The Cross-Border Biotech Blog

Biotechnology, Health and Business in Canada, the United States and Worldwide

Monthly Archives: February 2012

Friday Science Review: February 24, 2012

In my first contribution to the Cross-Border Biotech Blog’s Friday Science Review we have a promising advance in the treatment of Huntington’s disease by an international collaboration led by researchers from the Department of Pharmacology at the University of Alberta and another look at the surprisingly small number of critical genes in organisms by the Fraser lab at the University of Toronto.

A promising new avenue for Huntington’s Disease

The autosomal dominant genetic disorder Huntington’s disease (HD) is caused by the expansion of the CAG codon in the Huntingtin gene and the resulting inclusion of an abnormally long poly-glutamine stretch in the Huntingtin (Htt) protein. The expanded poly-glutamine stretch results in misfolding of the Htt protein and the formation of aggregates that are deposited as inclusion bodies within cells. As Htt protein is most highly expressed in neuronal cells, the aggregates lead to impaired neuronal transmission and ultimately to neuronal death, resulting in the loss of muscle coordination, cognitive impairment and psychiatric problems that are characteristic of HD.

Current treatments for HD are palliative in nature only, however, the paper by Di Pardo et al, published in PNAS, aimed to address the molecular mechanisms of HD by intraventricular infusion of the ganglioside GM1 – for those of us that like to picture molecules, GM1 is a glycosphingolipid with a headgroup attached sialic acid. GM1 levels have previously been shown to be reduced in HD animal models and post mortem HD patient brain samples. In this study, they show that infusion of GM1 reduces Htt toxicity and restores normal motor function in symptomatic HD mice models. GM1 appeared to exert its effect, by inducing an increase in DARPP-32 levels and phosphorylation, as well as by inducing the phosphorylation of the Htt protein at specific serine residues that reduce Htt toxicity.

While there is the chance that the rather invasive GM1 approach itself might represent a therapy, at the least, these results suggest new therapeutic pathways to research and target for the treatment of HD.

Eukaryotic genes: Necessary, but not required?

Previous studies in a range of model organisms have presented us with something of a paradox: eukaryotic genomes are highly conserved indicating that most genes are functionally important, yet only a minority of genes have detectable loss-of-function phenotypes. Models explaining this paradox range from suggestions that genetic networks have evolved to be robust and resistant to individual mutations, to suggestions that the targeted genes are essential, but just don’t happen to be required in the particular conditions used in the experiments.

The Fraser lab at the Donnelly Centre of the University of Toronto has helped resolve this paradox by showing in last weeks issue of Cell that, in fact, the majority of genes in C. elegans, if individually suppressed by RNA mediated interference, result in lower multi-generational fitness compared to wild-type. This result challenges the model that genetic networks are robust and instead suggests that the loss of most genes results in phenotypes that were too subtle to have been detected by previous assays. The complex interactions and subtle phenotypes, that manifested as decreased fitness, emphasizes that the development of a systems level understanding of gene function will be increasingly important to understand the molecular basis of diseases.

Other Publications

  • SKI-1 and Furin Generate Multiple RGMa Fragments that Regulate Axonal Growth. Developmental CellToronto Western Research Institute ♦ University of Toronto
  • Negative Supercoiling Creates Single-Stranded Patches of DNA That Are Substrates for AID-Mediated Mutagenesis. PLoS Genetics. University of Toronto
  • Genetic variation in cell death genes and risk of non-hodgkin lymphoma. PLoS One. Canada’s Michael Smith Genome Sciences Centre, BC Cancer Agency
  • Caffeic Acid phenethyl ester and its amide analogue are potent inhibitors of leukotriene biosynthesis in human polymorphonuclear leukocytes. PLoS One. Université de Moncton

Some Partnering Basics: Part 16 of Valuation and Other Biotech Mysteries

[Ed. This is the sixteenth part in Wayne's series. You can access the whole thing by clicking here. Please leave comments or questions on the blog and Wayne will address them in future posts in this series.]

