A national assessment of standards of care for inherited bleeding disorders in Canada.

AIM: The Canadian Integrated and Comprehensive Care Standards for Inherited Bleeding Disorders were adopted in June 2020. In early 2023, a self-assessment of each of the 26 Canadian inherited bleeding disorder treatment centre's (IBDTCs) capacity to meet the Standards was conducted. The goal was to validate the standards by assessing appropriateness and adherence. As a result, centres can compare their own practices and capacity against those of all centres, identify barriers to adherence, identify opportunities for remedial actions and use the results locally as evidence for needed resources. METHODS: Healthcare providers (HCPs) in the 26 IBDTCs were provided with a checklist to assess adherence to each of the 66 standards of care. Centre participation was voluntary but strongly encouraged by the healthcare provider and patient associations. RESULTS: All 26 centres completed the self-assessments. Collectively, centres reported meeting 88.8% of the standards. Adherence to each standard ranged from 40% to 100%. Forty-one (41) of the standards were adhered to by 90% or more of the centres, 12 by 80%-89% of the centres and 13 by fewer than 80% of the centres. A report consolidating all the assessments was sent to the 26 centres. CONCLUSION: None of the comments received in the self-assessment reports indicated that a given standard was irrelevant, unrealistic or unnecessary. These data are strong indicators that the standards, as written, are appropriate. The self-assessments, however, reveal alarming deficiencies in staffing levels, notably in physiotherapy, psychosocial support and data entry and data management. These constitute a barrier to comprehensive care for many centres. The findings echo similar conclusions from a previous assessment conducted in 2015.

Safety, pharmacokinetics, and antiviral activity of the capsid inhibitor AB-506 from Phase 1 studies in healthy subjects and those with hepatitis B.

AB-506 is a potent, pan-genotypic small molecule capsid inhibitor that inhibits hepatitis B virus (HBV) pregenomic RNA encapsidation. We assessed the safety, pharmacokinetics, and antiviral activity of AB-506 in two randomized, double-blinded Phase 1 studies in healthy subjects (HS) and subjects with chronic HBV infection (CHB). Single ascending and multiple doses of AB-506 or placebo (30-1000 mg or 400 mg daily for 10 days) were assessed in HS. AB-506 or placebo was assessed at either 160 mg or 400 mg daily for 28 days in subjects with CHB. A second follow-up study examined AB-506 or placebo at 400 mg daily for 28 days in 14 Caucasian and 14 East-Asian HS. Twenty-eight days of AB-506 at 160 mg and 400 mg produced mean HBV-DNA declines from baseline of 2.1 log10 IU/ml and 2.8 log10 IU/ml, respectively. Four subjects with CHB (all Asian) had Grade 4 alanine aminotransferase (ALT) elevations (2 at each dose) as HBV DNA was declining; three events led to treatment discontinuation. In the second follow-up study, 2 Asian HS had serious transaminitis events leading to treatment and study termination. No subjects had bilirubin elevations or signs of hepatic decompensation. Conclusion: AB-506 demonstrated mean HBV-DNA declines of >2 log10 ; however, transient but severe ALT flares were observed in 4 Asian subjects with CHB. In the follow-up study in HS, 2 additional Asian HS had Grade 4 flares, suggesting that AB-506 hepatotoxicity contributed to the ALT elevations. The AB-506 development program was terminated because of these findings.

Effects of a Fixed-Dose Co-Formulation of Daclatasvir, Asunaprevir, and Beclabuvir on the Pharmacokinetics of a Cocktail of Cytochrome P450 and Drug Transporter Substrates in Healthy Subjects.

