Type VI CRISPR enzymes have been developed as programmable RNA-guided Cas proteins for eukaryotic RNA editing. Notably, Cas13 has been utilized for site-targeted single base edits, demethylation, RNA cleavage or knockdown and alternative splicing. However, the ability to edit large stretches of mRNA transcripts remains a significant challenge. Here, we demonstrate that CRISPR-Cas13 systems can be repurposed to assist trans-splicing of exogenous RNA fragments into an endogenous pre-mRNA transcript, a method termed CR ISPR A ssisted mRNA F ragment T rans-splicing (CRAFT). Using split reporter-based assays, we evaluate orthogonal Cas13 systems, optimize guide RNA length and screen for optimal trans-splicing site(s) across a range of intronic targets. We achieve markedly improved editing of large 5’ and 3’ segments in different endogenous mRNAs across various mammalian cell types compared to other spliceosome-mediated trans-splicing methods. CRAFT can serve as a versatile platform for attachment of protein tags, studying the impact of multiple mutations/single nucleotide polymorphisms, modification of untranslated regions (UTRs) or replacing large segments of mRNA transcripts.

Over the past 5 years, our laboratory has systematically developed a structure-guided library approach to evolve new adeno-associated virus (AAV) capsids with altered tissue tropism, higher transduction efficiency and the ability to evade pre-existing humoral immunity. Here, we provide a detailed protocol describing two distinct evolution strategies using structurally divergent AAV serotypes as templates, exemplified by improving CNS gene transfer efficiency in vivo. We outline four major components of our strategy: (i) structure-guided design of AAV capsid libraries, (ii) AAV library production, (iii) library cycling in single versus multiple animal models, followed by (iv) evaluation of lead AAV vector candidates in vivo. The protocol spans ~95 d, excluding gene expression analysis in vivo, and can vary depending on user experience, resources and experimental design. A distinguishing attribute of the current protocol is the focus on providing biomedical researchers with 3D structural information to guide evolution of precise 'hotspots' on AAV capsids. Furthermore, the protocol outlines two distinct methods for AAV library evolution consisting of adenovirus-enabled infectious cycling in a single species and noninfectious cycling in a cross-species manner. Notably, our workflow can be seamlessly merged with other RNA transcript-based library strategies and tailored for tissue-specific capsid selection. Overall, the procedures outlined herein can be adapted to expand the AAV vector toolkit for genetic manipulation of animal models and development of human gene therapies.

Adeno-associated virus (AAV)-mediated gene therapies for central nervous system (CNS) disorders are experiencing a period of rapid development. A string of recent clinical trial outcomes for rare and complex CNS disorders complemented by preclinical studies in animal models continue to highlight both the prospects and challenges facing therapeutic gene transfer to the brain.1Hudry E. Vandenberghe L.H. Therapeutic AAV Gene Transfer to the Nervous System: A Clinical Reality.Neuron. 2019; 101: 839-862Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 2Rafii M.S. et al.Adeno-Associated Viral Vector (Serotype 2)–Nerve Growth Factor for Patients With Alzheimer Disease: A Randomized Clinical Trial.JAMA Neurol. 2018; 75: 834-841Crossref PubMed Scopus (83) Google Scholar, 3Deverman B.E. Ravina B.M. Bankiewicz K.S. Paul S.M. Sah D.W.Y. Gene therapy for neurological disorders: progress and prospects.Nat. Rev. Drug Discov. 2018; 17: 641-659Crossref PubMed Scopus (2) Google Scholar Notably, regional disparities in AAV vector spread within the brain parenchyma and transgene expression have spurred efforts in two key areas: optimizing physical methods of CNS dosing and better characterizing AAV biology in the brain.4Castle M.J. et al.Postmortem Analysis in a Clinical Trial of AAV2-NGF Gene Therapy for Alzheimer’s Disease Identifies a Need for Improved Vector Delivery.Hum. Gene Ther. 2020; 31: 415-422Crossref PubMed Scopus (24) Google Scholar In this issue of Molecular Therapy, Heller et al.5Heller G.J. Marshall M.S. Issa Y. Marshall J.N. Nguyen D. Rue E. Pathmasiri K.C. Domowicz M.S. van Breeman R.B. Tai L.M. et al.Waning efficacy in a long-term AAV-mediated gene therapy study in the murine model of Krabbe disease.Mol Ther. 2021; 29 (this issue): 1883-1902Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar highlight a third temporal component that shines a spotlight on the interface of CNS disease and AAV biology. Krabbe disease (KD), also known as globoid cell leukodystrophy, affects newborn children, often leading to death within the first few years of life.6Wenger D.A. Rafi M.A. Luzi P. Datto J. Costantino-Ceccarini E. Krabbe Disease: Genetic Aspects and Progress toward Therapy.Mol. Genet. Metab. 