NIH Awards Allele Collaboration with Grant to Fund the Development of Cell-Based Therapy for Alzheimer’s Disease

The NIH’s National Institute on Aging has awarded Allele Biotechnology and Pharmaceuticals (“Allele”) a Phase 1 SBIR grant to develop a stem cell-based therapy for the treatment of Alzheimer’s disease. The award includes funds for consortium activity with researchers at The Scintillon Institute, whose expertise in neurodegeneration leverages Allele’s expertise in stem cell technology.

Alzheimer’s disease is the most common form of dementia, affecting over 35 million people worldwide. Currently, there is no cure for this devastating disease. Patients with Alzheimer’s disease suffer from synaptic and neuronal loss, which is thought to be caused by the presence of a chemically “sticky” protein called amyloid beta (Aβ). The aggregates of Aβ may damage synaptic integrity and/or trigger immune cell activation, ultimately causing cell death.

Successful cell-replacement therapies would need to distribute cells to damaged areas in the brain and stimulate integration of new neurons into existing cellular networks. While the idea of replacing lost neurons sounds promising, even successfully transplanted neurons would face the same toxic environment that destroyed the original neurons.

Researchers at Allele and Scintillon propose a novel way to prevent further damage from Aβ to transplanted neural stem cells. They are collaborating to genetically modify human neural stem cells to express a small (58-amino acid) peptide derived from the protein called α1-takusan, which Scintillon researchers previously discovered to harbor a protective activity against Aβ-induced toxicity (1). The researchers will then transplant the cells expressing the α1-takusan fragment into transgenic mouse models to evaluate whether these cells can ameliorate or even rescue the neurological phenotypes related to Alzheimer’s disease.

The Allele-Scintillon team hopes that transplanting these cells will mitigate synaptic and neuronal damage from Aβ, ultimately leading to a novel cell-replacement therapy for Alzheimer’s disease. This is the second SBIR grant that Allele has received from the NIH on treating Alzheimer’s by combating Aβ toxicity; the first being the creation of nanoantibodies against Aβ, which has generated multiple single-domain antibodies now in early development.

(1) Nakanishi N, Ryan SD, Zhang X, Khan A, Holland T, Cho EG, Huang X, Liao FF, Xu H, Lipton SA, Tu S (2013) Synaptic protein alpha1-takusan mitigates amyloid-beta-induced synaptic loss via interaction with tau and postsynaptic density-95 at postsynaptic sites. J Neurosci 33:14170-14183. PMCID: PMC3756761

Prominent regulators of neurogenesis are also critical in maintaining eye health

A group of researchers at Scintillon Institute in San Diego, California and their collaborators identified important roles of myocyte enhancer factor 2 (MEF2) in the pathogenesis of stress-induced photoreceptor degeneration, a condition that is thought to contribute to eye diseases, such as retinitis pigmentosa and age-related macular degeneration, as described in two recent publications (1,2). MEF2 is an activity-dependent transcription factor which is expressed in various organs, such as the heart, lymphocytes and brain. Dr. Stuart Lipton’s group has continuously worked on MEF2 since 1993, when they first isolated MEF2C, one of four mammalian MEF2 isoforms, in the developing brain. These researchers made seminal discoveries that established the notion that MEF2 transcription factors are prominent regulators of neurogenesis and neuronal survival in the brain. More recently, their work on MEF2C mutant mice led to the recognition of the human disease called MEF2C haploinsufficiency syndrome, in which children with heterozygous loss-of-function MEF2C mutations suffer from severe neurological conditions, including autism spectrum disorders, developmental and intellectual disabilities and seizures.

Scientists at the Neural Center of the Scintillon Institute have been expanding on MEF2 research, most recently turning their eyes to eye diseases (pun intended). Retinal photoreceptor cells express two MEF2 isoforms: MEF2C and MEF2D, the latter apparently being the predominant form. In a recent study, the researchers examined mutant mice completely lacking MEF2C or MEF2D (MEF2C- or MEF2D- “null” mice). Interestingly, both mutant mice developed drastic retinal degenerations by postnatal day 30. They then took a candidate approach to identify the molecular pathways affected by the loss of MEF2D in MEF2D-null mice. Among the pathways they examined was the PGC1? pathway, which regulates mitochondrial biogenesis and thereby protects cells from degeneration. The Lipton group determined that transcription of PGC1? was indeed reduced in MEF2D-null mice. Yet by overexpressing PGC1? in the retina of MEF2D-null mice, the researchers found that the retinal degeneration could be rescued.