BACKGROUND: A fixed-dose combination of daclatasvir (DCV; hepatitis C virus NS5A inhibitor), asunaprevir (ASV; non-structural protein 3 inhibitor), and beclabuvir (BCV; non-structural protein 5B inhibitor) is approved in Japan for hepatitis C virus genotype 1. OBJECTIVE: The objective of this study was to assess the combination's drug-drug interaction potential in vivo using a validated cocktail of eight cytochrome P450 (CYP) and transporter probes. METHODS: We conducted an open-label single-sequence study in healthy adults (n = 20) given single-dose caffeine (CYP1A2 substrate), metoprolol (CYP2D6), flurbiprofen (CYP2C9), montelukast (CYP2C8), omeprazole (CYP2C19), midazolam (CYP3A4), digoxin (P-glycoprotein), and pravastatin (organic anion-transporting polypeptide), alone or with steady-state twice-daily DCV/ASV/BCV 30/200/75 mg (with or without additional BCV 75 mg to adjust for higher exposure in hepatitis C virus infection). RESULTS: Daclatasvir/asunaprevir/beclabuvir did not affect CYP1A2, CYP2C8, or CYP2C9; the probe maximum observed concentration and area under the concentration-time curve extrapolated to infinite time geometric mean ratios and 90% confidence intervals were all within the 0.8-1.25 bioequivalence range. Beclabuvir showed moderate dose-dependent CYP2C19 induction; omeprazole maximum observed concentration and area under the concentration-time curve from 0 to the last quantifiable concentration were lower with additional BCV [geometric mean ratio 0.36 (90% confidence interval 0.23-0.55) and 0.34 (0.25-0.46), respectively] than without [0.57 (0.42-0.78), 0.48 (0.39-0.59)]. Weak-to-moderate CYP3A4 induction was observed, plus weak CYP2D6, P-glycoprotein, and organic anion-transporting polypeptide inhibition [maximum observed concentration and area under the concentration-time curve extrapolated to infinite time without additional BCV: midazolam 0.57 (0.50-0.65), 0.53 (0.47-0.60); metoprolol 1.40 (1.20-1.64), 1.71 (1.49-1.97); digoxin 1.23 (1.12-1.35), 1.23 (1.17-1.29); pravastatin 2.01 (1.63-2.47), 1.68 (1.43-1.97)]. CONCLUSIONS: No dose adjustments with DCV/ASV/BCV are indicated for CYP1A2, CYP2C8, CYP2C9, or P-glycoprotein substrates. CYP3A4, CYP2D6, and OATP substrates should be co-administered with caution. Co-administration with agents solely metabolized by CYP2C19 is not recommended.

CYP2C19 Genotype-Dependent Pharmacokinetic Drug Interaction Between Voriconazole and Ritonavir-Boosted Atazanavir in Healthy Subjects.

Voriconazole, a broad-spectrum triazole antifungal agent, is metabolized by cytochrome P450 (CYP) 2C19 and, to a lesser extent, by CYP3A. Genetic polymorphism of CYP2C19 not only plays a prominent role in its disposition but may also influence potential drug interactions with CYP450 modulators such as ritonavir. This study assessed 2-way drug interactions of voriconazole added on to ritonavir-boosted atazanavir in both CYP2C19 extensive-metabolizer (EM) and poor-metabolizer (PM) healthy subjects. Each subject received voriconazole alone on days 1-3, followed by a 7-day washout. Atazanavir/ritonavir 300/100 mg once daily was given on days 11-30 and voriconazole on days 21-30. Voriconazole doses were 200 mg (400 mg on days 1 and 21) twice daily and 50 mg (100 mg on days 1 and 21) twice daily for CYP2C19 EM and PM subjects, respectively. On coadministration, voriconazole AUC and Cmin decreased by 33% (90%CI, 22%-42%) and 39% (90%CI, 28%-49%), respectively, in CYP2C19 EMs, whereas voriconazole Cmax and AUC increased 4.4-fold (90%CI, 3.6-fold to 5.4-fold) and 5.6-fold (90%CI, 4.5-fold to 7.0-fold), respectively, in PMs. Adding voriconazole resulted in a 20%-30% decrease in atazanavir Cmin in both EMs and PMs. Ritonavir exposure was generally unchanged in either population. The safety and tolerability profiles of the combination were comparable with atazanavir/ritonavir and voriconazole administered alone. The most frequent adverse events with voriconazole were visual disturbance and headache. Coadministration of voriconazole and atazanavir/ritonavir is not recommended unless the benefit/risk to the patient justifies the use of the combination.

NS5A Sequence Heterogeneity and Mechanisms of Daclatasvir Resistance in Hepatitis C Virus Genotype 4 Infection.

