequences and can be manufactured in a helper virus-free system. Skeletal muscle is an attractive target for gene transfer because of its abundance and easy accessibility, thereby allowing gene transfer with relatively noninvasive procedures. Muscle fibers are capable of expressing and secreting biologically active gene products that are normally not synthesized by this tissue. 3 However, sustained systemic expression of several therapeutic proteins has been limited by a neutralizing antibody response after intramuscular administration of recombinant AAV. Examples include coagulation factor IX (FIX) in treatment of hemophilia B, ␣ 1 -antitrypsin, and erythropoietin. [4][5][6] These responses were typically observed if a neo-antigen was expressed such as a human protein in a mouse or a species-specific transgene product in the context of a gene deletion or other type of null mutation. [7][8][9] Therefore, only subjects with missense mutations in the F9 gene were enrolled in a phase 1 clinical trial based on intramuscular administration of AAV-FIX vector. 10 Anti-FIX formation in the context of muscle gene transfer is the result of an adaptive CD4 ϩ T-helper cell-dependent immune response that is not observed in Rag-1-deficient or CD4-deficient mice and that may be blocked by transient immune suppression in immunocompetent animals. 5,8,11 Of interest, hepatic AAV-mediated expression of the same gene products was often sustained and has been shown to induce immune tolerance to [12][13][14][15][16][17] Adaptive immune responses after gene transfer to skeletal muscle have been shown to require antigen presentation to T cells by bone marrow-derived professional antigen-presenting cells (APCs). 18 Antigen presentation by dendritic cells is thought to be crucial for initiation of a primary immune response. In contrast to many other viral vectors, AAV vectors often fail to induce a cytotoxic T-lymphocyte (CTL) response to the transgene product, which is likely due to inefficiency of the vector to productively infect dendritic cells in vivo and to elicit a potent innate immune response, t...

To cite this article: Schlachterman A, Schuettrumpf J, Liu J-H, Freguia CF, Toso R, Poncz M, Camire RM, Arruda VR. Factor V Leiden improves in vivo hemostasis in murine hemophilia models. J Thromb Haemost 2005; 3: 2730-7.Summary. The role of factor V Leiden (FVL) as a modifier of the severe hemophilia phenotype is still unclear. We used mice with hemophilia A or B crossed with FVL to elucidate in vivo parameters of hemostasis. Real-time thrombus formation in the microcirculation was monitored by deposition of labeled platelets upon laser-induced endothelial injury using widefield microscopy in living animals. No thrombi formed in hemophilic A or B mice following vascular injuries. However, hemophilic mice, either heterozygous or homozygous for FVL, formed clots at all injured sites. Injection of purified activated FV into hemophilic A or B mice could mimic the in vivo effect of FVL. In contrast to these responses to a laser injury in a microvascular bed, FVL did not provide sustained hemostasis following damage of large vessels in a ferric chloride carotid artery injury model, despite of the improvement of clotting times and high circulating thrombin levels. Together these data provide evidence that FVL has the ability to improve the hemophilia A or B phenotype, but this effect is principally evident at the microcirculation level following a particular vascular injury. Our observations may partly explain the heterogeneous clinical evidence of the beneficial role of FVL in hemophilia.