Catherine Y.C. Ng

Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr-virus-related lymphoproliferation.

Reactivation of Epstein-Barr virus (EBV) after bone-marrow transplantation leads in many cases to lymphoproliferative disease that responds poorly to standard therapy and is usually fatal. To prevent or control this complication, we prepared EBV-specific cytotoxic T-lymphocyte (CTL) lines from donor leucocytes and infused them into ten allograft recipients. Three of the patients had shown signs of EBV reactivation, with or without overt lymphoproliferation, and the others received CTL infusions as prophylaxis. No patient developed any complication that could be attributed to the CTL infusions. In the three patients with EBV reactivation, EBV DNA concentrations (measured by semiquantitative polymerase chain reaction [PCR]), which had increased 1000-fold or more, returned to the control range within 3-4 weeks of immunotherapy. The most striking consequence was the resolution of immunoblastic lymphoma in a 17-year-old patient who received four CTL infusions (two 1 x 10(7)/m2 and two 5 x 10(7)/m2). Because the CTL had been genetically marked before infusion, we were able to show by PCR analysis that they persisted for 10 weeks after administration. EBV-specific donor-type T-cell lines seem to offer safe and effective therapy for control of EBV-associated lymphoproliferation.

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.).

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. Experimental Design: Established orthotopic neuroblastoma xenografts treated with bevacizumab were evaluated at serial time points for treatment-associated changes in intratumoral vascular physiology, penetration of systemically administered chemotherapy, and efficacy of combination therapy. Results: After a single bevacizumab dose, a progressive decrease in tumor microvessel density to <30% of control was observed within 7 days. Assessment of the tumor microenvironment revealed a rapid, sustained decrease in both tumor vessel permeability and tumor interstitial fluid pressure, whereas intratumoral perfusion, as assessed by contrast-enhanced ultrasonography, was improved, although this latter change abated by 1 week. Intratumoral drug delivery mirrored these changes; penetration of chemotherapy was improved by as much as 81% when given 1 to 3 days after bevacizumab, compared with when both drugs were given concomitantly, or 7 days apart. Finally, administering topotecan to tumor-bearing mice 3 days after bevacizumab resulted in greater tumor growth inhibition (36% of control size) than with monotherapy (88% bevacizumab, 54% topotecan) or concomitant administration of the two drugs (44%). Conclusions: Bevacizumab-mediated VEGF blockade effects alterations in tumor vessel physiology that allow improved delivery and efficacy of chemotherapy, although careful consideration of drug scheduling is required to optimize antitumor activity.

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. Peak hFIX expression following the highest dose of vector (2 × 10(12) pcr-vector genomes (vg)/kg) was 21 ± 3 µg/ml (~420% of normal). Fluorescent in-situ hybridization demonstrated scAAV provirus in almost 100% of hepatocytes at that dose. No perturbations of clinical or laboratory parameters were noted and vector genomes were cleared from bodily fluids by 10 days. Macaques transduced with 2 × 10(11) pcr-vg/kg were followed for the longest period (~5 years), during which time expression of hFIX remained >10% of normal level, despite a gradual decline in transgene copy number and the proportion of transduced hepatocytes. All macaques developed serotype-specific antibodies but no capsid-specific cytotoxic T lymphocytes were detected. The liver was preferentially transduced with 300-fold more proviral copies than extrahepatic tissues. Long-term biochemical, ultrasound imaging, and histologic follow-up of this large cohort of NHP revealed no toxicity. These data support further evaluation of this vector in hemophilia B patients.

Inhibition of neuroblastoma tumor growth by targeted delivery of microRNA-34a using anti-disialoganglioside GD2 coated nanoparticles.