BACKGROUND: Daclatasvir is an NS5A inhibitor approved for treatment of infection due to hepatitis C virus (HCV) genotypes (GTs) 1-4. To support daclatasvir use in HCV genotype 4 infection, we examined a diverse genotype 4-infected population for HCV genotype 4 subtype prevalence, NS5A polymorphisms at residues associated with daclatasvir resistance (positions 28, 30, 31, or 93), and their effects on daclatasvir activity in vitro and clinically. METHODS: We performed phylogenetic analysis of genotype 4 NS5A sequences from 186 clinical trial patients and 43 sequences from the European HCV database, and susceptibility analyses of NS5A polymorphisms and patient-derived NS5A sequences by using genotype 4 NS5A hybrid genotype 2a replicons. RESULTS: The clinical trial patients represented 14 genotype 4 subtypes; most prevalent were genotype 4a (55%) and genotype 4d (27%). Daclatasvir 50% effective concentrations for 10 patient-derived NS5A sequences representing diverse phylogenetic clusters were ≤0.080 nM. Most baseline sequences had ≥1 NS5A polymorphism at residues associated with daclatasvir resistance; however, only 3 patients (1.6%) had polymorphisms conferring ≥1000-fold daclatasvir resistance in vitro. Among 46 patients enrolled in daclatasvir trials, all 20 with baseline resistance polymorphisms achieved a sustained virologic response. CONCLUSIONS: Circulating genotype 4 subtypes are genetically diverse. Polymorphisms conferring high-level daclatasvir resistance in vitro are uncommon before therapy, and clinical data suggest that genotype 4 subtype and baseline polymorphisms have minimal impact on responses to daclatasvir-containing regimens.

Daclatasvir + asunaprevir + beclabuvir ± ribavirin for chronic HCV genotype 1-infected treatment-naive patients.

BACKGROUND AND AIMS: This phase-2b study examined the safety and efficacy of an all-oral, interferon-free combination of the NS5A replication complex inhibitor daclatasvir (DCV), the NS3 protease inhibitor asunaprevir (ASV), and the nonnucleoside NS5B polymerase inhibitor beclabuvir (BCV) with or without ribavirin in patients with HCV genotype (GT) 1 infection. METHODS: A total of 187 patients received 12 weeks of DCV 30 mg BID plus ASV 200 mg BID and BCV 150 mg BID (n = 86) or 75 mg BID with (n = 21) or without (n = 80) weight-based ribavirin BID. The primary endpoint was HCV RNA <25 IU/ml at post-treatment week 12 (SVR12). RESULTS: Overall, 90% of patients (169/187) in the combined treatment groups achieved SVR on or after post-treatment week 12. SVR rates were similar across subgroups (by mITT analysis), i.e. patients with cirrhosis (88%, 14/16), HCV GT-1a (90%, 137/155), and IL28B non-CC genotype (90%, 115/128). There were no drug-related serious AEs or grade 4 AEs. The most frequently reported AEs were headache, diarrhoea, fatigue and nausea. Addition of ribavirin to DCV+ASV+BCV was associated with decreased haemoglobin, compared with DCV+ASV+BCV alone. There were six grade 3/4 laboratory abnormalities noted, all unrelated to the study drugs. Viral breakthrough occurred in 2.5-4.8% of patients across groups and appeared unrelated to BCV dose or ribavirin inclusion. CONCLUSIONS: Results support phase 3 evaluation of a twice-daily, fixed-dose formulation of this DCV+ASV+BCV regimen with or without ribavirin in HCV GT-1-infected patients.

A randomized trial in healthy subjects to assess the bioequivalence of an atazanavir/cobicistat fixed-dose combination tablet versus administration as separate agents.

BACKGROUND: Cobicistat (COBI) is an alternative pharmacoenhancer to ritonavir. A fixed-dose combination (FDC) tablet containing atazanavir (ATV) and COBI has been developed for the treatment of HIV-1-infected patients. METHODS: This open-label, single-centre, single-dose, crossover study, randomized 64 healthy subjects to one of eight treatment sequences. Under light meal conditions, maximum plasma concentration (Cmax), area under the plasma concentration-time curve (AUC) to infinity (AUCINF) and AUC to the last measurable concentration (AUC0-T) for ATV and COBI administered as an FDC of ATV/COBI (300/150 mg) were compared to those following administration as separate agents given together; bioequivalence was concluded if the 90% CIs of the geometric mean ratios fell within the predetermined range of 0.80, 1.25. ATV and COBI pharmacokinetic parameters following administration as the FDC or as separate agents were also compared under fasted conditions. The effect of food (light and high-fat meals) on the pharmacokinetics of ATV and COBI for the FDC was also assessed. RESULTS: ATV and COBI administered in an FDC tablet were bioequivalent to the individual agents when given with a light meal. Under fasted conditions, pharmacokinetic parameters for ATV and COBI were similar for the individual components and the FDC. For the FDC, systemic exposure to ATV increased with a light meal compared to fasted conditions, and ATV concentration 24 h post-dose was similar with a light meal compared with a high-fat meal. CONCLUSIONS: ATV/COBI (300/150 mg) FDC tablet was bioequivalent to coadministration as separate agents with a light meal in healthy subjects. Clinicaltrials.gov identifier NCT01837719.

