Improving safety and establishing episomal maintenance of Adeno-associated viral vectors

Adeno-associated viral (AAV) vectors are among the most widely used gene transfer systems in basic and pre-clinical research and have been employed in more than 160 clinical trials. AAV vectors are commonly produced in producer cell lines like HEK293 by co-transfection with a so-called vector plasmid and one (in this work) or two so-called helper plasmids. The vector plasmid contains the transgene cassette of interest (TEC) flanked by AAV’s inverted terminal repeats (ITRs) which serve as packaging signals, whereas the helper plasmid provides the required AAV and helper virus functions in trans. A pivotal aspect of AAV vectorology is the manufacturing of AAV vectors free from impurities arising during the production process. These impurities include AAV vector preparations that contain capsids containing prokaryotic sequences, e.g. antibiotic resistance genes originating from the producer plasmids. In the first part of the thesis we aimed at improving the safety of AAV vectors. As we found that encapsidated prokaryotic sequences (using the ampicillin resistance gene as indicator) cannot be re-moved by standard purification methods we investigated whether the producer plasmids could be replaced by Minicircles (MCs). MCs are circular DNA constructs which contain no functional or coding prokaryotic sequences; they only consist of the TEC and a short sequence required for production and purification. MC counterparts of a vector plasmid encoding for enhanced green fluorescent (eGFP) protein and a helper plasmid encoding for AAV serotype 2 (AAV2) and helper Adenovirus (Ad) genes were designed and produced by PlasmidFactory (Bielefeld, Germany). Using all four possible combinations of plasmid and MCs, single-stranded AAV2 vectors (ssAAV) and self-complementary AAV vectors (scAAV) were produced and characterized for vector quantity, quality and functionality. The analyses showed that plasmids can be replaced by MCs without decreasing the efficiency of vector production and vector quality. MC-derived scAAV vector preparations even exceeded plasmid-derived preparations, as they displayed up to 30-fold improved transduction efficiencies. Using MCs as tools, we found that the vector plasmid is the main source of encapsidated prokaryotic sequences. Remarkably, we found that plasmid-derived scAAV vector preparations contained a much higher relative amount of prokaryotic sequences (up to 26.1 %, relative to TEC) compared to ssAAV vector preparations (up to 2.9 %). By replacing both plasmids by MCs the amount of functional prokaryotic sequences could be decreased to below the limit of quantification. Additional analyses for DNA impurities other than prokaryotic sequences showed that scAAV vectors generally contained a higher amount of non-vector DNA (e.g. adenoviral sequences) than ssAAV vectors. For both, ssAAV and scAAV vector preparations, MC-derived vectors tended to contain lower amounts of foreign DNA. None of the vectors tested could be shown to induce immunogenicity. In summary we could demonstrate that the quality of AAV vector preparations could be significantly improved by replacing producer plasmids by MCs. Upon transduction of a target tissue, AAV vector genomes predominantly remain in an episomal state, as duplex DNA circles or concatemers. These episomal forms mediate long-term transgene expression in terminally differentiated cells, but are lost in proliferating cells due to cell division. Therefore, in the second part of the thesis, in cooperation with Claudia Hagedorn and Hans J. Lipps (University Witten/Herdecke) an AAV vector genome was equipped with an autonomous replication element (Scaffold/matrix attachment region (S/MAR)). AAV-S/MAR encoding for eGFP and a blasticidin resistance gene and a control vector with the same TEC but lacking the S/MAR element (AAV-ΔS/MAR) were produced and transduced into highly proliferative HeLa cells. Antibiotic pressure was employed to select for cells stably maintaining the vector genome. AAV-S/MAR transduced cells yielded a higher number of colonies than AAV-ΔS/MAR-transduced cells. Colonies derived from each vector transduction were picked and cultured further. They remained eGFP-positive (up to 70 days, maximum cultivation period) even in the absence of antibiotic selection pressure. Interestingly, the mitotic stability of both AAV-S/MAR and control vector AAV-ΔS/MAR was found to be a result of episomal maintenance of the vector genome. This finding indicates that, under specific conditions such as the mild selection pressure we employed, “common” AAV vectors persist episomally. Thus, the S/MAR element increases the establishment frequency of stable episomes, but is not a prerequisite.

