John T. Gray

Exogenous control of mammalian gene expression through modulation of RNA self-cleavage.

Recent studies on the control of specific metabolic pathways in bacteria have documented the existence of entirely RNA-based mechanisms for controlling gene expression. These mechanisms involve the modulation of translation, transcription termination or RNA self-cleavage through the direct interaction of specific intracellular metabolites and RNA sequences. Here we show that an analogous RNA-based gene regulation system can effectively be designed for mammalian cells via the incorporation of sequences encoding self-cleaving RNA motifs into the transcriptional unit of a gene or vector. When correctly positioned, the sequences lead to potent inhibition of gene or vector expression, owing to the spontaneous cleavage of the RNA transcript. Administration of either oligonucleotides complementary to regions of the self-cleaving motif or a specific small molecule results in the efficient induction of gene expression, owing to inhibition of self-cleavage of the messenger RNA. Efficient regulation of transgene expression is shown in a variety of mammalian cell lines and live animals. In conjunction with other emerging technologies, this methodology may be particularly applicable to the development of gene regulation systems tailored to any small inducer molecule, and provide a novel means of biological sensing in vivo that may have an important application in the regulated delivery of protein therapeutics.

Climatic information from 18O/16O ratios of cellulose in tree rings

THE high precision of dendrochronology makes possible the construction of short (∼ 9,000 yr), detailed records of past climates. This paper explores the possibility of using the oxygen isotopic composition of cellulose from tree rings as a ‘thermometer’ to measure past temperatures. Using meteorological data from Edmonton we have shown that temperatures can be measured with a precision of ∼ ±0.15°C when averaged over a 5-yr period.

Good manufacturing practice production of self-complementary serotype 8 adeno-associated viral vector for a hemophilia B clinical trial.

To generate sufficient clinical-grade vector to support a phase I/II clinical trial of adeno-associated virus serotype 8 (AAV8)-mediated factor IX (FIX) gene transfer for hemophilia B, we have developed a large-scale, good manufacturing practice (GMP)-compatible method for vector production and purification. We used a 293T-based two-plasmid transient transfection system coupled with a three-column chromatography purification process to produce high-quality self-complementary AAV2/8 FIX clinical-grade vector. Two consecutive production campaigns using a total of 432 independent 10-stack culture chambers produced a total of ∼2u2009×u200910(15) vector genomes (VG) by dot-blot hybridization. Benzonase-treated microfluidized lysates generated from pellets of transfected cells were purified by group separation on Sepharose beads followed by anion-exchange chromatography. The virus-containing fractions were further processed by gel filtration and ultrafiltration, using a 100-kDa membrane. The vector was formulated in phosphate-buffered saline plus 0.25% human serum albumin. Spectrophotometric analysis suggested ∼20% full particles, with only low quantities of nonviral proteins were visible on silver-stained sodium dodecyl sulfate-polyacrylamide gels. A sensitive assay for the detection of replication-competent AAV was developed, which did reveal trace quantities of such contaminants in the final product. Additional studies have confirmed the long-term stability of the vector at -80°C for at least 24 months and for at least 24u2009hr formulated in the clinical diluent and stored at room temperature within intravenous bags. This material has been approved for use in clinical trials in the United States and the United Kingdom.

Developments in the treatment of hemophilia B: focus on emerging gene therapy

Hemophilia B is a genetic disorder that is characterized by a deficiency of clotting factor IX (FIX) and excessive bleeding. Advanced understanding of the pathophysiology of the disease has led to the development of improved treatment strategies that aim to minimize the acute and long-term complications of the disease. Patients with hemophilia B are ideal candidates for gene therapy, mostly because a small increase in protein production can lead to significantly decreased bleeding diathesis. Although human clotting FIX was cloned and sequenced over 30 years ago, progress toward achieving real success in human clinical trials has been slow, with long-term, therapeutically relevant gene expression only achieved in one trial published in 2011. The history of this extensive research effort has revealed the importance of the interactions between gene therapy vectors and multiple arms of the host immune system at multiple stages of the transduction process. Different viral vector systems each have unique properties that influence their ability to deliver genes to different tissues, and the data generated in several clinical trials testing different vectors for hemophilia have guided our understanding toward development of optimal configurations for treating hemophilia B. The recent clinical success implementing a novel adeno-associated virus vector demonstrated sufficient FIX expression in patients to convert a severe hemophilia phenotype to mild, an achievement which has the potential to profoundly alter the impact of this disease on human society. Continued research should lead to vector designs that result in higher FIX activity at lower vector doses and with reduced host immune responses to the vector and the transgene product.

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