2000; 70: 1-9Crossref PubMed Scopus (157) Google Scholar As KD is caused by loss-of-function mutations in the lysosomal enzyme galactosylceramidase (GALC), several groups have published promising results showing that AAV-delivered GALC significantly improves behavior and pathology in Twitcher mice, a murine model for KD.7Marshall M.S. et al.Long-Term Improvement of Neurological Signs and Metabolic Dysfunction in a Mouse Model of Krabbe Disease after Global Gene Therapy.Mol. Ther. 2018; 26: 874-889Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 8Karumuthil-Melethil S. et al.Intrathecal administration of AAV/GALC vectors in 10-11-day-old twitcher mice improves survival and is enhanced by bone marrow transplant.J. Neurosci. Res. 2016; 94: 1138-1151Crossref PubMed Scopus (30) Google Scholar, 9Rafi M.A. Rao H.Z. Luzi P. Wenger D.A. Long-term Improvements in Lifespan and Pathology in CNS and PNS After BMT Plus One Intravenous Injection of AAVrh10-GALC in Twitcher Mice.Mol Ther. 2015; 23: 1681-1690Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar These results have sparked optimism for treating KD via AAV gene therapy. In a recent long-term study, the Heller group found that AAV9-GALC-treated Twitcher mice showed little to no signs of disease for most of their significantly lengthened lifespans.7Marshall M.S. et al.Long-Term Improvement of Neurological Signs and Metabolic Dysfunction in a Mouse Model of Krabbe Disease after Global Gene Therapy.Mol. Ther. 2018; 26: 874-889Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar However, a wide range of lifespans were found to result from the treatment, suggesting differences in underlying factors affecting long-term survival. Upon deeper investigation, Heller et al.5Heller G.J. Marshall M.S. Issa Y. Marshall J.N. Nguyen D. Rue E. Pathmasiri K.C. Domowicz M.S. van Breeman R.B. Tai L.M. et al.Waning efficacy in a long-term AAV-mediated gene therapy study in the murine model of Krabbe disease.Mol Ther. 2021; 29 (this issue): 1883-1902Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar found that late-onset focal lesions of demyelination develop in white matter following AAV9-GALC treatment. Interestingly, the lesions developed in areas where, at earlier time points, the investigators observed healthy tissue with robust GALC expression, suggesting waning efficacy. Several groups have investigated whether Twitcher mice can be cured of disease using AAV-mediated GALC expression in the brain. Delivery of AAVrh10-GALC simultaneously via intravenous, intracerebellar, and intracerebroventricular injection in postnatal day 2 (P2) mice resulted in significantly extended lifespans up to 8 months. This approach, which requires three simultaneous injections, was further refined to a single AAVrh10-GALC injection at P10 and resulted in longer lifespans, although the lifespan was only extended by 1 month following injections.10Rafi M.A. et al.Intravenous injection of AAVrh10-GALC after the neonatal period in twitcher mice results in significant expression in the central and peripheral nervous systems and improvement of clinical features.Mol. Genet. Metab. 2015; 114: 459-466Crossref PubMed Scopus (32) Google Scholar Combining bone marrow transplant (BMT) with a single injection of AAVrh10 around P10 further improved lifespan, with many treated Twitcher mice living longer than 150 days. A similar strategy with multiple AAV serotypes led to the conclusion that AAV9-GALC delivered intrathecally in combination with BMT greatly increased the lifespan of Twitcher mice.8Karumuthil-Melethil S. et al.Intrathecal administration of AAV/GALC vectors in 10-11-day-old twitcher mice improves survival and is enhanced by bone marrow transplant.J. Neurosci. Res. 2016; 94: 1138-1151Crossref PubMed Scopus (30) Google Scholar Most recently, the Heller group demonstrated that intracranial delivery of AAV9-GALC or high-dose intravenous delivery in the first day after birth significantly improved average lifespan beyond 250 days and was not statistically different from a cohort receiving AAV9-GALC+BMT (via intravenous delivery of non-myeloablated syngenic bone marrow cells) at P1–P2.7Marshall M.S. et al.Long-Term Improvement of Neurological Signs and Metabolic Dysfunction in a Mouse Model of Krabbe Disease after Global Gene Therapy.Mol. Ther. 2018; 26: 874-889Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar These results demonstrate remarkable promise, yet posed a major question: what might be the underlying causes driving heterogeneity in long-term survival of Twitcher mice following AAV-GALC treatment? In this follow up study, Heller et al.5Heller G.J. Marshall M.S. Issa Y. Marshall J.N. Nguyen