Junfang Zhou

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). Study participants were enrolled sequentially in one of three cohorts (given a high, intermediate, or low dose of vector), with two participants in each group. Vector was administered without immunosuppressive therapy, and participants were followed for 6 to 16 months. RESULTS AAV-mediated expression of FIX at 2 to 11% of normal levels was observed in all participants. Four of the six discontinued FIX prophylaxis and remained free of spontaneous hemorrhage; in the other two, the interval between prophylactic injections was increased. Of the two participants who received the high dose of vector, one had a transient, asymptomatic elevation of serum aminotransferase levels, which was associated with the detection of AAV8-capsid-specific T cells in the peripheral blood; the other had a slight increase in liver-enzyme levels, the cause of which was less clear. Each of these two participants received a short course of glucocorticoid therapy, which rapidly normalized aminotransferase levels and maintained FIX levels in the range of 3 to 11% of normal values. CONCLUSIONS Peripheral-vein infusion of scAAV2/8-LP1-hFIXco resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype, with few side effects. Although immune-mediated clearance of AAV-transduced hepatocytes remains a concern, this process may be controlled with a short course of glucocorticoids without loss of transgene expression. (Funded by the Medical Research Council and others; ClinicalTrials.gov number, NCT00979238.).

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.).

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.

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.

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.

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.

IFN-β sensitizes neuroblastoma to the antitumor activity of temozolomide by modulating O6-methylguanine DNA methyltransferase expression

Although temozolomide has shown clinical activity against neuroblastoma, this activity is likely limited by the DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT). We hypothesized that IFN-β could sensitize neuroblastoma cells to the cytotoxic effects of temozolomide through its ability to down-regulate MGMT expression. In vitro proliferation of three neuroblastoma cell lines treated with IFN-β and temozolomide alone or in combination was examined. Antitumor activity was assessed in both localized and disseminated neuroblastoma xenografts using single-agent and combination therapy, with continuous delivery of IFN-β being established by a liver-targeted adeno-associated virus-mediated approach. Two neuroblastoma cell lines (NB-1691 and SK-N-AS) were found to have high baseline levels of MGMT expression, whereas a third cell line (CHLA-255) had low levels. Temozolomide had little effect on in vitro proliferation of the neuroblastoma cell lines with high MGMT expression, but pretreatment with IFN-β significantly decreased MGMT expression and cell counts (NB-1691: 36 ± 3% of control, P = 0.0008; SK-N-AS: 54 ± 7% control, P = 0.003). In vivo, NB-1691 tumors in CB17-SCID mice treated with the combination of IFN-β and temozolomide had lower MGMT expression and a significantly reduced tumor burden, both localized [percent initial tumor volume: 2,516 ± 680% (control) versus 1,272 ± 330% (temozolomide), P = 0.01; 1,348 ± 220%, P = 0.03 (IFN-β); 352 ± 110%, P = 0.0001 (combo)] and disseminated [bioluminescent signal: control (1.32e10 ± 6.5e9) versus IFN-β (2.78e8 ± 3.09e8), P = 0.025, versus temozolomide (2.06e9 ± 1.55e9), P = 0.1, versus combination (2.13e7 ± 7.67e6), P = 0.009]. IFN-β appears to sensitize neuroblastoma cells to the cytotoxic effects of temozolomide through attenuation of MGMT expression. Thus, IFN-β and temozolomide may be a useful combination for treating children with this difficult disease. [Mol Cancer Ther 2008;7(12):3852–8]

Continuous Local Delivery of Interferon-β Stabilizes Tumor Vasculature in an Orthotopic Glioblastoma Xenograft Resection Model

BACKGROUND High-grade glioblastomas have immature, leaky tumor blood vessels that impede the efficacy of adjuvant therapy. We assessed the ability of human interferon (hIFN)-β delivered locally via gene transfer to effect vascular stabilization in an orthotopic model of glioblastoma xenograft resection. METHODS Xenografts were established by injecting 3 grade IV glioblastoma cell lines (GBM6-luc, MT330-luc, and SJG2-luc) into the cerebral cortex of nude rats. Tumors underwent subtotal resection, and then had gel foam containing an adeno-associated virus vector encoding either hIFN-β or green fluorescence protein (control) placed in the resection cavity. The primary endpoint was stabilization of tumor vasculature, as evidenced by CD34, α-SMA, and CA IX staining. Overall survival was a secondary endpoint. RESULTS hIFN-β treatment altered the tumor vasculature of GBM6-luc and SJG2-luc xenografts, decreasing the density of endothelial cells, stabilizing vessels with pericytes, and decreasing tumor hypoxia. The mean survival for rats with these neoplasms was not improved, however. In rats with MT330-luc xenografts, hIFN-β resulted in tumor regression with a 6-month survival of 55% (INF-β group) and 9% (control group). CONCLUSION The use of AAV hIFN-β in our orthotopic model of glioblastoma resection stabilized tumor vasculature and improved survival in rats with MT330 xenografts.