Randomized, placebo-controlled, single-ascending-dose study of BMS-791325, a hepatitis C virus (HCV) NS5B polymerase inhibitor, in HCV genotype 1 infection.

BMS-791325 is a nonnucleoside inhibitor of hepatitis C virus (HCV) NS5B polymerase with low-nanomolar potency against genotypes 1a (50% effective concentration [EC50], 3 nM) and 1b (EC50, 7 nM) in vitro. BMS-791325 safety, pharmacokinetics, and antiviral activity were evaluated in a double-blind, placebo-controlled, single-ascending-dose study in 24 patients (interferon naive and experienced) with chronic HCV genotype 1 infection, randomized (5:1) to receive a single dose of BMS-791325 (100, 300, 600, or 900 mg) or placebo. The prevalence and phenotype of HCV variants at baseline and specific posttreatment time points were assessed. Antiviral activity was observed in all cohorts, with a mean HCV RNA decline of ≈2.5 log10 copies/ml observed 24 h after a single 300-mg dose. Mean plasma half-life among cohorts was 7 to 9 h; individual 24-hour levels exceeded the protein-adjusted EC90 for genotype 1 at all doses. BMS-791325 was generally well tolerated, with no serious adverse events or discontinuations. Enrichment for resistance variants was not observed at 100 to 600 mg. At 900 mg, variants (P495L/S) associated with BMS-791325 resistance in vitro were transiently observed in one patient, concurrent with an observed HCV RNA decline of 3.4 log10 IU/ml, but were replaced with wild type by 48 h. Single doses of BMS-791325 were well tolerated; demonstrated rapid, substantial, and exposure-related antiviral activity; displayed dose-related increases in exposure; and showed viral kinetic and pharmacokinetic profiles supportive of once- or twice-daily dosing. These results support its further development in combination with other direct-acting antivirals for HCV genotype 1 infection. (This trial has been registered at ClinicalTrials.gov under registration no. NCT00664625.).

Efficacy of an interferon- and ribavirin-free regimen of daclatasvir, asunaprevir, and BMS-791325 in treatment-naive patients with HCV genotype 1 infection.

BACKGROUND & AIMS: The combination of peginterferon and ribavirin with telaprevir or boceprevir is the standard treatment of hepatitis C virus (HCV) genotype 1 infection. However, these drugs are not well tolerated because of their side effects and suboptimal virologic responses. In a phase 2a, open-label study, we examined the safety and efficacy of an interferon-free, ribavirin-free regimen of direct-acting antivirals, comprising daclatasvir (an NS5A replication complex inhibitor), asunaprevir (an NS3 protease inhibitor), and BMS-791325 (a non-nucleoside NS5B inhibitor), in patients with chronic HCV infection. METHODS: We analyzed data from 66 treatment-naive patients with HCV genotype 1 infection without cirrhosis who were assigned randomly to groups given daclatasvir (60 mg, once daily), asunaprevir (200 mg, twice daily), and BMS-791325 (75 or 150 mg, twice daily) for 12 or 24 weeks. The primary end point was an HCV-RNA level less than 25 IU/mL at 12 weeks after treatment (sustained virologic response at 12 weeks [SVR12]). RESULTS: In 64 patients, HCV-RNA levels were less than 25 IU/mL by week 4 of treatment (including 48 of 49 patients with HCV genotype 1a infection and 45 of 46 patients with the non-CC interleukin 28B genotype). Sixty-one patients (92%) achieved SVR12, based on a modified intention-to-treat analysis. Virologic responses were similar between 12 and 24 weeks of treatment. During the study, 2 patients experienced viral breakthrough and 1 patient relapsed. There were no grade 3-4 increases in levels of alanine or aspartate aminotransferases or bilirubin; there were no deaths or discontinuations resulting from serious adverse events or adverse events related to the treatment regimen. The most common adverse events were headache, asthenia, and gastrointestinal symptoms. CONCLUSIONS: In a phase 2a study, the all-oral, interferon-free, and ribavirin-free regimen of daclatasvir, asunaprevir, and BMS-791325 was well tolerated and achieved high rates of SVR12 in patients with HCV genotype 1 infection. Further studies of this regimen are warranted. ClinicalTrials.gov, number NCT01455090.

