Aslam Abbasi Akhtar

In Vivo CRISPR/Cas9 Gene Editing Corrects Retinal Dystrophy in the S334ter-3 Rat Model of Autosomal Dominant Retinitis Pigmentosa

Reliable genome editing via Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 may provide a means to correct inherited diseases in patients. As proof of principle, we show that CRISPR/Cas9 can be used in vivo to selectively ablate the rhodopsin gene carrying the dominant S334ter mutation (RhoS334) in rats that model severe autosomal dominant retinitis pigmentosa. A single subretinal injection of guide RNA/Cas9 plasmid in combination with electroporation generated allele-specific disruption of RhoS334, which prevented retinal degeneration and improved visual function.

Ets Factors Regulate Neural Stem Cell Depletion and Gliogenesis in Ras Pathway Glioma

As the list of putative driver mutations in glioma grows, we are just beginning to elucidate the effects of dysregulated developmental signaling pathways on the transformation of neural cells. We have employed a postnatal, mosaic, autochthonous glioma model that captures the first hours and days of gliomagenesis in more resolution than conventional genetically engineered mouse models of cancer. We provide evidence that disruption of the Nf1-Ras pathway in the ventricular zone at multiple signaling nodes uniformly results in rapid neural stem cell depletion, progenitor hyperproliferation, and gliogenic lineage restriction. Abolishing Ets subfamily activity, which is upregulated downstream of Ras, rescues these phenotypes and blocks glioma initiation. Thus, the Nf1-Ras-Ets axis might be one of the select molecular pathways that are perturbed for initiation and maintenance in glioma.

A Transposon-Mediated System for Flexible Control of Transgene Expression in Stem and Progenitor-Derived Lineages

Summary Precise methods for transgene regulation are important to study signaling pathways and cell lineages in biological systems where gene function is often recycled within and across lineages. We engineered a genetic toolset for flexible transgene regulation in these diverse cellular contexts. Specifically, we created an optimized piggyBac transposon-based system, allowing for the facile generation of stably transduced cell lineages in vivo and in vitro. The system, termed pB-Tet-GOI (piggyBac-transposable tetracycline transactivator-mediated flexible expression of a genetic element of interest), incorporates the latest generation of tetracycline (Tet) transactivator and reverse Tet transactivator variants—along with engineered mutants—in order to provide regulated transgene expression upon addition or removal of doxycycline (dox). Altogether, the flexibility of the system allows for dox-induced, dox-suppressed, dox-resistant (i.e., constitutive), and dox-induced/constitutive regulation of transgenes. This versatile strategy provides reversible temporal regulation of transgenes with robust inducibility and minimal leakiness.

Lost highway(s): barriers to postnatal cortical neurogenesis and implications for brain repair

The genesis of the cerebral cortex is a highly complex and tightly-orchestrated process of cell division, migration, maturation, and integration. Developmental missteps often have catastrophic consequences on cortical function. Further, the cerebral cortex, in which neurogenesis takes place almost exclusively prenatally, has a very poor capacity for replacement of neurons lost to injury or disease. A multitude of factors underlie this deficit, including the depletion of radial glia, the gliogenic switch which mitigates continued neurogenesis, diminished neuronal migratory streams, and inflammatory processes associated with disease. Despite this, there are glimmers of hope that new approaches may allow for more significant cortical repair. Herein, we review corticogenesis from the context of regeneration and detail the strategies to promote neurogenesis, including interneuron transplants and glial reprogramming. Such strategies circumvent the “lost highways” which are critical for cortical development but are absent in the adult. These new approaches may provide for the possibility of meaningful clinical regeneration of elements of cortical circuitry lost to trauma and disease.

Organoid and Organ-on-a-Chip Systems: New Paradigms for Modeling Neurological and Gastrointestinal Disease

Purpose of ReviewThe modeling of biological processes in vitro provides an important tool to better understand mechanisms of development and disease, allowing for the rapid testing of therapeutics. However, a critical constraint in traditional monolayer culture systems is the absence of the multicellularity, spatial organization, and overall microenvironment present in vivo. This limitation has resulted in numerous therapeutics showing efficacy in vitro, but failing in patient trials. In this review, we discuss several organoid and “organ-on-a-chip” systems with particular regard to the modeling of neurological diseases and gastrointestinal disorders.Recent FindingsRecently, the in vitro generation of multicellular organ-like structures, coined organoids, has allowed the modeling of human development, tissue architecture, and disease with human-specific pathophysiology. Additionally, microfluidic “organ-on-a-chip” technologies add another level of physiological mimicry by allowing biological mediums to be shuttled through 3D cultures.SummaryOrganoids and organ chips are rapidly evolving in vitro platforms which hold great promise for the modeling of development and disease.

