Preventing packaging of translatable P5-associated DNA contaminants in recombinant AAV vector preps

Recombinant adeno-associated virus (rAAV) vectors are increasingly being used for clinical gene transfer and have shown great potential for the treatment of several monogenic disorders. However, contaminant DNA from producer plasmids can be packaged into rAAV alongside the intended expression cassette-containing vector genome. The consequences of this are unknown. Our analysis of rAAV preps revealed abundant contaminant sequences upstream of the AAV replication (Rep) protein driving promoter, P5, on the Rep-Cap producer plasmid. Characterization of P5-associated contaminants after infection showed transfer, persistence, and transcriptional activity in AAV-transduced murine hepatocytes, in addition to in vitro evidence suggestive of integration. These contaminants can also be efficiently translated and immunogenic, revealing previously unrecognized side effects of rAAV-mediated gene transfer. P5-associated contaminant packaging and activity were independent of an inverted terminal repeat (ITR)-flanked vector genome. To prevent incorporation of these potentially harmful sequences, we constructed a modified P5-promoter (P5-HS), inserting a DNA spacer between an Rep binding site and an Rep nicking site in P5. This prevented upstream DNA contamination regardless of transgene or AAV serotype, while maintaining vector yield. Thus, we have constructed an rAAV production plasmid that improves vector purity and can be implemented across clinical rAAV applications. These findings represent new vector safety and production considerations for rAAV gene therapy

Effects of insurance status on children's access to specialty care: a systematic review of the literature

<p>Abstract</p> <p>Background</p> <p>The current climate of rising health care costs has led many health insurance programs to limit benefits, which may be problematic for children needing specialty care. Findings from pediatric primary care may not transfer to pediatric specialty care because pediatric specialists are often located in academic medical centers where institutional rules determine accepted insurance. Furthermore, coverage for pediatric specialty care may vary more widely due to systematic differences in inclusion on preferred provider lists, lack of availability in staff model HMOs, and requirements for referral. Our objective was to review the literature on the effects of insurance status on children's access to specialty care.</p> <p>Methods</p> <p>We conducted a systematic review of original research published between January 1, 1992 and July 31, 2006. Searches were performed using Pubmed.</p> <p>Results</p> <p>Of 30 articles identified, the majority use number of specialty visits or referrals to measure access. Uninsured children have poorer access to specialty care than insured children. Children with public coverage have better access to specialty care than uninsured children, but poorer access compared to privately insured children. Findings on the effects of managed care are mixed.</p> <p>Conclusion</p> <p>Insurance coverage is clearly an important factor in children's access to specialty care. However, we cannot determine the structure of insurance that leads to the best use of appropriate, quality care by children. Research about specific characteristics of health plans and effects on health outcomes is needed to determine a structure of insurance coverage that provides optimal access to specialty care for children.</p

The influence of contextual factors on healthcare quality improvement initiatives:what works, for whom and in what setting? Protocol for a realist review

Background&nbsp; Context shapes the effectiveness of knowledge implementation and influences health improvement. Successful healthcare quality improvement (QI) initiatives frequently fail to transfer to different settings, with local contextual factors often cited as the cause. Understanding and overcoming contextual barriers is therefore crucial to implementing effective improvement; yet context is still poorly understood. There is a paucity of information on the mechanisms underlyinghowandwhyQI projects succeed or fail in given settings. A realist review of empirical studies of healthcare QI initiatives will be undertaken to examine the influence and impact of contextual factors on quality improvement in healthcare settings and explore whether QI initiatives can work in all contexts.&nbsp; Methods&nbsp; The review will explore which contextual factors are important, and how, why, when and for whom they are important, within varied settings. The dynamic nature of context and change over time will be explored by examining which aspects of context impact at key points in the improvement trajectory. The review will also consider the influence of context on improvement outcomes (provider- and patient-level), spread and sustainability. The review process will follow five iterative steps: (1) clarify scope, (2) search for evidence, (3) appraise primary studies and extract data, (4) synthesise evidence and draw conclusions and (5) disseminate findings. The reviewers will consult with experts and stakeholders in the early stages to focus the review and develop a programme theory consisting of explanatory &lsquo;context&ndash;mechanism&ndash;outcome&rsquo; configurations. Searches for primary evidence will be conducted iteratively. Data will be extracted and tested against the programme theory. A review advisory group will oversee the review process. Review findings will follow RAMESES guidelines and will be disseminated via a report, presentations and peer-reviewed publications.&nbsp; Discussion&nbsp; The review will update and consolidate evidence on the contextual conditions for effective improvement and distil new knowledge to inform the design and development of context-sensitive QI initiatives. This review ties in with the study of improvement programmes as vehicles of change and the development of an evidence base around healthcare improvement by addressing whether QI initiatives can work in all contexts.&nbsp; Systematic review registration&nbsp; PROSPERO&nbsp;CRD4201706213

Inhibition of Neuroblastoma Tumor Growth by Targeted Delivery of MicroRNA-34a Using Anti-Disialoganglioside GD2 Coated Nanoparticles

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 (GD(2)), providing a target for tumor-specific delivery.Nanoparticles encapsulating miR-34a and conjugated to a GD(2) 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.These novel findings highlight the potential of anti-GD(2)-nanoparticle-mediated targeted delivery of miR-34a for both the treatment of GD(2)-expressing tumors, and as a basic discovery tool for elucidating biological effects of novel miRNAs on tumor growth

Syndromics: A Bioinformatics Approach for Neurotrauma Research

Substantial scientific progress has been made in the past 50 years in delineating many of the biological mechanisms involved in the primary and secondary injuries following trauma to the spinal cord and brain. These advances have highlighted numerous potential therapeutic approaches that may help restore function after injury. Despite these advances, bench-to-bedside translation has remained elusive. Translational testing of novel therapies requires standardized measures of function for comparison across different laboratories, paradigms, and species. Although numerous functional assessments have been developed in animal models, it remains unclear how to best integrate this information to describe the complete translational “syndrome” produced by neurotrauma. The present paper describes a multivariate statistical framework for integrating diverse neurotrauma data and reviews the few papers to date that have taken an information-intensive approach for basic neurotrauma research. We argue that these papers can be described as the seminal works of a new field that we call “syndromics”, which aim to apply informatics tools to disease models to characterize the full set of mechanistic inter-relationships from multi-scale data. In the future, centralized databases of raw neurotrauma data will enable better syndromic approaches and aid future translational research, leading to more efficient testing regimens and more clinically relevant findings