A rare chromosome 3 imbalance and its clinical implications.

The duplication of chromosome 3q is a rare disorder with varying chromosomal breakpoints and consequently symptoms. Even rarer is the unbalanced outcome from a parental inv(3) resulting in duplicated 3q and a deletion of 3p. Molecular karyotyping should aid in precisely determining the length and breakpoints of the 3q+/3p- so as to better understand a child's future development and needs. We report a case of an infant male with a 57.5 Mb duplication from 3q23-qter. This patient also has an accompanying 1.7 Mb deletion of 3p26.3. The duplicated segment in this patient encompasses the known critical region of 3q26.3-q27, which is implicated in the previously reported 3q dup syndrome; however, the accompanying 3p26.3 deletion is smaller than the previously reported cases. The clinical phenotype of this patient relates to previously reported cases of 3q+ that may suggest that the accompanying 1.7 Mb heterozygous deletion is not clinically relevant. Taken together, our data has refined the location and extent of the chromosome 3 imbalance, which will aid in better understanding the molecular underpinning of the 3q syndrome.

Common infections in the lung transplant recipient.

Infections are a major cause of morbidity and mortality after lung transplantation. Pretransplant assessments for infection risk and immunization updates may help reduce posttransplant infections. In addition careful choice of posttransplant prophylaxis for cytomegalovirus and fungal infections is critical. Because of the potential association of infections such as respiratory viral infections and gram-negative bacterial infections with bronchiolitis obliterans syndrome, prompt attention to these pathogens is critical. Choice of antimicrobials for prophylaxis and treatment should take into consideration both adverse effects and drug interactions associated with antimicrobial choice.

Effect of single vs bilateral lung transplantation on plasma surfactant protein D levels in idiopathic pulmonary fibrosis.

BACKGROUND: Serum levels of surfactant protein D (SP-D) have been suggested as reflecting epithelial damage in acute lung injury, COPD, and idiopathic pulmonary fibrosis (IPF). However, little is known about SP-D levels in the setting of lung transplantation. METHODS: We examined plasma SP-D levels in 104 subjects from a prospective, multicenter cohort study of lung allograft recipients. Plasma SP-D was measured by enzyme-linked immunosorbent assay prior to transplant and daily for 3 days after transplant. RESULTS: Subjects undergoing transplant for IPF had higher baseline SP-D levels (median, 325 ng/mL) compared with subjects with cystic fibrosis, COPD, and pulmonary hypertension (median, 100, 80, and 82 ng/mL, respectively; P = .0001). Among subjects with IPF undergoing bilateral transplant, SP-D levels declined rapidly postoperatively. In contrast, SP-D levels in subjects undergoing single lung transplant for IPF remained significantly higher than those of bilateral allograft recipients. Among subjects undergoing single lung transplant for IPF, the development of primary graft dysfunction (PGD) was associated with a subsequent rise in SP-D levels, whereas SP-D levels in IPF subjects undergoing bilateral transplant declined, even in the presence of grade 3 PGD. Importantly, single lung allograft recipients without PGD had higher postoperative SP-D levels than bilateral allograft recipients with PGD. CONCLUSIONS: Subjects undergoing lung transplant for IPF have significantly higher baseline plasma SP-D levels compared with those with other diagnoses. Plasma SP-D is likely a biomarker of the air-blood barrier integrity in the native IPF lung, but may be less useful as a biomarker of PGD after transplant.

Targeting choroid plexus epithelia and ventricular ependyma for drug delivery to the central nervous system.