Schools' guide : How to select providers of road safety education programs for senior school students

Road safety education is not just about safe driving. Best practice road safety education seeks to improve knowledge and change attitudes relating to being safe, and making sure others are safe on the road. Typical topics might include: • Strengthening attitudes toward safe road use behaviours and avoiding risks • Supporting behaviours to ensure others are safe • Promoting knowledge of traffic rules.

Safer construction : the development of a voluntary code of practice to improve safety performance in the Australian construction industry

In Australia, between 1994 and 2000, 50 construction workers were killed each year as a result of their work, the industry fatality rate, at 10.4 per 100,000 persons, is similar to the national road toll fatality rate and the rate of serious injury is 50% higher than the all industries average. This poor performance represents a significant threat to the industry’s social sustainability. Despite the best efforts of regulators and policy makers at both State and Federal levels, the incidence of death, injury and illness in the Australian construction industry has remained intransigently high, prompting an industry-led initiative to improve the occupational health and safety (OHS) performance of the Australian construction industry. The ‘Safer Construction’ project involves the development of an evidence-based Voluntary Code of Practice for OHS in the industry.

Targeting injury prevention strategies to adolescents of Pacific Islander descent : what will work?

Background: Ethnicity is rarely considered in injury prevention program development, even though this is known to impact on participation in injury risk behaviour. An understanding of injury, risk behaviour and risk and protective factors specific to adolescents of Pacific Islander descent will inform the development of prevention strategies appropriate to this group.----- Aims: To determine patterns of injury and associated risk behaviour among adolescents of Pacific Islander descent, and to understand the risk and protective factors that influence injury rates among this group.----- Methods: A total of 875 Year 9 students from five Queensland high schools completed a survey during health classes. Seventy-one students (n = 38 male) identified as Pacific Islander. The survey consisted of scales examining injury, risk taking behaviour, and relationships with family, school and police.----- Results: The leading causes of injury among adolescents of Pacific Islander descent were sports (48%) and transport (e.g. 45% reported bicycle injuries). Interpersonal violence related injuries were also relatively frequent, with 28% having been injured in a fight. Reports of alcohol use were relatively low (20% c.f. 40% of the remaining sample), however reports of other risk behaviours were relatively high (e.g. 43% c.f. 25% of remaining sample reported a group fight).----- Discussion and conclusions: Conclusions will be drawn regarding risk-related injuries reported by adolescents of Pacific Islander descent and those of other ethnic backgrounds. Additionally, risk and protective factors relating to family, school and police will be explored, in order to inform prevention strategies appropriate to this group.

The importance of school connectedness in adolescent risk taking and injury prevention

Background: Injury is the leading cause of mortality for young people in Australia (AIHW, 2008). Adolescent injury mortality is consistently associated with risk taking behaviour, including transport and interpersonal violence (AIHW, 2003), which often occurs in the context of alcohol and other substance use. A rapid increase in risk taking and injury through early to late adolescence highlights the need for effective school based interventions. Aim: The aim of the current research was to examine the relationship between school connectedness and adolescent risk and injury, in order to inform effective prevention approaches. School connectedness, or students’ feelings of belongingness to school, has been shown to be a critical protective factor in adolescence which can be targeted effectively through teacher interventions. Despite evidence linking low school connectedness with increased health risk behaviour, including substance use and violence, research has not yet addressed possible links between connectedness and a broader range of risk taking behaviours (e.g. transport risks) or injury. Method: This study involved background data collection to inform the development of an intervention. A total of 595 Year 9 students (aged 13-14 years) from 5 Southeast Queensland high schools completed questionnaires that included measures of school connectedness, risk taking behaviour, alcohol and other substance use, and injuries. Results: Increased school connectedness was found to be associated with fewer transport risk behaviours and with decreased alcohol and other substance use for both males and females. Similarly, increased school connectedness was associated with fewer passenger and motorcycle injuries for male participants. Both males and females with increased school connectedness reported fewer alcohol related injuries. Implications: These results indicate that school connectedness appears to have protective effects for early adolescence. These findings may also hold for older adolescents and indicate that it may be an important factor to target in school based risk and injury prevention programs. A school connectedness intervention is currently being designed, focusing on teacher professional development. The intervention will be implemented in conjunction with a curriculum based injury prevention program for Year 9 students and will be evaluated through a large scale cluster randomised trial involving 26 schools.

