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).

Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant.

Recombinant adeno-associated virus (rAAV) vectors encoding human factor VIII (hFVIII) were systematically evaluated for hemophilia A (HA) gene therapy. A 5.7-kb rAAV-expression cassette (rAAV-HLP-codop-hFVIII-N6) containing a codon-optimized hFVIII cDNA in which a 226 amino acid (aa) B-domain spacer replaced the entire B domain and a hybrid liver-specific promoter (HLP) mediated 10-fold higher hFVIII levels in mice compared with non-codon-optimized variants.

Adenovirus-associated virus vector-mediated gene transfer in hemophilia B.

Background: Hemophilia B, an X-linked disorder, is ideally suited for gene therapy. We investigated the use of a new gene therapy in patients with the disorder.

Methods: We infused a single dose of a serotype-8-pseudotyped, self-complementary adenovirus-associated virus (AAV) vector expressing a codon-optimized human factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco) in a peripheral vein in six patients with severe hemophilia B (FIX activity, <1% of normal values).

Long-term safety and efficacy following systemic administration of a self-complementary AAV vector encoding human FIX pseudotyped with serotype 5 and 8 capsid proteins.

Adeno-associated virus vectors (AAV) show promise for liver-targeted gene therapy. In this study, we examined the long-term consequences of a single intravenous administration of a self-complementary AAV vector (scAAV2/ 8-LP1-hFIXco) encoding a codon optimized human factor IX (hFIX) gene in 24 nonhuman primates (NHPs). A dose-response relationship between vector titer and transgene expression was observed.

In vivo bioluminescence imaging for early detection and monitoring of disease progression in a murine model of neuroblastoma.

Background: We evaluated the potential of bioluminescence imaging (BLI) for early tumor detection, demonstrating occult sites of disseminated disease and assessing disease progression in a murine model of neuroblastoma.

Methods: Neuroblastoma cells engineered to express the enzyme firefly luciferase were used to establish localized tumors and disseminated disease in SCID mice. Bioluminescent signal intensity was measured at serial time points, and compared with traditional methods of evaluating tumor growth.

Bevacizumab-induced transient remodeling of the vasculature in neuroblastoma xenografts results in improved delivery and efficacy of systemically administered chemotherapy.

Purpose: Dysfunctional tumor vessels can be a significant barrier to effective cancer therapy. However, increasing evidence suggests that vascular endothelial growth factor (VEGF) inhibition can effect transient "normalization" of the tumor vasculature, thereby improving tumor perfusion and, consequently, delivery of systemic chemotherapy. We sought to examine temporal changes in tumor vascular function in response to the anti-VEGF antibody, bevacizumab.

Continuous delivery of IFN-beta promotes sustained maturation of intratumoral vasculature.

IFNs have pleiotropic antitumor mechanisms of action. The purpose of this study was to further investigate the effects of IFN-beta on the vasculature of human xenografts in immunodeficient mice. We found that continuous, systemic IFN-beta delivery, established with liver-targeted adeno-associated virus vectors, led to sustained morphologic and functional changes of the tumor vasculature that were consistent with vessel maturation.

Interferon beta-mediated vessel stabilization improves delivery and efficacy of systemically administered topotecan in a murine neuroblastoma model.

Background: We have recently demonstrated that continuous delivery of interferon beta (IFN-beta) stabilizes solid tumor vasculature and improves tumor perfusion. In this study, we have further investigated the functional consequences of this effect by assessing delivery and efficacy of conventional chemotherapy against neuroblastoma xenografts when used in combination with IFN-beta.

Methods: Mice with established retroperitoneal tumors received adeno-associated virus vector encoding IFN-beta (AAV IFN-beta) or control vector.

Intravascular administration of tumor tropic neural progenitor cells permits targeted delivery of interferon-beta and restricts tumor growth in a murine model of disseminated neuroblastoma.

Background: Interferon-beta (IFN-beta) has potent antitumor activity; however, systemic toxicity has limited its clinical use. We investigated the potential of targeted delivery using tumor-tropic neural progenitor cells (NPCs) transduced to express human IFN-beta (hIFN-beta).

Methods: Disseminated neuroblastoma was established in SCID mice by tail vein injection of tumor cells.

