9 research outputs found
Producer cell line engineering for large volume manufacturing of therapeutic AAV
Recombinant adeno-associated viruses (rAAV) are among the most promising gene therapy delivery vectors for treating patients with genetic abnormalities. rAAV can safely deliver long-lasting expression of a therapeutic transgene to target cells. Multiple studies using rAAV have demonstrated sustained transgene expression in cultured cells and pre-clinical models, suggesting that rAAV could provide a cure for certain diseases. Moreover, bioengineering advancements have expanded the viral tropism beyond the constraints of naturally occurring AAV capsids, increasing the cell types that can be thought of as targets. Taken together, rAAV therapies have attractive qualities to safely address the needs of patients where other modalities may fall short. One challenge with therapeutic rAAV is the ability to generate enough virus for clinical trials and commercial supply. This challenge is particularly true with neuromuscular or hemophilia patients in which doses can exceed 1 x 1014 viral genomes per patient. Typical yields from a rAAV production are around 104 viral genomes per cell, meaning batch cell numbers would need to exceed 1010 for a single dose. These doses require a robust, scalable platform to generate quantities of rAAV to meet patient demand. Biogen has selected the producer cell line (PCL) platform to meet the large demand for therapeutic rAAV. A PCL is a stable cell line engineered to contain the ITR flanked transgene of interest and AAV sequences needed to produce rAAV upon addition of helper virus. We will present our rationale for selecting the PCL platform as a cost-effective manufacturing strategy for gene therapy programs as well as current technical improvements and our vision for the next generation PCL platform
Novel Pancreatic Endocrine Maturation Pathways Identified by Genomic Profiling and Causal Reasoning
<div><p>We have used a previously unavailable model of pancreatic development, derived <em>in vitro</em> from human embryonic stem cells, to capture a time-course of gene, miRNA and histone modification levels in pancreatic endocrine cells. We investigated whether it is possible to better understand, and hence control, the biological pathways leading to pancreatic endocrine formation by analysing this information and combining it with the available scientific literature to generate models using a casual reasoning approach. We show that the embryonic stem cell differentiation protocol is highly reproducible in producing endocrine precursor cells and generates cells that recapitulate many aspects of human embryonic pancreas development, including maturation into functional endocrine cells when transplanted into recipient animals. The availability of whole genome gene and miRNA expression data from the early stages of human pancreatic development will be of great benefit to those in the fields of developmental biology and diabetes research. Our causal reasoning algorithm suggested the involvement of novel gene networks, such as NEUROG3/E2F1/KDM5B and SOCS3/STAT3/IL-6, in endocrine cell development We experimentally investigated the role of the top-ranked prediction by showing that addition of exogenous IL-6 could affect the expression of the endocrine progenitor genes NEUROG3 and NKX2.2.</p> </div
Induction of insulin expression is not accompanied with epigenetic changes.
<p>(A) Gene expression (blue) and H3K4me<sup>3</sup> level (red) profiles for SOX17. The horizontal dashed line indicates the background H3K4me<sup>3</sup> level. (B) H3K4me<sup>3</sup> reads piled up over the SOX17 gene body at days 0, 2 and 11. The start and end points of SOX17 are indicated by dashed lines. (C&D) As for (A&B) but for Insulin.</p
Expression of cholesterol metabolizing genes shows a strong time dependence.
<p>(A) The cholesterol biosynthesis pathway from squalene-PP (top left) to cholesterol (top right) showing all enzymes and some intermediates. Enzymes whose gene expression patterns fall into the largest cluster seen in the heatmap of the expression levels for these enzymes (B) are colored purple, other enzymes are light blue. LIPA and SOAT1/2 interconvert cholesterol and cholesterol ester. The cholesterol sensitive miRNA-33a shows a similar expression pattern (C) to the main cluster.</p
Epigenetic changes can be used to identify novel regulatory miRNAs.
<p>(A–C) Gene expression (blue), miRNA expression (green) and H3K4me<sup>3</sup> levels for CD47, ITGB1 and ANP32B and the miRNAs associated with them. In all 3 cases the miRNA is predicted to regulate the relevant gene and is also anti-correlated in level. In (A) H3K4me<sup>3</sup> levels correlate closely with the gene expression, whilst in (B & C) there is no correlation, suggesting a stronger role for the miRNA regulation.</p
Transcriptome analysis shows the reproducibility of the differentiation protocol.
<p>(A) Genome wide gene expression correlation heatmap between samples. Samples are clustered by the Euclidean distance between rows/columns and single linkage clustering. The colored bar along the top of the heatmap indicates the timepoint at which the sample was taken (pink: day 0, maroon: day 11). (B) Heatmap of expression of selected markers. The developmental stage is indicated by the labels to the left of the heatmap.</p
IL6, the top CRE prediction, has effects on expression of endocrine markers.
<p>(A) Treatment of pancreatic aggregates with IL-6 induces de novo gene expression of the pro-endocrine transcription factors NEUROG3 and NKX2.2, indicating commitment of pancreatic progenitor cells into the endocrine lineage. Noggin induction of these genes resulted in 8-fold increases (data not shown) (B) Gene expression in response to IL-6 was compared between whole aggregates (mixture of pancreatic progenitors and endocrine cells) and cultures of enriched endocrine cells (depleted of pancreatic progenitors). Induction of NKX2.2 expression was only seen in whole aggregates, consistent with the role of IL-6 in converting pancreatic progenitors into new endocrine cells. Enhanced expression of NEUROD1, IAPP, and SOMATOSTATIN seen in response to IL-6 in purified endocrine cells, suggesting IL-6 has additional roles in committed endocrine cells. No significant differences seen in INSULIN or GCG gene expression. Statistical testing using a standard t-test was performed.</p
Top 20 protein causal drivers of early pancreatic endoderm formation between day 8 and day 11.
<p>The number of correctly, incorrectly and ambiguously explained gene expression observations are given for each gene as well as the predicted direction of regulation (up meaning activation/down meaning inhibition). The notes for each gene indicate that in cases where the gene is already associated with beta cell function whether it is generally considered a positive or negative regulator of beta cell differentiation, proliferation (growth) or apoptosis. All hypotheses pass correctness and enrichment p-value thresholds of 10<sup>−5</sup>.</p