In another related study, they examined mice lacking one copy of MEF2D (MEF2D-heteretozygous or “het” mice). Unlike MEF2D-null mice, MEF2D-het mice did not show any retinal regeneration when they were raised under normal housing environment. The researchers then exposed MEF2D-het mice to a strong white fluorescent light for 2 hours. While this light exposure did not induce any retinal degeneration in the wild-type mice, it did cause significant retinal cell death in MEF2D-het mice. The light exposure massively produced reactive oxygen species (ROS), which appeared to be the toxic cause. When searching for affected downstream pathways, they found that the transcription factor NRF2, a regulator of the cellular antioxidant defense response, fails to be induced by light exposure in MEF2D mutant mice. The researchers attempted to reverse light-induced retinal cell death by treating the MEF2D-het mice with carnosic acid, a chemical they had previously identified as a potent antioxidant and NRF2 activator. Intriguingly, treatment of carnosic acid drastically ameliorated the amount of light-induced retinal cell death in the mutant mice.

Together, these studies from the Scintillon Institute identify MEF2 transcription factors as crucial molecules in maintaining eye health. Importantly, they have shown that MEF2 and its downstream pathways can be targeted by drugs such as carnosic acid. Incidentally, carnosic acid is a naturally occurring chemical that is contained in herbs such as rosemary and sage. So, there may be a health benefit in cooking chicken and turkey with rosemary!

1. Proc Natl Acad Sci USA 114, E4048

2. Inv Opthal Vis Sci 58, 3741

Cellular Control – at the Flick of a Light Switch

What if you could turn on an enzyme inside a living cell—or release a cellular factor from its anchor—with the flick of a light switch?

Researchers at the University of Alberta’s Department of Chemistry have developed a new tool for manipulating biochemical processes within cells using light. By applying the unique properties of a photoconvertible fluorescent protein called mMaple, the team created such a light switch, a photocleavable protein called PhoCl (pronounced “focal”).

mMaple, whose name was inspired by the green-to-red color change of maple leaves as seasons transition, undergoes a light-dependent conformational change. Dr. Robert E. Campbell’s team engineered PhoCl to cleave into two pieces when exposed to light.

This novel optogenetic tool is especially useful for applications that involve manipulating cellular processes. For example, PhoCl can be used to create “caged” proteins that will not become activated until exposed to light. Researchers link one terminus of PhoCl to a cellular enzyme and the other terminus to an inhibitor, “caging” the enzyme and preventing it from performing its function. Upon exposure to violet light, PhoCl is cleaved to separate the inhibitor from the enzyme, thus activating the enzyme at the user’s command.

The cleavage mechanism of PhoCl is particularly useful for the activation of proteins within a specific location of a cell. Because intact PhoCl is fluorescent, researchers can visualize its location and movement within the cell and have control over when it cleaves. Upon cleavage, the fluorescence is quenched, enabling users to visually determine where the event took place.

As Allele Biotechnology & Pharmaceuticals is a licensed distributor of plasmids containing the gene for mMaple, the development of PhoCl is particularly exciting news to us and our customers. Interested readers can learn more about PhoCl in their paper published in Nature Methods.

Ablynx Develops Nano Antibody for Treatment of Rare Clotting Disorder

Last week, Ablynx announced substantial progress in the development of the nano antibody drug caplicizumab to treat acquired thrombotic thrombocytopenic purpura (aTTP), a rare, but life-threatening autoimmune disease. The Belgian biopharmaceutical company has submitted a Marketing Authorization Application (MAA) to the European Medicines Agency (EMA) for approval. If accepted, caplicizumab will not only be the first therapeutic specifically indicated for the treatment of aTTP, but also the first approved nano antibody drug on the market.

aTTP is characterized by the autoimmune impairment of ADAMTS13, an enzyme that normally cleaves multimeric von Willebrand factor (vWF) into its functional form. Without the function of ADAMTS13, multimeric vWF forms aggregates with platelets in the blood. Low free platelet count and excess clotting result in thrombotic complications and a significant risk of organ damage due to the blockages of blood flow to tissues.

