FAK-Src signalling is important to renal collecting duct morphogenesis: discovery using a hierarchical screening technique

Summary This report describes a hierarchical screening technique for identification of pathways that control the morphogenesis of the renal collecting duct system. The multi-step screen involves a first round using a 2-dimensional, cell-line-based scrape-healing assay, then a second round using a 3-dimensional tubulogenesis assay; both of these rounds use new cell lines described in this report. The final stage is ex vivo organ culture. We demonstrate the utility of the screen by using it to identify the FAK–Src-pathway signalling as being important for collecting duct development, specifically for the cell proliferation on which this development depends

Viral vector platforms within the gene therapy landscape

Throughout its 40-year history, the field of gene therapy has been marked by many transitions. It has seen great strides in combating human disease, has given hope to patients and families with limited treatment options, but has also been subject to many setbacks. Treatment of patients with this class of investigational drugs has resulted in severe adverse effects and, even in rare cases, death. At the heart of this dichotomous field are the viral-based vectors, the delivery vehicles that have allowed researchers and clinicians to develop powerful drug platforms, and have radically changed the face of medicine. Within the past 5 years, the gene therapy field has seen a wave of drugs based on viral vectors that have gained regulatory approval that come in a variety of designs and purposes. These modalities range from vector-based cancer therapies, to treating monogenic diseases with life-altering outcomes. At present, the three key vector strategies are based on adenoviruses, adeno-associated viruses, and lentiviruses. They have led the way in preclinical and clinical successes in the past two decades. However, despite these successes, many challenges still limit these approaches from attaining their full potential. To review the viral vector-based gene therapy landscape, we focus on these three highly regarded vector platforms and describe mechanisms of action and their roles in treating human disease

Bone-targeting AAV-mediated silencing of Schnurri-3 prevents bone loss in osteoporosis

RNAi-based bone anabolic gene therapy has demonstrated initial success, but many practical challenges are still unmet. Here, we demonstrate that a recombinant adeno-associated virus 9 (rAAV9) is highly effective for transducing osteoblast lineage cells in the bone. The adaptor protein Schnurri-3 (SHN3) is a promising therapeutic target for osteoporosis, as deletion of shn3 prevents bone loss in osteoporotic mice and short-term inhibition of shn3 in adult mice increases bone mass. Accordingly, systemic and direct joint administration of an rAAV9 vector carrying an artificial-microRNA that targets shn3 (rAAV9-amiR-shn3) in mice markedly enhanced bone formation via augmented osteoblast activity. Additionally, systemic delivery of rAAV9-amiR-shn3 in osteoporotic mice counteracted bone loss and enhanced bone mechanical properties. Finally, we rationally designed a capsid that exhibits improved specificity to bone by grafting the bone-targeting peptide motif (AspSerSer)6 onto the AAV9-VP2 capsid protein. Collectively, our results identify a bone-targeting rAAV-mediated gene therapy for osteoporosis

Gene Delivery to Nonhuman Primate Preimplantation Embryos Using Recombinant Adeno-Associated Virus

Delivery of genome editing tools to mammalian zygotes has revolutionized animal modeling. However, the mechanical delivery method to introduce genes and proteins to zygotes remains a challenge for some animal species that are important in biomedical research. Here, an approach to achieve gene delivery and genome editing in nonhuman primate embryos is presented by infecting zygotes with recombinant adeno-associated viruses (rAAVs). Together with previous reports from the authors of this paper and others, this approach is potentially applicable to a broad range of mammals. In addition to genome editing and animal modeling, this rAAV-based method can facilitate gene function studies in early-stage embryos

Low-Dose Recombinant Adeno-Associated Virus-Mediated Inhibition of Vascular Endothelial Growth Factor Can Treat Neovascular Pathologies Without Inducing Retinal Vasculitis

The wet form of age-related macular degeneration is characterized by neovascular pathologies that, if untreated, can result in edemas followed by rapid vision loss. Inhibition of vascular endothelial growth factor (VEGF) has been used to successfully treat neovascular pathologies of the eye. Nonetheless, some patients require frequent intravitreal injections of anti-VEGF drugs, increasing the burden and risk of complications from the procedure to affected individuals. Recombinant adeno-associated virus (rAAV)-mediated expression of anti-VEGF proteins is an attractive alternative to reduce risk and burden to patients. However, controversy remains as to the safety of prolonged VEGF inhibition in the eye. Here, we show that two out of four rAAV serotypes tested by intravitreal delivery to express the anti-VEGF drug conbercept lead to a dose-dependent vascular sheathing pathology that is characterized by immune cell infiltrates, reminiscent of vasculitis in humans. We show that this pathology is accompanied by increased expression in vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1), both of which promote extravasation of immune cells from the vasculature. While formation of the vascular sheathing pathology is prevented in immunodeficient Rag-1 mice that lack B and T cells, increased expression of VACM1 and ICAM1 still occurs, indicating that inhibition of VEGF function leads to expression changes in cell adhesion molecules that promote extravasation of immune cells. Importantly, a 10-fold lower dose of one of the vectors that cause a vascular sheathing pathology is still able to reduce edemas resulting from choroidal neovascularization without causing any vascular sheathing pathology and only a minimal increase in VCAM1 expression. The data suggest that treatments of neovascular eye pathologies with rAAV-mediated expression of anti VEGF drugs can be developed safely. However, viral load needs to be adjusted to the tropisms of the serotype and the expression pattern of the promoter

Precision Cas9 Genome Editing in vivo with All-in-one, Self-targeting AAV Vectors [preprint]

Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits, especially for large effectors like Cas9s. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. The use of compact effectors has enabled single-AAV delivery of Cas9s with 1-3 guides for edits that use end-joining repair pathways, but many precise edits that correct disease-causing mutations in vivo require homology-directed repair (HDR) templates. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs to produce segmental deletions, or a single sgRNA with an HDR template. We also examine the utility of Nme2Cas9 target sites in the vector for self-inactivation. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice. These results will enable single-vector AAVs to achieve diverse therapeutic genome editing outcomes

Electrically stimulated cell migration and its contribution to wound healing.

Naturally occurring electric fields are known to be morphogenetic cues and associated with growth and healing throughout mammalian and amphibian animals and the plant kingdom. Electricity in animals was discovered in the eighteenth century. Electric fields activate multiple cellular signaling pathways such as PI3K/PTEN, the membrane channel of KCNJ15/Kir4.2 and intracellular polyamines. These pathways are involved in the sensing of physiological electric fields, directional cell migration (galvanotaxis, also known as electrotaxis), and possibly other cellular responses. Importantly, electric fields provide a dominant and over-riding signal that directs cell migration. Electrical stimulation could be a promising therapeutic method in promoting wound healing and activating regeneration of chronic and non-healing wounds. This review provides an update of the physiological role of electric fields, its cellular and molecular mechanisms, its potential therapeutic value, and questions that still await answers