If partnering is basically the process of selling an asset, the first step is to let people know more about the asset which is for sale. Three initial questions to be answered are:

  • What companies do you talk to;
  • Who do you talk to at these companies; and
  • What do you tell them?

The answer to the first question is easy – you talk to all companies which may have an interest in commercializing your asset. The list of companies should be easy to prepare by looking at information sources already discussed in this blog series, including:

  • Annual reports and pipeline reviews by pharma and biotech companies;
  • Searching for all companies running clinical trials in the therapeutic fields targeted by your asset; and
  • Subscription newsletters and databases.

The people who need to be targeted are those who are both directly and indirectly involved in the partnering process. Since you can never predict which initial contact will lead to the deal, you have to target all potential contacts at all potential partners. Read more of this post

Monday Biotech Deal Review: February 13, 2012

Welcome to your Monday Biotech Deal Review for February 13, 2012.  There was some interesting biotech activity over the previous two weeks, including the closing by Oncolytics of its $18.5M equity financing and the withdrawal by Valeant of its offer to acquire ISTA Pharmaceuticals and its acquisition of a Brazilian food and sport supplement company.  Read on to learn more.   Read more of this post

Friday Science Review: February 10, 2012

Automated Regulation of Inhibitory Feedback Signalling Leads to Rapid and Robust Expansion of Cord Blood-Derived Hematopoietic Stem Cells

University of Toronto ♦ Genomics Institute of the Novartis Research Foundation ♦ Heart and Stroke/Richard Lewar Centre of Excellence ♦ McEwen Centre for Regenerative Medicine

Published in Cell Stem Cell, February 3, 2012

The greatest current justification for the storage of cord blood stem cells, in the setting of both the private and public cord blood bank, is for hematopoietic stem cell transplantation (HSCT) for reconstitution of the bone marrow compartment; most often following intensive chemotherapy regimens that ablate the bone marrow completely.

Cord blood is a viable source of hematopoietic stem cells (HSCs), however a single cord blood unit in its original form only contains a small quantity of these cells. It has been found that the most critical factor in patient survival following HSCT is administering a threshold cell dose (roughly 30 million cells per kilogram patient) that must be met or surpassed in order to achieve successful engraftment and reconstitution of the bone marrow. As a result, the majority of clinical studies utilizing cord blood stem cells for HSCT have been reserved for the paediatric population. However, even in children, successful engraftment and recovery is by no means a given. Despite the widespread storage of cord blood units in public banks that can be HLA-matched to recipients, and the numerable benefits of this source of HSCs over others, cord blood has yet to become a viable solution for HSCT.

One treatment paradigm under current investigation in the clinic is double cord blood transplant, wherein two units are transplanted simultaneously in order to boost cell number. The shortcoming of this approach is the difficulty in acquiring two units that are HLA-matched to the recipient. Although cord blood is better tolerated by the host’s immune system following transplantation, relatively speaking, and typically exhibits lower levels of graft versus host disease than other sources of HSCs, finding two adequately safe units for a single patient is no trivial task. With a cord blood unit ringing in at $30,000 a hit (from a public bank) the economics of this approach are also prohibitive.

A second approach has been to expand cord blood units in the lab prior to transplantation. This process increases the number of HSCs in the unit by several-fold; the results depending on the protocol and hands at work. Preclinical studies show that expanded cord blood stem cells can reconstitute the bone marrow compartment in immune compromised mice. The medical community has begun preliminary studies in the clinic mixing expanded cord blood stem cells with unexpanded (mixed because there is currently not enough definitive evidence to suggest that expanded cord blood stem cells retain repopulating activity). The results of these studies so far have failed to show that the expanded product contributes significantly to engraftment and recovery. Expansion protocols have become iteratively better, however limitations on HSC number, and their ability to accurately home to the bone marrow compartment and engraft, have prevented expanded units from reaching their potential in the clinic.