BACKGROUND: Because the choroid plexus (CP) is uniquely suited to control the composition of cerebrospinal fluid (CSF), there may be therapeutic benefits to increasing the levels of biologically active proteins in CSF to modulate central nervous system (CNS) functions. To this end, we sought to identify peptides capable of ligand-mediated targeting to CP epithelial cells reasoning that they could be exploited to deliver drugs, biotherapeutics and genes to the CNS. METHODS: A peptide library displayed on M13 bacteriophage was screened for ligands capable of internalizing into CP epithelial cells by incubating phage with CP explants for 2 hours at 37C and recovering particles with targeting capacity. RESULTS: Three peptides, identified after four rounds of screening, were analyzed for specific and dose dependent binding and internalization. Binding was deemed specific because internalization was prevented by co-incubation with cognate synthetic peptides. Furthermore, after i.c.v. injection into rat brains, each peptide was found to target phage to epithelial cells in CP and to ependyma lining the ventricles. CONCLUSION: These data demonstrate that ligand-mediated targeting can be used as a strategy for drug delivery to the central nervous system and opens the possibility of using the choroid plexus as a portal of entry into the brain.

Epidermal growth factor targeting of bacteriophage to the choroid plexus for gene delivery to the central nervous system via cerebrospinal fluid.

Because the choroid plexus normally controls the production and composition of cerebrospinal fluid and, as such, its many functions of the central nervous system, we investigated whether ligand-mediated targeting could deliver genes to its secretory epithelium. We show here that when bacteriophages are targeted with epidermal growth factor, they acquire the ability to enter choroid epithelial cells grown in vitro as cell cultures, ex vivo as tissue explants or in vivo by intracerebroventricular injection. The binding and internalization of these particles activate EGF receptors on targeted cells, and the dose- and time-dependent internalization of particles is inhibited by the presence of excess ligand. When the phage genome is further reengineered to contain like green fluorescent protein or firefly luciferase under control of the cytomegalovirus promoter, gene expression is detectable in the choroid plexus and ependymal epithelium by immunohistochemistry or by noninvasive imaging, respectively. Taken together, these data support the hypothesis that reengineered ligand-mediated gene delivery should be considered a viable strategy to increase the specificity of gene delivery to the central nervous system and bypass the blood-brain barrier so as to exploit the biological effectiveness of the choroid plexus as a portal of entry into the brain.

Why did passion wane? A qualitative study of married women's attributions for declines in sexual desire.

The high prevalence of sexual desire complaints in women have led a number of researchers and theorists to argue for a reconceptualization of female sexual desire that deemphasizes the drive model and places more focus on relational factors. Lacking in this effort has been a critical mass of qualitative research that asks women to report on their causal attributions for low desire. In this study, the authors conducted open-ended interviews with 19 married women who had lost desire in their marriage and asked what causal attributions they made for their loss of sexual desire and what barriers they perceived to be blocking its reinstatement. Three core themes emerged from the data, all of which represented forces dragging down on sexual desire in the present sample: (a) institutionalization of the relationship, (b) over-familiarity, and (c) the de-sexualization of roles in these relationships. Interpersonal and intrapersonal sexual dynamics featured more prominently than did relationship problems in women's attributions. The authors discuss the results in terms of clinical implications in the psychosocial component of treatment for hypoactive sexual desire disorder.

The deployment of adenovirus-containing gene activated matrices onto severed axons after central nervous system injury leads to transgene expression in target neuronal cell bodies.

BACKGROUND: In previous studies, we showed that gene activated matrices (GAMs) containing nonviral vectors successfully deliver genes to neurons after optic nerve and spinal cord injury. In the present study, we evaluated whether adenoviral vectors delivered within a GAM increase the efficiency of local gene delivery to injured CNS neurons. Lyophilized GAMs containing collagen and adenoviral vectors were assessed in vitro and in vivo. METHODS: We evaluated viral vector stability, release kinetics and efficiency of transduction for this GAM formulation in vitro using the quantitative polymerase chain reaction (qPCR), flow cytometry and fluorescence microscopy. Using PCR, reverse transcriptase-PCR and confocal microscopy, we assessed viral DNA retrograde axonal transport, green fluorescent protein (GFP) expression in retinal ganglion cells (RGCs) after GAM implantation into the wound of the rat transected optic nerve. RESULTS: qPCR analyses demonstrated that 100% of viral particles were retained within the collagen after lyophilization. In vitro studies demonstrated that 60% of the particles within the GAM were infective and not released from the collagen matrix when placed in water. By 24 h, GFP expression was detected within cells that have invaded the GAM. In vivo studies demonstrated that adenoviral particles were retrogradely transported in axons from the GAM implanted at the lesion site to the RGC in the retina where the corresponding mRNA was expressed. Analysis of the efficiency of cell transduction indicated that 69% of RGC express GFP. CONCLUSIONS: These studies demonstrate that lyophilized GAMs containing adenoviral particles within collagen are stable, retain a significant proportion of their infectivity and successfully and efficiently deliver genes to neurons after central nervous system injury.

