NIH CRM is a Common Fund-supported initiative that was established in 2010. The efforts of Mahendra Rao, the NIH CRM Director appointed in August 2011, are split between the Center and his intramural laboratory. Since his appointment, Dr. Rao has completed recruitment of the key positions in the laboratory. This comprises two Staff Scientists, Nasir Malik and Jizhong Zou. They in turn have assisted Dr. Rao in hiring two biologists, Xiantao Wang and Yongquan Luo. The laboratory group has also included non-permanent staff in the past year, including post-doctoral and post-baccalaureate fellows. Efforts in neural development have focused on developing and optimizing protocols for the generation of differentiated cell types that can have applications in translational medicine. Several screening projects with neural cells are currently underway with collaborators at NCATS and NINDS. A screen that is in progress with NCATS is examining whether compounds that protect astrocytes from cell death can be identified. The screen has identified several target compounds and current efforts are aimed at validating these targets. Another screen with NINDS is attempting to identify drugs that are selectively toxic to human neural stem cells. This screen is an extension of work that was published at the beginning of 2014 to establish a platform to conduct viability and high content screens of human neural (NSCs) and the neurons differentiated from them (Efthymiou et al., 2014). The NINDS screen has identified 100 such compounds and found that a group of these show no toxicity to mixed cultures of rat cortical neurons. Of particular interest, in this respect is a group of compounds belonging to the cardiac glycoside family which have also recently been shown to kill cultured glioblastoma cells. The manuscript related to this screen is currently being revised for resubmission (Malik et al., manuscript in revision). The lab is also conducting more targeted screens with human NSCs and neurons with compounds effecting selected signaling pathways including WNT, NOTCH, and JAK/STAT to identify compounds that enhance proliferation, neurogenesis, and gliogenesis. The work that has been performed in this area sets the stage for screens in cells derived from patients with neurological disorders to identify new compounds that may be of therapeutic value. In addition to the screening projects the lab is also attempting to better understand the mechanisms underlying the transition from a multipotent neural stem cell stage to terminally differentiated neurons and astrocytes. Microarray analysis is being used to identify genes and signaling pathways critical for astrocyte development and maintenance (Malik et al., 2014). Ongoing work is also looking at how neural differentiation is affected in iPSC-derived neural stem cells from patients with amytrophic lateral sclerosis and Niemann-Pick type C disease (NPC). In the case of NPC the analysis indicates premature glial differentiation and misregulation of the Wnt signaling pathway providing a possible assay for a drug screen to identify novel compounds that could have therapeutic value (Efthymiou, manuscript in revision). The laboratory also has an active gene engineering program focusing on human iPSCs and their derivatives. The lab has established its own open-source TALEN assembly platform. Using AAVS1 safe harbor as our first target, we compared different AAVS1-ZFN/TALEN efficiency, and by both targeted mutagenesis and homologous recombination assays, our open-source TALENs showed the highest efficiency (4-fold higher than commercial AAVS1-ZFN) in human cells. We also developed novel TALENs, targeting new safe harbor on Chromosome 13, which showed 25% gene-editing efficiency. In terms of genome engineering using human iPSCs and neural stem cells (NSCs), we designed two sets of universal donors to accommodate various gene targeting tasks. One set is designed to target safe harbor loci such as AAVS1 on chr. 19 (AAVS1 safe harbor) or CLYBL2 on chr. 13 (C13 safe harbor). We have used these TALENs and donors to generate several reporter cell lines driven by constitutive or tissue-specific promoters. We have achieved simultaneous gene knock-in at double safe harbor alleles to engineer multiplex reporter iPSC lines. Using same genome editing TALENs and donors described above, we have achieved single-/double-safe harbor reporter knock-in in human NSCs. From this exciting work we have filed 6 EIRs on 2 pairs of safe harbor TALENs, 11 AAVS1 donors, 2 gene-specific donors, 9 safe harbor engineered human iPSC lines, and the methods and compositions of Chr. 13 safe harbor locus for targeted genome modification, which we are also filing patents on. These materials have been transferred to 32 academic labs worldwide and 10 companies through SLA, MTA or BMLA. We are also depositing three engineered human iPSC lines to Rutgers, Sigma and other cell repositories for broader distribution. Two of the companies, SystemBiosciences and Geneocopeia, have launched the commercial products based on our AAVS1 TALENs and donor vectors. We have also provided consultation and support to several dozen academic labs inside or outside NIH to learn TALEN and other genome engineering technologies through an NIH CRM sponsored Bio-Trac training course. Collectively, these efforts in neural development and genome engineering are all part of NIH CRMs overarching goal of translating cell-based therapy to the clinic. The laboratory has maintained its focused on neural derivatives, and these initial reports showcase an efficient processes for manufacture of iPSC and engineering of pluripotent cells which brings us one step closer towards clinical translation.
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