Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types

Recombinant adeno-associated viruses (rAAVs) are efficient gene delivery vectors via intravenous delivery; however, natural serotypes display a finite set of tropisms. To expand their utility, we evolved AAV capsids to efficiently transduce specific cell types in adult mouse brains. Building upon our Cre-recombination-based AAV targeted evolution (CREATE) platform, we developed Multiplexed-CREATE (M-CREATE) to identify variants of interest in a given selection landscape through multiple positive and negative selection criteria. M-CREATE incorporates next-generation sequencing, synthetic library generation and a dedicated analysis pipeline. We have identified capsid variants that can transduce the central nervous system broadly, exhibit bias toward vascular cells and astrocytes, target neurons with greater specificity or cross the blood–brain barrier across diverse murine strains. Collectively, the M-CREATE methodology accelerates the discovery of capsids for use in neuroscience and gene-therapy applications

Rare variants with large effects provide functional insights into the pathology of migraine subtypes, with and without aura

Migraine is a complex neurovascular disease with a range of severity and symptoms, yet mostly studied as one phenotype in genome-wide association studies (GWAS). Here we combine large GWAS datasets from six European populations to study the main migraine subtypes, migraine with aura (MA) and migraine without aura (MO). We identified four new MA-associated variants (in PRRT2, PALMD, ABO and LRRK2) and classified 13 MO-associated variants. Rare variants with large effects highlight three genes. A rare frameshift variant in brain-expressed PRRT2 confers large risk of MA and epilepsy, but not MO. A burden test of rare loss-of-function variants in SCN11A, encoding a neuron-expressed sodium channel with a key role in pain sensation, shows strong protection against migraine. Finally, a rare variant with cis-regulatory effects on KCNK5 confers large protection against migraine and brain aneurysms. Our findings offer new insights with therapeutic potential into the complex biology of migraine and its subtypes.</p

Chitosan and Chitin Hexamers affect expansion and differentiation of mesenchymal stem cells differently.

To access publisher's full text version of this article. Please click on the hyperlink in Additional Links field.Chitooligosaccharides are of interest as potential drugs due to their bioactivity and water solubility. We compared the effect of acetylated and deacetylated chitooligomers (Hexamers) on short-term expansion (7 days) and osteogenic differentiation of bone-marrow derived, human mesenchymal stem cells in terms of gene expression, cytokine secretion and quality of osteogenic differentiation. We show that chitooligomers affect hMSC gene expression and cytokine secretion, but not mineralization. The effect of chitooligomers was shown to be dependent on the acetylation degree, with significantly stronger effects when cells are stimulated with chitin-derived Hexamers (N-Acetyl Chitohexaose) than with Chitosan Hexamers (Chitohexaose).Icelandic Centre for Research Landspitali University Hospita

Endotoxins affect bioactivity of chitosan derivatives in cultures of bone marrow-derived human mesenchymal stem cells.

To access publisher's full text version of this article. Please click on the hyperlink in Additional Links field.Biomaterials research has been expanding over the last decade, in part to provide improved medical devices for the treatment of orthopedic tissue injuries. In the quest to provide the best performance combined with low cost for medical implants, an increasing number of non-chemists have entered the field of biomaterials research without the profound knowledge of chemistry needed to understand the complex interaction mechanisms and characteristics of natural substances. Likewise, non-biologists often lack understanding when it comes to the presence of the contaminating biota frequently found in natural substances. This lack of knowledge by researchers in the field, combined with sensitive in vitro cell-based assays, can lead to inaccurate evaluation of biomaterials. Hence, there should be both an active effort to assemble multi-disciplinary teams and a genuine concern for the possible effects of contamination on in vitro assays. Here, we show that the presence of bacterial endotoxins in chitosan derivatives can result in false-positive results, profoundly altering product performance in in vitro assays. False-positive results through uncritical use of natural substances in vitro can be avoided by proper endotoxin testing and careful evaluation of cytokine secretion patterns.Technology Development Fund Icelandic Research Fund Landspitali University Hospital research fun

Multiplexed-CREATE Selection Yields AAV Vectors Targeting Different Cell Types of the Central Nervous System Following Systemic Delivery

Recombinant adeno-associated viral (rAAV) capsids are widely accepted as safe gene delivery vehicles in research laboratories and in gene therapy clinical trials and there is potential to further improve their usage by evolving the surface of the capsids to enhance their affinity to specific cell-types or tissues after intravenous (IV) delivery. To this end, we built upon our previous Cre recombination-based AAV targeted evolution (CREATE) method (Deverman et al, Nat. Biotech., 2016; Chan et al., Nat. Neurosci., 2017) to develop Multiplexed-CREATE (M-CREATE). M-CREATE facilitates both positive and negative selections and minimizes the propagation of biases from successive rounds of selection via synthetic library generation. In addition to increasing the confidence in the selections, M-CREATE enables a detailed characterization of the selection process, improving our understanding of selection progression and outcome

Multiplexed-CREATE Selection Yields AAV Vectors Targeting Different Cell Types of the Central Nervous System Following Systemic Delivery

Recombinant adeno-associated viral (rAAV) capsids are widely accepted as safe gene delivery vehicles in research laboratories and in gene therapy clinical trials and there is potential to further improve their usage by evolving the surface of the capsids to enhance their affinity to specific cell-types or tissues after intravenous (IV) delivery. To this end, we built upon our previous Cre recombination-based AAV targeted evolution (CREATE) method (Deverman et al, Nat. Biotech., 2016; Chan et al., Nat. Neurosci., 2017) to develop Multiplexed-CREATE (M-CREATE). M-CREATE facilitates both positive and negative selections and minimizes the propagation of biases from successive rounds of selection via synthetic library generation. In addition to increasing the confidence in the selections, M-CREATE enables a detailed characterization of the selection process, improving our understanding of selection progression and outcome