Background Neuroblastoma is one of the most challenging malignancies of childhood, being associated with the highest death rate in paediatric oncology, underlining the need for novel therapeutic approaches. Typically, patients with high risk disease undergo an initial remission in response to treatment, followed by disease recurrence that has become refractory to further treatment. Here, we demonstrate the first silica nanoparticle-based targeted delivery of a tumor suppressive, pro-apoptotic microRNA, miR-34a, to neuroblastoma tumors in a murine orthotopic xenograft model. These tumors express high levels of the cell surface antigen disialoganglioside GD2 (GD2), providing a target for tumor-specific delivery. Principal Findings Nanoparticles encapsulating miR-34a and conjugated to a GD2 antibody facilitated tumor-specific delivery following systemic administration into tumor bearing mice, resulted in significantly decreased tumor growth, increased apoptosis and a reduction in vascularisation. We further demonstrate a novel, multi-step molecular mechanism by which miR-34a leads to increased levels of the tissue inhibitor metallopeptidase 2 precursor (TIMP2) protein, accounting for the highly reduced vascularisation noted in miR-34a-treated tumors. Significance These novel findings highlight the potential of anti-GD2-nanoparticle-mediated targeted delivery of miR-34a for both the treatment of GD2-expressing tumors, and as a basic discovery tool for elucidating biological effects of novel miRNAs on tumor growth.

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. A further twofold improvement in potency was achieved by replacing the 226-aa N6 spacer with a novel 17-aa peptide (V3) in which 6 glycosylation triplets from the B domain were juxtaposed. The resulting 5.2-kb rAAV-HLP-codop-hFVIII-V3 cassette was more efficiently packaged within AAV virions and mediated supraphysiologic hFVIII expression (732 ± 162% of normal) in HA knock-out mice following administration of 2 × 10(12) vector genomes/kg, a vector dose shown to be safe in subjects with hemophilia B. Stable hFVIII expression at 15 ± 4% of normal was observed at this dose in a nonhuman primate. hFVIII expression above 100% was observed in 3 macaques that received a higher dose of either this vector or the N6 variant. These animals developed neutralizing anti-FVIII antibodies that were abrogated with transient immunosuppression. Therefore, rAAV-HLP-codop-hFVIII-V3 substantially improves the prospects of effective HA gene therapy.

Improved Intratumoral Oxygenation Through Vascular Normalization Increases Glioma Sensitivity to Ionizing Radiation

PURPOSE Ionizing radiation, an important component of glioma therapy, is critically dependent on tumor oxygenation. However, gliomas are notable for areas of necrosis and hypoxia, which foster radioresistance. We hypothesized that pharmacologic manipulation of the typically dysfunctional tumor vasculature would improve intratumoral oxygenation and, thus, the antiglioma efficacy of ionizing radiation. METHODS AND MATERIALS Orthotopic U87 xenografts were treated with either continuous interferon-beta (IFN-beta) or bevacizumab, alone, or combined with cranial irradiation (RT). Tumor growth was assessed by quantitative bioluminescence imaging; the tumor vasculature using immunohistochemical staining, and tumor oxygenation using hypoxyprobe staining. RESULTS Both IFN-beta and bevaziumab profoundly affected the tumor vasculature, albeit with different cellular phenotypes. IFN-beta caused a doubling in the percentage of area of perivascular cell staining, and bevacizumab caused a rapid decrease in the percentage of area of endothelial cell staining. However, both agents increased intratumoral oxygenation, although with bevacizumab, the effect was transient, being lost by 5 days. Administration of IFN-beta or bevacizumab before RT was significantly more effective than any of the three modalities as monotherapy or when RT was administered concomitantly with IFN-beta or bevacizumab or 5 days after bevacizumab. CONCLUSION Bevacizumab and continuous delivery of IFN-beta each induced significant changes in glioma vascular physiology, improving intratumoral oxygenation and enhancing the antitumor activity of ionizing radiation. Additional investigation into the use and timing of these and other agents that modify the vascular phenotype, combined with RT, is warranted to optimize cytotoxic activity.

