Environmental Health and Safety Offers a Biosafety Risk Assessment for a Theoretical Model of a Gene Therapy Process Transfer from Research and Development to Large-Scale Manufacturing.

Introduction: This article provides a strategy by which a manufacturing process with a Biosafety Level 2 (BL2) designation can be downgraded to Biosafety Level 1 (BL1). The principles of the downgrading process are based on the robust contamination controls in clinical and commercial manufacturing, which typically are not part of Research and Development processes. These strict requirements along with the application of current Good Manufacturing Practice (cGMP) principles provide a framework by which processes can be suitably managed and controlled to mitigate biohazard risk, specifically for cell lines that may be contaminated with human pathogenic viral agents. Purpose: We demonstrate how a risk assessment guide was used to define the risk profile of a theoretical process with a human cell line intended for clinical/commercial application. Based on the risk assessment, key BL2 elements were identified as suitable for downgrading, including facility containment controls, emergency spill response plans, and storage and shipping requirements. For various reasons, some aspects of the systems were deemed unsuitable for downgrading due to the severity of the control risk and, therefore, remained at BL2. Summary and Conclusions: We have used an established risk assessment guide to show how cGMP compliments and augments biosafety containment. We provide justification for downgrading from BL2 to BL1 for clinical and commercial cell and gene therapy manufacturing with human cell lines.

Dysfunction of basal ganglia functional connectivity associated with subjective and cognitive fatigue in multiple sclerosis.

Objectives: Central fatigue is one of the most common symptoms in multiple sclerosis (MS). It has a profound impact on quality of life and a negative effect on cognition. Despite its widespread impact, fatigue is poorly understood and very difficult to measure. Whilst the basal ganglia has been implicated in fatigue the nature of its role and involvement with fatigue is still unclear. The aim of the present study was to establish the role of the basal ganglia in MS fatigue using functional connectivity measures. Methods: The present study examined the functional connectivity (FC) of the basal ganglia in a functional MRI study with 40 female participants with MS (mean age = 49.98 (SD = 9.65) years) and 40 female age-matched (mean age = 49.95 (SD = 9.59) years) healthy controls (HC). To measure fatigue the study employed the subjective self-report Fatigue Severity Scale and a performance measure of cognitive fatigue using an alertness-motor paradigm. To distinguish physical and central fatigue force measurements were also recorded. Results: The results suggest that decreased local FC within the basal ganglia plays a key role in cognitive fatigue in MS. Increased global FC between the basal ganglia and the cortex may sub serve a compensatory mechanism to reduce the impact of fatigue in MS. Conclusion: The current study is the first to show that basal ganglia functional connectivity is associated with both subjective and objective fatigue in MS. In addition, the local FC of the basal ganglia during fatigue inducing tasks could provide a neurophysiological biomarker of fatigue.

Environmental Health and Biosafety Risk Assessment Guidance for Commercial-Scale Cell and Gene Therapy Manufacturing.

Introduction: This article aims to identify best practices, improve risk controls, and aid regulatory agencies in developing guidance for environmental and biosafety risk assessment for commercial-scale cell and gene therapy manufacturing. Methods: A cross-functional team should start with hazard classification and testing requirements for materials used or generated by the process and process hazard characterization. Results: The team develops a safety profile of the process to mitigate risks, including: product biological contamination risk and process controls, including raw materials, facilities, operator and environmental controls, and method of detection;a technical review of the process to evaluate the operational and engineering controls;monitoring systems to mitigate the risk of failure and/or breach of the system, preventing the release of material to the facility or operator exposure;site sanitization strategy and facility containment measures, including engineering designs, air handling systems, spill containment measures, surface cleanability, waste flows, and decontamination practices;a review of site practices, including process, employee, material and waste flows, staff training, controlled access, operator gowning, and emergency response plans/measures. Discussion: The cross-functional team should regularly reconvene to provide solutions for enhanced process control, process life-cycle management, monitor assumptions, and track performance. The plan must be revised following any relevant failure event or process change. Conclusion: A risk assessment template is shared to bring to the reader's attention the complexity of commercial-scale manufacturing, areas to assess, potential questions to ask, and other pertinent parties who may input to the risk assessment.

An extended model for culture-dependent heterogenous gene expression and proliferation dynamics in mouse embryonic stem cells.