Safe and efficient transduction of the liver after peripheral vein infusion of self-complementary AAV vector results in stable therapeutic expression of human FIX in nonhuman primates.

The safety and efficacy of peripheral venous administration of a self-complementary adeno-associated viral vector encoding the human FIX gene (scAAV-LP1-hFIXco) was evaluated in nonhuman primates for gene therapy of hemophilia B. Peripheral vein infusion of 1x10(12) vg/kg scAAV-LP1-hFIXco pseudotyped with serotype 8 capsid, in 3 macaques, resulted in stable therapeutic expression (more than 9 months) of human FIX (hFIX) at levels (1.1+/-0.

Efficacy of zoledronate against neuroblastoma.

Background: We investigated the antitumor and antiosteoclastic effects of zoledronate against human neuroblastoma in vitro and in a murine model of bone metastasis.

Methods: Antitumor activity of zoledronate against neuroblastoma cell lines was assessed by evaluating proliferation, apoptosis, and cell-cycle progression. A murine model of bone invasion was used to assess antiosteoclastic and antitumor activity in vivo.

Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver.

Transduction with recombinant adeno-associated virus (AAV) vectors is limited by the need to convert its single-stranded (ss) genome to transcriptionally active double-stranded (ds) forms. For AAV-mediated hemophilia B (HB) gene therapy, we have overcome this obstacle by constructing a liver-restricted mini-human factor IX (hFIX) expression cassette that can be packaged as complementary dimers within individual AAV particles. Molecular analysis of murine liver transduced with these self-complementary (sc) vectors demonstrated rapid formation of active ds-linear genomes that persisted stably as concatamers or monomeric circles.

Adeno-associated virus vector-mediated systemic delivery of IFN-beta combined with low-dose cyclophosphamide affects tumor regression in murine neuroblastoma models.

Purpose: Type I IFNs (IFN-alpha/beta) have shown significant antitumor activity in preclinical models but limited efficacy and significant toxicity in clinical trials. We hypothesized that the antitumor activity of type I IFNs could be enhanced by chronic, low-dose systemic delivery and sought to test this in murine neuroblastoma models.

Experimental Design: Continuous liver-generated expression of human IFN-beta (hINF-beta) was achieved through a gene therapy-mediated approach using adeno-associated virus vectors encoding hIFN-beta (AAV hINF-beta).

Interferon-mediated anti-angiogenic therapy for neuroblastoma.

Angiogenesis appears to be a fundamental requirement for tumor growth, invasion and metastasis. Evidence also exists to suggest that inhibition of tumor-associated angiogenesis can retard tumor growth and prevent tumor spread. Several naturally occurring angiogenesis inhibitors have been identified, including type I interferons (alpha/beta).

Comparison of the ability of adeno-associated viral vectors pseudotyped with serotype 2, 5, and 8 capsid proteins to mediate efficient transduction of the liver in murine and nonhuman primate models.

A detailed comparison of recombinant adeno-associated viral (rAAV) vectors of serotypes 2, 5, and 8 was performed in mice and nonhuman primates. Differences within the capsid proteins and viral terminal repeats of rAAV-2 and -5 did not significantly influence their ability to transduce murine liver. However, vectors pseudotyped with AAV-8 capsid (rAAV-2/8) mediated transgene expression more rapidly and from lower doses than possible with rAAV-2 and -5, although expression declined from peak values in a distinct dose-dependent manner prior to reaching steady-state levels.

Careful decoy receptor titering is required to inhibit tumor angiogenesis while avoiding adversely altering VEGF bioavailability.

To inhibit tumor-induced angiogenesis, the VEGF signaling pathway was targeted using AAV vectors encoding a VEGF decoy receptor, a truncated, soluble form of the murine VEGF receptor-2 (tsFlk-1). This approach initially had significant anti-neuroblastoma efficacy in murine xenograft models of local and metastatic disease, but when higher circulating levels of tsFlk-1 were established, tumor growth was more aggressive than even in control mice. Part of the mechanism for this apparent tumor resistance was increased human VEGF expression by the tumor cells.

Purification of recombinant adeno-associated virus type 8 vectors by ion exchange chromatography generates clinical grade vector stock.