Safety effectiveness indicators project workbook

The Safety Effectiveness Indicators (SEI) Project has used extensive research to determine what safety effectiveness measures can be developed by industry, for industry use to improve its safety performance. These indicators can measure how effectively the 13 safety management tasks1 (SMTs) selected for this workbook are undertaken. Currently, positive performance indicators (PPIs) are only able to measure the number of activities undertaken. They do not provide information on whether each activity is being undertaken effectively, and therefore do not provide data which can be used by industry to target areas of focus and improvement. The initial workbook contained six SMTs, and was piloted on various construction sites during August 2008. The workbook was refined through feedback from the pilot, and 13 SMTs were used in a field trial during the months of October, November and December 2008. The project team also carried out 12 focus groups in Brisbane, Canberra, Sydney and Melbourne during April, May and June 2008, and developed an initial format of this workbook through these groups and team workshops. Simplification of the language was a recurring theme, and we have attempted to do this throughout the project. The challenge has been to ensure we keep the descriptions short, to the point and relevant to all companies, without making them too specific. The majority of the construction industry participants also requested an alteration to the scale used, so a ‘Yes’/‘No’/’Not applicable’ format is used in this workbook. This workbook, based on industry feedback, is for use on site by various construction companies and contains 13 SMTs. However, you are invited to personalise the SEI tools to better suit your individual company and workplaces.

Measuring safety effectiveness : literature review

Cohen (1977) reviewed the then current research on occupational safety and stated that both strong company commitment to safety, and communication between all levels of a company are the most influential factors to improving safety. Other relevant factors included careful selection of staff, and early and continuous training throughout the lifetime with the company. These continue to be important factors in OHS today. There has been a continued decrease in the injury rates since Cohen’s review within the Australian construction industry, however, the construction industry has far more injuries and ill-health than the Australian average, with one fatality occurring on average per week in the Australian Construction Industry. The Fatality rate in the building and construction industry remains three times higher than the national average, and 15% of all industry fatalities are in the building and construction industry. In addition the construction industry pays one of the highest workers’ compensation premium rates – in 2001 alone approximately 0.5% ($267 million) of revenue would have to be allocated to the direct cost of 1998/99 compensations (Office of the Federal Safety Commissioner, 2006). Based on these statistics there is a need to measure and improve safety performance within the construction industry.

Friends looking out for friends as an intervention strategy for young adults' risky driving

Young adults are at the greatest risk of experiences road trauma disproportionately to those in other age groups. While the influence of peers is commonly associated with motor vehicle crashes and injury few studies examine whether their influence can be positive. In particular friends may be able to actively intervene to reduce the likelihood of risky driving (e.g. speeding, drink driving or drug driving) and alcohol use. The aim of this paper is to conduct a systematic review on intervening in risky driving behaviour including the situations in which it is likely or unlikely to occur, factors associated with individuals who might or report having intervened and any evaluated programs that make use of such strategies. In addition a study was conducted with 247 first year university students (32% males) to examine whether young adults report engaging in protective behaviour with their peers in South-east Queensland. In particular, if they intervene if their friends are about to drive after drinking, drive after taking illicit drugs or when speeding. It examines any differences in reported likelihood of discouraging such illegal and dangerous behaviour (in the past 12 months prior to the survey). Findings showed that young adults (17-25 years) did indeed report protective behaviour in relation to friends’ drink driving, drug driving, speeding and binge drinking. Conclusions will be drawn regarding important considerations in developing positive strategies and advertising campaigns that encourage positive behaviours (e.g. ‘don’t let mates drink and drive’).