During development, pluripotency is a transient state describing a cell's ability to give rise to all three germ layers and germline. Recent studies have shown that, in vitro, pluripotency is highly dynamic: exogenous stimuli provided to cultures of mouse embryonic stem cells, isolated from pre-implantation blastocysts, significantly affect the spectrum of pluripotency. 2i/LIF, a recently defined serum-free medium, forces mouse embryonic stem cells into a ground-state of pluripotency, while serum/LIF cultures promote the co-existence of ground-like and primed-like mouse embryonic stem cell subpopulations. The latter heterogeneity correlates with temporal fluctuations of pluripotency markers, including the master regulator Nanog, in single cells. We propose a mathematical model of Nanog dynamics in both media, accounting for recent experimental data showing the persistence of a small Nanog Low subpopulation in ground-state pluripotency mouse embryonic stem cell cultures. The model integrates into the core pluripotency Gene Regulatory Network both inhibitors present in 2i/LIF (PD and Chiron), and feedback interactions with genes found to be differentially expressed in the two media. Our simulations and bifurcation analysis show that, in ground-state cultures, Nanog dynamics result from the combination of reduced noise in gene expression and the shift of the system towards a monostable, but still excitable, regulation. Experimental data and agent-based modelling simulations indicate that mouse embryonic stem cell proliferation dynamics vary in the two media, and cannot be reproduced by accounting only for Nanog-dependent cell-cycle regulation. We further demonstrate that both PD and Chiron play a key role in regulating heterogeneity in transcription factor expression and, ultimately, mouse embryonic stem cell fate decision.

Development of a platform process for adenovirus purification that removes human SET and nucleolin and provides high purity vector for gene delivery.

The manufacturing of virus occurs at a modest scale in comparison to many therapeutic proteins mainly because a gene therapy dose is typically only µg of vector. Although modest in scale the generation of high purity virus is challenging due to low viral expression levels and the difficulties in adequately characterizing such a large and complex molecule. A 100 L bioreactor might produce only 100 mg of virus that must be separated from host and process impurities that are typically greater by several orders of magnitude. Furthermore, in the later purification stages the main milieu component is often virus at low concentration (µg/mL) which may non-specifically adsorb to purification surfaces resulting in a lowered virus recovery. This study describes our approach to develop a scalable, manufacturable robust process for an Adenovirus (Ad) gene therapy vector. A number of analytical tools were developed to guide the purification design. During process development, two human proteins, SET and nucleolin, were identified in viral preparations. To our knowledge, this is the first time that SET and nucleolin have been described in Ad. In this report we detail a process for their removal and the robust removal of all process, product and host cell impurities.

Thermal stability of adenovirus type 2 as a function of pH.

Although several recent studies have focused on the characterization and formulation of adenovirus type 5, similar efforts focusing on adenovirus type 2 (Ad2) have been lacking. To this end, multiple biophysical techniques were employed to investigate the thermal stability of Ad2 as a function of pH. Highly cooperative thermally induced changes in capsid conformation were detected near 45 and 65 degrees C under neutral conditions. The first transition is attributed to the loss of the penton bases and their associated fibers followed by more complete physical degradation at higher temperatures. Data in this work as well as previous studies suggest that a common mechanism of icosahedral virus thermal degradation exists. Conformational changes detected in these studies occurred at increasingly higher temperatures with decreasing pH from 8 to 5 suggesting that the physical stability of Ad2 is enhanced under mildly acidic conditions. To consolidate the data generated in these studies, a multi-dimensional Eigenvector approach was employed to generate an empirical phase diagram (EPD) of Ad2. The EPD identifies conditions, or "phase boundaries," where structural integrity is altered, in addition to providing a tool that can be used to identify conditions under which forced degradation and excipient-screening studies can be conducted.

Dual therapeutic utility of proteasome modulating agents for pharmaco-gene therapy of the cystic fibrosis airway.