D. Rue E. Pathmasiri K.C. Domowicz M.S. van Breeman R.B. Tai L.M. et al.Waning efficacy in a long-term AAV-mediated gene therapy study in the murine model of Krabbe disease.Mol Ther. 2021; 29 (this issue): 1883-1902Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar tackled this challenging question and reported that waning efficacy can be attributed to psychosine accumulation and decreased GALC activity in specific demyelinated areas of the CNS following treatment. Notably, the peripheral nervous system (PNS)/spinal cord and other areas of the CNS do not exhibit these focal lesions or deficits at later time points. Rather than gray matter, focal demyelination is observed in the CNS white matter, accompanied by surrounding gliotic scarring, local inflammation, and accumulating Iba1+ microglia. In addition, extravasation of serum factors and fibrinogen into focal lesions appears to recruit microglia, resulting in axonal damage. The authors go on to show that dilution of AAV episomes due to proliferation of oligodendrocyte precursor cells (OPCs) is associated with these focal lesions. These latter findings turn our attention to an underappreciated area in CNS gene therapy, i.e., gliogenesis. Astrocytes are known to proliferate in response to injury, concomitantly with the process of astrogliosis.11Shimada I.S. LeComte M.D. Granger J.C. Quinlan N.J. Spees J.L. Self-renewal and differentiation of reactive astrocyte-derived neural stem/progenitor cells isolated from the cortical peri-infarct area after stroke.J. Neurosci. 2012; 32: 7926-7940Crossref PubMed Scopus (101) Google Scholar Both OPCs and astrocytes display robust proliferative potential post-specification.12Richardson W.D. Young K.M. Tripathi R.B. McKenzie I. NG2-glia as multipotent neural stem cells: fact or fantasy?.Neuron. 2011; 70: 661-673Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar While still unclear, NG2+ glia can potentially serve as OPCs and retain the ability to proliferate in response to injury.12Richardson W.D. Young K.M. Tripathi R.B. McKenzie I. NG2-glia as multipotent neural stem cells: fact or fantasy?.Neuron. 2011; 70: 661-673Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar Further, myelination, which follows synaptic pruning and gliogenesis, has been shown to occur robustly during postnatal development, and myelin turnover requirements may be greatest at sites of high neural activity.13Gibson E.M. et al.Neuronal Activity Promotes Oligodendrogenesis and Adaptive Myelination in the Mammalian Brain.Science. 2014; 344: 1252304Crossref PubMed Scopus (721) Google Scholar Linking these aspects of CNS biology to that of AAV genome persistence, it is plausible that proliferation of OPCs early in development, in adulthood in areas of frequent myelin remodeling, or both could account for the loss of AAV-mediated GALC activity and re-emergence of KD phenotypes over time. Importantly, the authors conclude that, since other areas of the PNS/CNS in AAV9-GALC treated Twitcher mice remain healthy, the emergence of focal demyelinated lesions is not likely to arise due to AAV transgene silencing, but rather from local myelin turnover, generation of new oligodendrocytes, and dilution of AAV episomes on a per-cell basis. The exact reason behind the focal nature of these lesions is unclear, although the authors propose that such might be seeded by accelerated glial proliferation in regions undergoing myelin remodeling. Taken together, the findings from the Heller et al.5Heller G.J. Marshall M.S. Issa Y. Marshall J.N. Nguyen D. Rue E. Pathmasiri K.C. Domowicz M.S. van Breeman R.B. Tai L.M. et al.Waning efficacy in a long-term AAV-mediated gene therapy study in the murine model of Krabbe disease.Mol Ther. 2021; 29 (this issue): 1883-1902Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar study investigating long-term effects of AAV gene therapy in a mouse model for KD highlight a previously under-appreciated temporal component in CNS gene transfer. Envisioning successful gene therapy in KD invariably includes gene therapy very early after birth. Ongoing efforts will undoubtedly help improve CNS coverage through AAV gene transfer. However, we may discover potential trade-offs to early treatment arising from proliferative events and resulting in AAV genome dilution. These observations in the Twitcher mouse model have important implications toward clinical trial design and assessment of different endpoints. The implications also extend beyond KD and, in general, underscore the need to assess long-term AAV-mediated gene expression in the brain. Overall, as we continue to evaluate the potential of AAV-mediated gene therapy for CNS disorders, the importance of pursuing a multidimensional approach that hinges at the interface of physical delivery methods, AAV vectorology, and CNS biology is clear.