Generating in vivo somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

Viral vectors and electroporation (EP)-mediated gene transfers are efficient means of inducing somatic mosaicism in mice, but they lack the exquisite control over transgene copy number, gene zygosity, and genomic-locus specificity that genetically engineered mouse models (GEMMs) provide. Here, we develop and demonstrate a simple and generalizable in vivo method, mosaic analysis by dual recombinase-mediated cassette exchange (MADR). MADR allows for stable labeling of mutant cells express transgenic elements from a precisely-defined chromosomal locus. To test our method, we generated reporter-labeled lineages from stem and progenitor cells in a well-defined Rosa26mTmG mouse. We demonstrate the power and versatility of MADR by creating novel glioma models with mixed, reporter-defined zygosity or with “personalized” driver mutations from pediatric glioma—each manipulation altering the profile of resulting tumors. Thus, MADR provides a high-throughput genetic platform for the dissection of development and disease, and this rapid method can be applied to the thousands of existing gene-trap mice.

Mutations within the pathogenic region of herpes simplex virus 1 gK signal sequences alter cell surface expression and neurovirulence.

ABSTRACT To investigate the role of the signal sequences of herpes simplex virus 1 (HSV-1) gK on virus replication and viral pathogenesis, we constructed recombinant viruses with or without mutations within the signal sequences of gK. These recombinant viruses expressed two additional copies of the mutated (MgK) or native (NgK) form of the gK gene in place of the latency-associated transcript with a myc epitope tag to facilitate detection at their 3′ ends. The replication of MgK virus was similar to that of NgK both in vitro and in vivo, as well as in the trigeminal ganglia (TG) of latently infected mice. The levels of gB and gK transcripts in the corneas, TG, and brains of infected mice on days 3 and 5 postinfection were markedly virus and time dependent, as well as tissue specific. Mutation in the signal sequence of gK in MgK virus blocked cell surface expression of gK-myc in rabbit skin cells, increased 50% lethal dose, and decreased corneal scarring in ocularly infected mice compared to the NgK or revertant (RgK) virus. MgK and NgK viruses, and not the RgK virus, showed a reduced extent of explant reactivation at the lower dose of ocular infection but not at the higher dose. However, the time of reactivation was not affected by overexpression of the different forms of gK. Taken together, these results strongly suggest that the 8mer peptide (ITAYGLVL) within the signal sequence of gK promotes cell surface expression of gK in infected cells and ocular pathogenesis in infected mice. IMPORTANCE In this study, we show for the first time that mutations within the signal sequence of gK blocked cell surface expression of inserted recombinant gK in vitro. Furthermore, this blockage in cell surface expression was correlated with higher 50% lethal dose and less corneal scarring in vivo. Thus, these studies point to a key role for the 8mer within the signal sequence of gK in HSV-1-induced pathogenicity.

Inducible Expression of GDNF in Transplanted iPSC-Derived Neural Progenitor Cells

Summary Trophic factor delivery to the brain using stem cell-derived neural progenitors is a powerful way to bypass the blood-brain barrier. Protection of diseased neurons using this technology is a promising therapy for neurodegenerative diseases. Glial cell line-derived neurotrophic factor (GDNF) has provided benefits to Parkinsonian patients and is being used in a clinical trial for amyotrophic lateral sclerosis. However, chronic trophic factor delivery prohibits dose adjustment or cessation if side effects develop. To address this, we engineered a doxycycline-regulated vector, allowing inducible and reversible expression of a therapeutic molecule. Human induced pluripotent stem cell (iPSC)-derived neural progenitors were stably transfected with the vector and transplanted into the adult mouse brain. Doxycycline can penetrate the graft, with addition and withdrawal providing inducible and reversible GDNF expression in vivo, over multiple cycles. Our findings provide proof of concept for combining gene and stem cell therapy for effective modulation of ectopic protein expression in transplanted cells.

Tetracycline‐Inducible and Reversible Stable Gene Expression in Human iPSC‐Derived Neural Progenitors and in the Postnatal Mouse Brain

The pB-tet-GOI plasmid system allows for stable piggyBac transposition-mediated integration into cells, a fluorescent nuclear reporter to identify cells that have been transfected, and robust transgene activation or suppression upon the addition of dox to the cell culture or diet of the animal. Furthermore, the addition of luciferase downstream of the target gene allows for quantitative assessment of gene activity in a non-invasive manner. The protocols herein provide instructions for the use of this system in cell lines and in the neonatal mouse brain. Specifically, a detailed protocol is provided to illustrate: (1) cloning of the respective GOI (genetic element(s) of interest); (2) nucleofection of the plasmid system into human induced pluripotent stem cell (iPSC)-derived neural progenitors; (3) dox-induced activation in vitro or in vivo; and (4) non-invasive assessment of gene activity in vivo by bioluminescence imaging.