Expansion protocols modulate molecular mechanisms that regulate stem cell fate and proliferation in order to maximize the number of HSCs produced. Two approaches have primarily been used. The first, cytokine-driven expansion, utilizes molecular messengers relevant to the bone marrow niche. These proteins interact with surface markers on HSCs, triggering pathways that help reinforce cell fate decision towards the HSC identity. Cytokines are amenable to expansion as cells can be grown in 3-dimensional space, often in bags, which allows for easier scale-up. This being said, it is arguable that the approach is flawed, as it fails to truly recapitulate signaling mechanisms in the bone marrow niche where cell-to-cell contact is critical for the maintenance of different HSC pools.

The second approach, stromal-driven expansion, expands HSCs in the presence of a second population of cells known as stromal cells. While this cell culture approach provides a microenvironment that more accurately reflects the niche an HSC would experience inside the body, it is exceedingly difficult to scale-up to produce clinically relevant cell numbers.

Both approaches struggle in producing large numbers of clinically relevant populations of HSCs. Emerging data supports the idea that not all HSCs in the bone marrow compartment are equivalent. Slight differences in their gene and protein expression stratify them into classes that behave differently in terms of their capability to home and engraft. Some HSCs exhibit the classical HSC markers but only retain repopulating capacity transiently. Other rarer HSC populations exhibit a unique and specialized capacity to form colonies over the long-term. These stem cells, known as long-term repopulating HSCs (LTR-HSCs), are the cells that expansion protocols must produce if they are to create a cord blood product that is useful to humans in the clinic. In addition, neither approach accounts for the production of HSC progeny that amass within the cell culture system. These differentiated cells produce high concentrations of inhibitory feedback molecules that prevent HSC proliferation.

A transformative development in this space is the advent of a cell culture platform that enables rapid expansion of hematopoietic stem cells from a cord blood unit at some of the highest levels ever achieved. Developed by Peter Zandstra at the University of Toronto, this closed-system approach utilizes a controlled fed-batch media dilution strategy to reduce concentrations of proteins that inhibit stem cell proliferation. Within 12 days, LTR-HSC populations can be scaled-up to 11 times their original number while retaining their capacity to self-renew and differentiate into cells of multiple lineages. At its core, the platform hinges on the concept that HSC self-renewal and differentiation are regulated tightly by secreted factors that either promote or inhibit stem cell proliferation.

Zandstra’s group took a computational approach based on the effects of feedback signaling to design the expansion protocol. Measurements of secreted factors were taken from previously established in vitro growth conditions to identify factors that had inhibitory effects on HSCs. Computational simulations were then performed to model the effects that inhibitory proteins would have on stem cell population dynamics. Simulations depicted an accumulation of predominantly inhibitory proteins within the system. Hence, the group rationalized that media exchange would be a key advance in stimulating stem cell expansion. Further investigation with the simulation predicted that a fed-batch process (continuous input of new media) would outperform both perfusion (continuous input of new media and output of old media) and frequent full or partial media exchange. The hypothesis generated was then tested utilizing an automated media delivery system, which confirmed that the fed-batch approach led to a significant increase in the absolute numbers of various HSC subpopulations over other media exchange processes.

A critical component of this study was evaluating the increase in expansion of LTR-HSCs. The only means in which to do this is to carry out transplantation studies in immune compromised mice. Repopulation of the bone marrow compartment was quantified by judging the extent of the contribution of human cells to hematopoietic reconstitution. All of the mice that experienced successful repopulation of the bone marrow exhibited multilineage differentiation, as indicated by the presence of human cells from the myeloid, lymphoid, and erythroid lineages, and the presence of T-cells. Importantly, human cells from repopulated mice could be transplanted to reconstitute the bone marrow of secondary mouse recipients, confirming the long-term engraftment potential of the HSCs at hand.