The current standard of care for aTTP involves immunosuppression and daily plasma exchange transfusion, in which a patient’s plasma is replaced with donor plasma to remove platelet-vWF aggregates. Caplicizumab is an anti-vWF nano antibody that prevents the formation of aggregates by blocking the interaction of multimeric vWF complexes with platelets.

While dozens of monoclonal antibodies have been approved by the FDA for therapeutic use (with hundreds more undergoing clinical trials), caplicizumab is the first therapeutic nano antibody. Nano antibodies are single-domain antibody fragments that bear full antigen binding capacity like monoclonal antibodies, but have a smaller size and unique structure, giving them features of small-molecule drugs. Nano antibodies are more stable than conventional monoclonal antibodies, allowing for multiple administration routes, and can be humanized to lower toxicity and immunogenicity. Because they are encoded by single genes, nano antibodies are easier and more cost-effective than traditional antibodies to engineer and manufacture.

Currently, caplicizumab is undergoing Phase III clinical trials and a three-year follow-up study has been initiated to determine the long-term safety and efficacy of this drug. Ablynx aims to commercialize caplicizumab in North America and Europe upon the trial’s conclusion and approval of BLA filing in 2018.

With the obvious advantages of nano antibodies over conventional monoclonal antibodies as biological drugs, caplicizumab is likely only the first of many to come.

Allele Researchers Engineer Modified Nanoantibodies to Increase Sensitivity in Biochemical Assays

Researchers at Allele have published new work demonstrating a novel application for nanoantibodies (nAbs) in direct signal amplification. nAbs have distinguishable qualities that set them apart from their traditional IgG counterparts, including significantly smaller size, better stability, and excellent specificity. However, because of their small size, there are no suitable secondary antibodies for traditional assays like immunohistochemistry, immunofluorescence, and other biochemical assays that require an enhanced signal.

The researchers engineered a modified nAb, termed “nAb Plus,” to directly amplify nAb signal detection through the addition of a small scaffolding protein containing numerous reporter binding sites. nAb Plus bypasses the need for secondary antibodies or additional amplification steps, streamlining biochemical assays and decreasing costs of reagents. The authors demonstrate the use of nAb Plus using immunohistochemistry, an assay typically requiring one or more signal amplification steps. However, nAb Plus could also be incorporated in any biochemical assay needing signal enhancement.

Abstract: Revealing the spatial arrangement of molecules within a tissue through immunohistochemistry (IHC) is an invaluable tool in biomedical research and clinical diagnostics. Choosing both the appropriate antibody and amplification system is paramount to the pathologic interpretation of the tissue at hand. The use of single domain VHH nanoantibodies (nAbs) promise more robust and consistent results in IHC, but are rarely used as an alternative to conventional immunoglobulin G (IgG) antibodies. nAbs are originally obtained from llamas and are the smallest antigen-binding fragments available. To determine whether the unique biophysical properties of nAbs give them an advantage in IHC, we first compared a basic fibroblast growth factor nAb to polyclonal IgG antibodies using tissue isolated from pancreatic adenocarcinoma. The nAb was extremely effective in antigen signal detection and allowed for a more streamlined and reproducible protocol. Furthermore, because nAbs are expressed in Escherichia coli from a single gene, they are quite amenable to genetic engineering. As such, we then covalently bound a highly biotinylated amplifier protein to basic fibroblast growth factor and p16 nAbs (termed nAb Plus), resulting in improved IHC sensitivity. The use of a biotinylated nAb Plus not only achieved local, covalent signal amplification, but also eliminated the need for a secondary antibody and subsequent amplification steps. These results highlight nAbs as valuable alternatives to conventional IgG antibodies, decreasing overall processing time and costs of reagents while increasing sensitivity and reproducibility across individual IHC assays.

Link to full text

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