In vitro evaluation of a 'stealth' adenoviral vector for targeted gene delivery to adult mammalian neurones.

BACKGROUND: Polymer coating of adenovirus type 5 (Ad5) particles produces a 'stealth' Ad5 (sAd5) that confers protection from immune recognition, blocks receptor-mediated uptake, and favours uptake into pinocytic cells. METHODS: In mixed cultures of primary adult rat dorsal root ganglion neurones (DRGN), rat C6 glioma cells, A9 non-Coxsackie and Ad Receptor (CAR)- and CAR-expressing fibroblasts, reporter gene expression after sAd5 pinocytotic uptake was monitored using the green fluorescent protein (gfp) gene, and viral particle trafficking and polymer coat dismantling was followed using Yoyo-1 tagged Ad5 DNA and Texas Red (TR) to label the coat. RESULTS: sAd5.gfp was pinocytosed by significantly higher proportions of neurones, than other cells, but GFP was not expressed. The TR-labelled coat remained co-localised with tagged viral DNA within transfected DRGN, showing that sAd5 did not uncoat and viral DNA did not traffic to the nucleus. Noncoated Ad5 transduced non-neuronal DRG cells more efficiently than DRGN, whereas A9(CAR) cells were more significantly transduced than any other cell type. Retargeting of the sAd5.gfp with either fibroblast growth factor-2 or nerve growth factor (NGF) enhanced internalisation by DRGN into endocytic vesicles allowing uncoating and thus GFP expression. Retargeting with NGF resulted in significantly higher numbers of DRGN expressing GFP than non-neuronal DRG cells. CONCLUSIONS: These findings indicate that DRGN pinocytose atropic genetic particles at higher levels than non-neuronal DRG cells and the environment of pinocytic vesicles is not conducive to sAd5 uncoating and capsid dismantling, requiring reformulation of sAd5 with either a neurone specific ligand or a self-dismantling coat to target sAd5 transgene expression to neurones.

Epstein-Barr virus nuclear antigen 3C augments Mdm2-mediated p53 ubiquitination and degradation by deubiquitinating Mdm2.

Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is one of the essential latent antigens for primary B-cell transformation. Previous studies established that EBNA3C facilitates degradation of several vital cell cycle regulators, including the retinoblastoma (pRb) and p27(KIP) proteins, by recruitment of the SCF(Skp2) E3 ubiquitin ligase complex. EBNA3C was also shown to be ubiquitinated at its N-terminal residues. Furthermore, EBNA3C can bind to and be degraded in vitro by purified 20S proteasomes. Surprisingly, in lymphoblastoid cell lines, EBNA3C is extremely stable, and the mechanism for this stability is unknown. In this report we show that EBNA3C can function as a deubiquitination enzyme capable of deubiquitinating itself in vitro as well as in vivo. Functional mapping using deletion and point mutational analysis showed that both the N- and C-terminal domains of EBNA3C contribute to the deubiquitination activity. We also show that EBNA3C efficiently deubiquitinates Mdm2, an important cellular proto-oncogene, which is known to be overexpressed in several human cancers. The data presented here further demonstrate that the N-terminal domain of EBNA3C can bind to the acidic domain of Mdm2. Additionally, the N-terminal domain of EBNA3C strongly stabilizes Mdm2. Importantly, EBNA3C simultaneously binds to both Mdm2 and p53 and can form a stable ternary complex; however, in the presence of p53 the binding affinity of Mdm2 toward EBNA3C was significantly reduced, suggesting that p53 and Mdm2 might share a common overlapping domain of EBNA3C. We also showed that EBNA3C enhances the intrinsic ubiquitin ligase activity of Mdm2 toward p53, which in turn facilitated p53 ubiquitination and degradation. Thus, manipulation of the oncoprotein Mdm2 by EBNA3C potentially provides a favorable environment for transformation and proliferation of EBV-infected cells.