547. AAV Preparations Contain Contamination from DNA Sequences in Production Plasmids Directly Outside of the ITRs

Despite having the best safety profile of any current clinical viral vector, it is known that AAV preps contain contaminating sequences that are packaged alongside the expression cassette at a low rate. These sequences can originate from production plasmid DNA, or chromosomal DNA from producer cell lines. It has been reported that sequences from the expression cassette producer plasmid are more likely to be packaged into AAV than DNA from other producer plasmids, or chromosomal DNA. We hypothesised that in the expression cassette plasmid, backbone sequences directly flanking the ITRs might be packaged into AAV at a higher rate than sequences further away from either ITR. We first confirmed the presence of these sequences via PCR amplification of non-expression cassette DNA flanking the ITR sequence up to 1.5kb in length in an AAV prep. Sequential qPCR assays showed that plasmid sequences at a range of distances up to 2kb from the ITR make up between 1% and 9% of AAV particles. Most significantly, there was an observable decreasing trend in contaminant titer as distance from the ITR increased. Contaminant sequences closer to the ITRs (within 1kb) are detected at a 100 fold greater rate than distal plasmid DNA (9kb from ITRs on the same plasmid). The disparity in the levels of ITR adjacent DNA sequences, compared to sequences 9kb from either ITR, suggest that the origin of this DNA is from within AAV particles rather than residual plasmid DNA remaining after purification procedures. ITR adjacent contamination is present at both a TRS mutated ITR (required for self-complementary vectors) and non TRS mutated ITRs. Contaminating plasmid sequences were present when the transgene was half of the packaging capacity (2.3kb FIX prep) and at the full capacity (5kb FVIII prep) at comparable levels, suggesting that increasing the transgene size with stuffer DNA to create a full genome will not solve this issue. Previous studies have concluded that increasing the size of the backbone with stuffer DNA reduces the level of plasmid backbone contamination, as the two ITRs are then not in range of each other to facilitate reverse packaging. However, the total plasmid size in our studies was >20kb. Therefore, the ITRs should not be in range for this to occur. We hypothesise that these ITR adjacent sequences are either a product of read-through from the expression cassette into flanking sequences, due to inefficient cleavage at the ITR breakpoint, or from reverse priming mediated by only 1 functional ITR. In the current expression cassette plasmids examined, the Kanr gene and bacterial f1origin of replication are within the range of the flanking sequences that could be packaged. With current, unsolved clinical challenges for AAV, including transaminitis post high dose infection, it is clear that clinical AAV vectors should be designed to contain as little contamination as possible. We conclude that newly designed AAV production plasmids should contain significant lengths of stuffer DNA flanking each ITR (at least 2kb) to ensure that bacterial sequences are not packaged into AAV preps. Further research into vector design is required to eliminate this source of non-functional DNA from AAV produced for the clinic.View Large Image | Download PowerPoint Slide

Targeting histone demethylases in MYC-driven neuroblastomas with ciclopirox

Histone lysine demethylases facilitate the activity of oncogenic transcription factors, including possibly MYC. Here we show that multiple histone demethylases influence the viability and poor prognosis of neuroblastoma cells, where MYC is often overexpressed. We also identified the approved small-molecule antifungal agent ciclopirox as a novel pan-histone demethylase inhibitor. Ciclopirox targeted several histone demethylases, including KDM4B implicated in MYC function. Accordingly, ciclopirox inhibited Myc signaling in parallel with mitochondrial oxidative phosphorylation, resulting in suppression of neuroblastoma cell viability and inhibition of tumor growth associated with an induction of differentiation. Our findings provide new insights into epigenetic regulation of MYC function and suggest a novel pharmacologic basis to target histone demethylases as an indirect MYC-targeting approach for cancer therapy. Cancer Res; 77(17); 4626-38. ©2017 AACR.