ENHANCING TRANSDUCTION OF THE LIVER BY ADENO-ASSOCIATED VIRAL VECTORS

A number of distinct factors acting at different stages of the adeno-associated virus vector (AAV)-mediated gene transfer process were found to influence murine hepatocyte transduction. Foremost among these was the viral capsid protein. Self-complementary (sc) AAV pseudotyped with capsid from serotype 8 or rh.10 mediated fourfold greater hepatocyte transduction for a given vector dose when compared with vector packaged with AAV7 capsid. An almost linear relationship between vector dose and transgene expression was noted for all serotypes with vector doses as low as 1 × 107 vg per mouse (4 × 108 vg kg−1) mediating therapeutic levels of human FIX (hFIX) expression. Gender significantly influenced scAAV-mediated transgene expression, with twofold higher levels of expression observed in male compared with female mice. Pretreatment of mice with the proteasome inhibitor bortezomib increased scAAV-mediated hFIX expression from 4±0.6 to 9±2 μg ml−1 in female mice, although the effect of this agent was less profound in males. Exposure of mice to adenovirus 10–20 weeks after gene transfer with AAV vectors augmented AAV transgene expression twofold by increasing the level of proviral mRNA. Hence, optimization of individual steps in the AAV gene transfer process can further enhance the potency of AAV-mediated transgene expression, thus increasing the probability of successful gene therapy.

Antitumor efficacy of AAV-mediated systemic delivery of interferon-beta.

Type I interferons (α/β) have significant antitumor activity although their short half-life and systemic side effects have limited their clinical utility. An alternative dosing schedule of continuous, low-level delivery, as is achieved by gene therapy, rather than intermittent, high concentration pulsed-dosing, might avoid the toxicity of interferon while maintaining its antitumor efficacy. We have tested a gene therapy approach in murine tumor models to treat malignancies that have shown responsiveness to interferon in clinical trials. The tumor cell lines used were moderately sensitive to the direct effects of human interferon-β (hIFN-β) in vitro. For in vivo testing, systemic delivery of hIFN-β was generated following liver-targeted delivery of adeno-associated virus (AAV) vector carrying the hIFN-β transgene. This prevented engraftment of subcutaneous human gliomas, and orthotopic, localized (intrarenal) and disseminated (primarily pulmonary) human renal cell carcinomas; and caused regression of established tumors at these sites. In a syngeneic, immunocompetent model of melanoma, AAV IFN-β treatment limited subcutaneous tumor growth and prevented disseminated disease. A significant decrease in mean intratumoral vessel density was demonstrated in hIFN-β-treated tumors, suggesting that in addition to a direct tumoricidal effect, the antitumor efficacy of AAV IFN-β in this study was due to its ability to inhibit angiogenesis.

Adeno-Associated Virus Vector-Mediated Systemic Delivery of IFN-β Combined with Low-Dose Cyclophosphamide Affects Tumor Regression in Murine Neuroblastoma Models

Purpose: Type I IFNs (IFN-α/β) 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-β (hINF-β) was achieved through a gene therapy–mediated approach using adeno-associated virus vectors encoding hIFN-β (AAV hINF-β). Orthotopic localized retroperitoneal and disseminated models of neuroblastoma were established using three different xenografts. Immunohistochemical analysis and ELISA were used to evaluate the antiangiogenic effect of therapy. Results: The development of both localized orthotopic (retroperitoneal) and disseminated neuroblastoma was prevented in all mice expressing hINF-β. Continued growth of established retroperitoneal tumors, treated with AAV hINF-β as monotherapy, was significantly restricted, and survival for mice with established, disseminated disease was significantly prolonged following administration of AAV hINF-β. Analysis of treated tumors revealed a significant antiangiogenic effect. Mean intratumoral vessel density was diminished and expression of the angiogenic factors vascular endothelial growth factor and basic fibroblast growth factor were both decreased. Finally, combination therapy in which AAV hIFN-β was used together with low-dose cyclophosphamide resulted in regression of both established retroperitoneal and disseminated disease. Conclusions: AAV-mediated delivery of hIFN-β when used as monotherapy was able to restrict neuroblastoma growth due in part to inhibition of angiogenesis. When used in combination with conventional chemotherapy, AAV hIFN-β was able to effect complete tumor regression.