Pharmacologic- and gene-based therapies have historically been developed as two independent therapeutic platforms for cystic fibrosis (CF) lung disease. Inhibition of the dysregulated epithelial Na channel (ENaC) is one pharmacologic approach to enhance airway clearance in CF. We investigated pharmacologic approaches to enhance CFTR gene delivery with recombinant adeno-associated virus (rAAV) and identified compounds that significantly improved viral transduction while simultaneously inhibiting ENaC activity through an unrelated mechanism. Treatment of human CF airway epithelia with proteasome modulating agents (LLnL and doxorubicin) at the time of rAAV2 or rAAV2/5 infection dramatically enhanced CFTR gene delivery and correction of CFTR-mediated short-circuit currents. Surprisingly, these agents also facilitated long-term (15-day) functional inhibition of ENaC currents independent of CFTR vector administration. Inhibition of ENaC activity was predominantly attributed to a doxorubicin-dependent decrease in gamma-ENaC subunit mRNA expression and an increase in gamma-ENaC promoter methylation. This is the first report to describe the identification of compounds with dual therapeutic action that are able to enhance the efficacy of CFTR gene therapy to the airway while simultaneously ameliorating primary aspects of CF disease pathophysiology. The identification of such compounds mark a new area for drug development, not only for CF, but also for other gene therapy disease targets.

Distinct classes of proteasome-modulating agents cooperatively augment recombinant adeno-associated virus type 2 and type 5-mediated transduction from the apical surfaces of human airway epithelia.

Tripeptidyl aldehyde proteasome inhibitors have been shown to effectively increase viral capsid ubiquitination and transduction of recombinant adeno-associated virus type 2 (rAAV-2) and rAAV-5 serotypes. In the present study we have characterized a second class of proteasome-modulating agents (anthracycline derivatives) for their ability to induce rAAV transduction. The anthracycline derivatives doxorubicin and aclarubicin were chosen for analysis because they have been shown to interact with the proteasome through a mechanism distinct from that of tripeptidyl aldehydes. Our studies demonstrated that doxorubicin and aclarubicin also significantly augmented rAAV transduction in airway cell lines, polarized human airway epithelia, and mouse lungs. Both tripeptidyl aldehyde and anthracycline proteasome-modulating agents similarly augmented nuclear accumulation of rAAV in A549 and IB3 airway cell lines. However, these two cell types demonstrated cell specificity in the ability of N-acetyl-L-leucyl-L-leucyl-L-norleucine (LLnL) or doxorubicin to augment rAAV transduction. Interestingly, the combined administration of LLnL and doxorubicin resulted in substantially increased transduction (>2,000-fold) following apical infection of human polarized epithelia with either rAAV-2 or rAAV-5. In summary, the cell type specificity of LLnL and doxorubicin to induce rAAV transduction, together with the ability of these compounds to synergistically enhance rAAV transduction in polarized airway epithelial induction, suggests that these two classes of compounds likely modulate different proteasome functions that affect rAAV transduction. Findings from this study provide new insights into how modulation of proteasome function can be effectively used to augment rAAV transduction in airway epithelia for gene therapy of cystic fibrosis.

AAV2 vector harboring a liver-restricted promoter facilitates sustained expression of therapeutic levels of alpha-galactosidase A and the induction of immune tolerance in Fabry mice.

The successful application of gene therapy for the treatment of genetic diseases such as Fabry is reliant on the development of vectors that are safe and that facilitate sustained expression of therapeutic levels of the transgene product. Here, we report that intravenous administration of a recombinant AAV2 vector encoding human alpha-galactosidase A under the transcriptional control of a liver-restricted enhancer/promoter (AAV2/DC190-alphagal) generated significantly higher levels of expression in BALB/c and Fabry mice than could be realized using the ubiquitous CMV promoter (AAV2/CMVHI-alphagal). Moreover, AAV2/DC190-alphagal-mediated hepatic expression of alpha-galactosidase A was sustained for 12 months in BALB/c mice and was associated with a significantly reduced immune response to the expressed enzyme. Subsequent challenge of the AAV2/DC190-alphagal-treated animals with recombinant human alpha-galactosidase A at 6 months failed to elicit the production of anti-alpha-galactosidase A antibodies, suggesting the induction of immune tolerance in these animals. The levels of expression attained with AAV2/DC190-alphagal in the Fabry mice were sufficient to reduce the abnormal accumulation of globotriaosylceramide in the liver, spleen, and heart to basal levels and in the kidney by approximately 40% at 8 weeks. Together, these results demonstrate that AAV2-mediated gene transfer that limits the expression of alpha-galactosidase A to the liver may be a viable strategy for treating Fabry disease.