Recombinant vectors based on the recently isolated AAV serotype 8 (rAAV-8) shows great promise for gene therapy, particularly for disorders affecting the liver. Transition of this vector system to the clinic, however, is limited by the lack of an efficient scaleable purification method. In this report, we describe a simple method for purification of rAAV-8 vector particles based on ion exchange chromatography that generates vector stocks with greater than 90% purity.

Restriction of neuroblastoma angiogenesis and growth by interferon-alpha/beta.

Purpose: We tested the hypothesis that the antiangiogenic activity of the type I interferons (IFNs), could affect tumor engraftment and growth in murine xenograft models of neuroblastoma.

Methods: Subcutaneous and retroperitoneal human neuroblastoma xenografts were established in SCID mice. Five days after tumor cell inoculation, daily subcutaneous injections of human IFN-alpha at several different doses were initiated and continued for 30 days.

Endostatin-mediated concomitant resistance in neuroblastoma.

Purpose: Concomitant resistance, the phenomenon whereby a primary malignancy inhibits the growth of metastatic lesions, is likely caused by the production of endogenous anti-angiogenic factors. The purpose of this study was to evaluate the influence of the angiogenesis inhibitor, endostatin, expressed by primary sites of neuroblastoma, on synchronous disease.

Methods: Two neuroblastoma models were used.

Retroviral vector-producer cell-mediated in vivo gene transfer of TIMP-3 restricts angiogenesis and neuroblastoma growth in mice.

Destruction and remodeling of the extracellular matrix occurs during the formation of new blood vessels that are required for tumor growth. We sought to determine whether gene-therapy mediated in vivo delivery of tissue inhibitor of matrix metalloproteinase-3 (TIMP-3), using retroviral vector-producer cells, could suppress angiogenesis and subsequent tumor growth in a murine neuroblastoma model. Tumor volume 28 days after coinjection of tumor cells with producer cells generating TIMP-3-encoding retroviral vectors was 21% that of controls, as was the mean tumor vascular index, a measure of blood vessel maturity.

Sex significantly influences transduction of murine liver by recombinant adeno-associated viral vectors through an androgen-dependent pathway.

A systematic evaluation of the influence of sex on transduction by recombinant adeno-associated viral vector (rAAV) indicated that transgene expression after liver-targeted delivery of vector particles was between 5- to 13-fold higher in male mice compared with female mice, irrespective of the proviral promoter or cDNA and mouse strain. Molecular analysis revealed that the rAAV genome was stably retained in male liver at levels that were 7-fold higher than those observed in females. Further, the sex difference in transduction was observed with AAV-2- and AAV-5-based vectors, which use distinct receptor complexes for infection.

Enforced expression of tissue inhibitor of matrix metalloproteinase-3 affects functional capillary morphogenesis and inhibits tumor growth in a murine tumor model.

Homeostasis of the extracellular matrix is a delicate balance between degradation and remodeling, the balance being maintained by the interaction of activated matrix metalloproteinases (MMPs) and specific tissue inhibitors of matrix metalloproteinases (TIMPs). Up-regulation of MMP activity, favoring proteolytic degradation of the basement membrane and extracellular matrix, has been linked to tumor growth and metastasis, as well as tumor-associated angiogenesis, whereas inhibition of MMP activity appears to restrict these processes. We have used retroviral-mediated gene delivery to effect sustained autocrine expression of TIMP-3 in murine neuroblastoma and melanoma tumor cells in order to further examine the ability of TIMPs to inhibit angiogenesis in vivo.

Sustained high-level expression of human factor IX (hFIX) after liver-targeted delivery of recombinant adeno-associated virus encoding the hFIX gene in rhesus macaques.

The feasibility, safety, and efficacy of liver-directed gene transfer was evaluated in 5 male macaques (aged 2.5 to 6.5 years) by using a recombinant adeno-associated viral (rAAV) vector (rAAV-2 CAGG-hFIX) that had previously mediated persistent therapeutic expression of human factor IX (hFIX; 6%-10% of physiologic levels) in murine models.

rAAV-mediated long-term liver-generated expression of an angiogenesis inhibitor can restrict renal tumor growth in mice.

It is now well established that tumor growth is angiogenesis dependent. Inhibition of angiogenesis, therefore, is likely to be an effective anticancer approach. A gene therapy-mediated approach to the delivery of antiangiogenic agents using adeno-associated virus (AAV) vectors has a number of advantages, including the potential for sustained expression.