Background/Aims: Prenatal microcephaly is posited to arise from aberrant mitosis of neural progenitors, which disrupts both neuronal production and survival. Although microcephaly has both a genetic and environmental etiology, the mechanisms by which dysregulation of mitosis causes microcephaly are poorly understood. We previously discovered that prolonged mitosis of mouse neural progenitors, either ex vivo or in vitro, directly alters progeny cell fate, ­resulting in precocious differentiation and apoptosis. This raises questions as to whether prolonged progenitor mitosis affects cell fate and neurogenesis in vivo, and what are the underlying mechanisms? Methods/Results: Towards addressing these knowledge gaps, we developed an in vivo model of mitotic delay. This uses pharmacological inhibition to acutely and reversibly prolong mitosis during cortical development, and fluorescent dyes to label direct progeny. Using this model, we discovered that a causal relationship between mitotic delay of neural progenitors and altered progeny cell fate is evident in vivo. Using transcriptome analyses to investigate the state of delayed cells and their progeny, we uncovered potential molecular mechanisms by which prolonged mitosis induces altered cell fates, including DNA damage and p53 signaling. We then extended our studies to human neural progenitors, demonstrating that lengthened mitosis duration also directly alters neuronal cell fate. Conclusions: This study establishes a valuable new experimental paradigm towards understanding mechanisms whereby lengthened mitosis duration may explain some cases of microcephaly.

Zika virus (ZIKV) is a re-emerging flavivirus that is transmitted to humans through the bite of an infected mosquito or through sexual contact with an infected partner. ZIKV infection during pregnancy has been associated with numerous fetal abnormalities, including prenatal lethality and microcephaly. However, until recent outbreaks in the Americas, ZIKV has been relatively understudied, and therefore the biology and pathogenesis of ZIKV infection remain incompletely understood. Better methods to study ZIKV infection in live cells could enhance our understanding of the biology of ZIKV and the mechanisms by which ZIKV contributes to fetal abnormalities. To this end, we developed a fluorescent cell-based reporter system allowing for live imaging of ZIKV-infected cells. This system utilizes the protease activity of the ZIKV non-structural proteins 2B and 3 (NS2B-NS3) to specifically mark virus-infected cells. Here, we demonstrate the utility of this fluorescent reporter for identifying cells infected by ZIKV strains of two lineages. Further, we use this system to determine that apoptosis is induced in cells directly infected with ZIKV in a cell-autonomous manner. Ultimately, approaches that can directly track ZIKV-infected cells at the single cell-level have the potential to yield new insights into the host-pathogen interactions that regulate ZIKV infection and pathogenesis.

Throughout development, neural stem cells (NSCs) give rise to differentiated neurons, astrocytes, and oligodendrocytes which together modulate perception, memory, and behavior in the adult nervous system. To understand how NSCs contribute to postnatal/adult brain remodeling and repair after injury, the lateral ventricular (LV) neurogenic niche in the rodent postnatal brain serves as an excellent model system. It is a specialized area containing self-renewing GFAP+ astrocytes functioning as NSCs generating new neurons throughout life. In addition to this now well-studied regenerative process, the LV niche also generates differentiated astrocytes, playing an important role for glial scar formation after cortical injury. While LV NSCs can be clearly distinguished from their neuroblast and oligodendrocyte progeny via molecular markers, the astrocytic identity of NSCs has complicated their distinction from terminally-differentiated astrocytes in the niche. Our current models of postnatal/adult LV neurogenesis do not take into account local astrogenesis, or the possibility that cellular markers may be similar between non-dividing GFAP+ NSCs and their differentiated astrocyte daughters. Postnatal LV neurogenesis is regulated by NSC-intrinsic mechanisms interacting with extracellular/niche-driven cues. It is generally believed that these local effects are responsible for sustaining neurogenesis, though behavioral paradigms and disease states have suggested possibilities for neural circuit-level modulation. With recent experimental findings that neuronal stimulation can directly evoke responses in LV NSCs, it is possible that this exciting property will add a new dimension to identifying postnatal/adult NSCs. Here, we put forth a notion that neural circuit-level input can be a distinct characteristic defining postnatal/adult NSCs from non-neurogenic astroglia.

Long-range integration of transcriptional inputs is critical for gene expression, yet the mechanisms remain poorly understood. We investigated the molecular determinants that confer fidelity to expression of the heart identity gene even-skipped (eve). Targeted deletion of regions bound by the repressor Yan defined two novel enhancers that contribute repressive inputs to stabilize tissue-specific output from a third enhancer. Deletion of any individual enhancer reduced Yan occupancy at the other elements, impacting eve expression, cell fate specification, and cardiac function. These long-range interactions may be stabilized by three-dimensional chromatin contacts that we detected between the elements. Our work provides a new paradigm for chromatin-level integration of general repressive inputs with specific patterning information to achieve robust gene expression.