How do other people influence your driving speed? Exploring the 'who' and the 'how' of social influences on speeding from a qualitative perspective

Using only legal sanctions to manage the speed at which people drive ignores the potential benefits of harnessing social factors such as the influence of others. Social influences on driver speeds were explored in this qualitative examination of 67 Australian drivers. Focus group interviews with 8 driver types (young, mid-age and older males and females, and self-identified Excessive and Rare speeders) were guided by Akers’ social learning theory (Akers, 1998). Findings revealed two types of influential others: people known to the driver (passengers and parents), and unknown other drivers. Passengers were generally described as having a slowing influence on drivers: responsibility for the safety of people in the car and consideration for passenger comfort were key themes. In contrast, all but the Rare speeders reported increasing their speed when driving alone. Parental role modelling was also described. In relation to other drivers, key themes included speeding to keep up with traffic flow and perceived pressure to drive faster. This ‘pressure’ from others to ‘speed up’ was expressed in all groups and reported strategies for managing this varied. Encouragingly, examples of actual or anticipated social rewards for speeding were less common than examples of social punishments. Three main themes relating to social punishments were embarrassment, breaching the trust of others, and presenting an image of a responsible driver. Impression management and self-presentation are discussed in light of these findings. Overall, our findings indicate scope to exploit the use of social sanctions for speeding and social praise for speed limit compliance to enhance speed management strategies.

Alcohol and other drugs in the Australian construction industry : A national evaluation and the best way forward

Anecdotal evidence highlights issues of alcohol and other drugs (AODs) and its association with safety risk on construction sites. Information is limited however regarding the prevalence of AODs in the workplace and there is limited evidential guidance regarding how to effectively address it. This research aimed to scientifically evaluate the use of AODs within the Australian construction industry in order to reduce the potential resulting safety and performance impacts and engender a cultural change in the workforce. A national qualitative and quantitative evaluation of the use of AODs was conducted with approximately 500 employees. Results indicate that as in the general population, a proportion of those sampled in the construction sector may be at risk of hazardous alcohol consumption and support the need for evidence-based, tailored responses. This is the first known study to scientifically evaluate the use of AODs and potential workplace safety impacts in the construction sector.

"There's no way in hell I would pull up" : Deterrent and other effects of vehicle impoundment laws for hooning

Traffic law enforcement is based on deterrence principles, whereby drivers control their behaviour in order to avoid an undesirable sanction. For “hooning”-related driving behaviours in Queensland, the driver’s vehicle can be impounded for 48 hours, 3 months, or permanently depending on the number of previous hooning offences. It is assumed that the threat of losing something of value, their vehicle, will discourage drivers from hooning. While official data shows that the rate of repeat offending is low, an in-depth understanding of the deterrent effects of these laws should involve qualitative research with targeted drivers. A sample of 22 drivers who reported engaging in hooning behaviours participated in focus group discussions about the vehicle impoundment laws as applied to hooning offences in Queensland. The findings suggested that deterrence theory alone cannot fully explain hooning behaviour, as participants reported hooning frequently, and intended to continue doing so, despite reporting that it is likely that they will be caught, and perceiving the vehicle impoundment laws to be extremely severe. The punishment avoidance aspect of deterrence theory appears important, as well as factors over and above legal issues, particularly social influences. A concerning finding was drivers’ willingness to flee from police in order to avoid losing their vehicle permanently for a third offence, despite acknowledging risks to their own safety and that of others. This paper discusses the study findings in terms of the implications for future research directions, enforcement practices and policy development for hooning and other traffic offences for which vehicle impoundment is applied.