Limiting dilution analysis was used to quantify the expansion of LTR-HSCs. In fresh cord blood the frequency of LTR-HSCs was roughly 1 in 14,700. After 8 days of growth in the fed-batch system this expansion was increased by 7.6-fold to a frequency of 1 in 1,940. And finally, after 12 days the expansion had increased by 11-fold to a frequency of 1 in 1,334.

Zandstra has created a cell culture technology that rapidly and cost-effectively expands clinically relevant populations of blood stem cells that retain the ability to engraft and contribute to hematopoietic reconstitution over the long-term. A technology of this nature is truly enabling. Not only does it provide new potential to the hundreds of thousands of cord blood units currently stored in public stem cell banks, it ensures, at least in the eyes of HSCT with cord blood stem cells, that cord blood units stored in the future will be put to good medical use. On a high level, the technology is also a platform approach to the problem of scaling up any number of different stem cell types for cell therapies in the future.

An interesting innovation for the future of this technology would be the addition of a device that can measure the concentration of inhibitory proteins within the system as cell growth occurs. If this could be achieved in real-time, it is conceivable that media input could be regulated to create a dynamic stem cell expansion environment. This would be highly fitting for the expansion of stem cells from cord blood units, as the protocol would be tailored to every expanded unit; all of which are different in cellular composition and genetic make-up.

Friday Science Review: February 3, 2012

Pathogenesis of Paediatric Glioblastoma Multiforme

McGill University ♦ Genome Quebec Innovation Centre

Published in Nature, January 29, 2012

Researchers have not only discovered the first recurring mutation in a human histone but have uncovered a key pathway involved in the formation of paediatric glioblastoma multiforme (GBM). This highly aggressive form of cancer is almost always lethal. Previously acquired gene expression patterns suggest that the mechanisms underlying GBM formation in children and adults are different. Mutations involved in the pathogenesis of GBM were identified by sequencing the exomes of 48 paediatric GBM samples. A chromatin remodelling pathway involving the histone H3.3 and the genes ATRX and DAXX seems to be at the heart of transformation. Researchers found that mutations in H3F3A, the gene encoding H3.3, lead to amino acid substitutions in the histone tail, a portion responsible for key regulatory post-translational modifications. In addition, mutations in ATRX and DAXX, both part of a chromatin remodelling complex that incorporates H3.3 histones at telomeres, were also identified in many of the patient samples. Subsequent screening of a large cohort (n=748) of gliomas showed that H3F3A mutation is frequently found in GBM and is highly specific to children.

The mutations identified in this study were associated with elongated telomeres. During the normal aging process telomeres shorten over time, and in a sense are a ‘biological clock’ that dictates the length of a cell’s life. Eventually, after many cell divisions, telomeres reach a critically short length and the cell undergoes senescence and/or programmed cell death. This is one mechanism by which the human body has evolved to prevent cells from accruing enough genomic mutations to undergo malignant transformation. Telomere elongation allows cells to live beyond their normal biological lifespan. Cells with elongated telomeres become dangerous as they are allowed to continue to live in the presence of mutational ‘build up’. Upregulation of the enzyme telomerase, which helps maintain telomere length through the addition of DNA to the ends of chromosomes, is also associated with cellular immortalization.

Partner, Sell, or Go it alone: Part 15 of Valuation and Other Biotech Mysteries

[Ed. This is the fifteenth part in Wayne's series. You can access the whole thing by clicking here. Please leave comments or questions on the blog and Wayne will address them in future posts in this series.]

This is a discussion that the management and board of a company need to start as the company is being formed and continue throughout the development of a new drug product. The decision on any specific drug product is probably as unique as that drug product and can change along with the market in which that drug will compete. There is a fourth option – stop all product development – which also needs to be assessed at each review. In this blog, we will assume that the information is positive and the product merits further development.

There are many interested parties in this decision, each of which may have different objectives. Read more of this post


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