Unique aspects of IFN-γ/STAT1 signaling in neurons.

The IFN-γ/STAT1 immune signaling pathway impacts many homeostatic and pathological aspects of neurons, beyond its canonical role in controlling intracellular pathogens. Well known for its potent pro-inflammatory and anti-viral functions in the periphery, the IFN-γ/STAT1 pathway is rapidly activated then deactivated to prevent excessive inflammation; however, neurons utilize unique IFN-γ/STAT1 activation patterns, which may contribute to the non-canonical neuron-specific downstream effects. Though it is now well-established that the immune system interacts and supports the CNS in health and disease, many aspects regarding IFN-γ production in the CNS and how neurons respond to IFN-γ are unclear. Additionally, it is not well understood how the diversity of the IFN-γ/STAT1 pathway is regulated in neurons to control homeostatic functions, support immune surveillance, and prevent pathologies. In this review, we discuss the neuron-specific mechanisms and kinetics of IFN-γ/STAT1 activation, the potential sources and entry sites of IFN-γ in the CNS, and the diverse set of homeostatic and pathological effects IFN-γ/STAT1 signaling in neurons has on CNS health and disease. We will also highlight the different contexts and conditions under which IFN-γ-induced STAT1 activation has been studied in neurons, and how various factors might contribute to the vast array of downstream effects observed.

Improving Gene Editing Outcomes in Human Hematopoietic Stem and Progenitor Cells by Temporal Control of DNA Repair.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated system (Cas9)-mediated gene editing of human hematopoietic stem cells (hHSCs) is a promising strategy for the treatment of genetic blood diseases through site-specific correction of identified causal mutations. However, clinical translation is hindered by low ratio of precise gene modification using the corrective donor template (homology-directed repair, HDR) to gene disruption (nonhomologous end joining, NHEJ) in hHSCs. By using a modified version of Cas9 with reduced nuclease activity in G1 phase of cell cycle when HDR cannot occur, and transiently increasing the proportion of cells in HDR-preferred phases (S/G2), we achieved a four-fold improvement in HDR/NHEJ ratio over the control condition in vitro, and a significant improvement after xenotransplantation of edited hHSCs into immunodeficient mice. This strategy for improving gene editing outcomes in hHSCs has important implications for the field of gene therapy, and can be applied to diseases where increased HDR/NHEJ ratio is critical for therapeutic success. Stem Cells 2019;37:284-294.

Editing the Sickle Cell Disease Mutation in Human Hematopoietic Stem Cells: Comparison of Endonucleases and Homologous Donor Templates.

Site-specific correction of a point mutation causing a monogenic disease in autologous hematopoietic stem and progenitor cells (HSPCs) can be used as a treatment of inherited disorders of the blood cells. Sickle cell disease (SCD) is an ideal model to investigate the potential use of gene editing to transvert a single point mutation at the ß-globin locus (HBB). We compared the activity of zinc-finger nucleases (ZFNs) and CRISPR/Cas9 for editing, and homologous donor templates delivered as single-stranded oligodeoxynucleotides (ssODNs), adeno-associated virus serotype 6 (AAV6), integrase-deficient lentiviral vectors (IDLVs), and adenovirus 5/35 serotype (Ad5/35) to transvert the base pair responsible for SCD in HBB in primary human CD34+ HSPCs. We found that the ZFNs and Cas9 directed similar frequencies of nuclease activity. In vitro, AAV6 led to the highest frequencies of homology-directed repair (HDR), but levels of base pair transversions were significantly reduced when analyzing cells in vivo in immunodeficient mouse xenografts, with similar frequencies achieved with either AAV6 or ssODNs. AAV6 also caused significant impairment of colony-forming progenitors and human cell engraftment. Gene correction in engrafting hematopoietic stem cells may be limited by the capacity of the cells to mediate HDR, suggesting additional manipulations may be needed for high-efficiency gene correction in HSPCs.

Prolonged STAT1 activation in neurons drives a pathological transcriptional response.

Neurons require physiological IFN-γ signaling to maintain central nervous system (CNS) homeostasis, however, pathological IFN-γ signaling can cause CNS pathologies. The downstream signaling mechanisms that cause these drastically different outcomes in neurons has not been well studied. We hypothesized that different levels of IFN-γ signaling in neurons results in differential activation of its downstream transcription factor, signal transducer and activator of transduction 1 (STAT1), causing varying outcomes. Using primary cortical neurons, we showed that physiological IFN-γ elicited brief and transient STAT1 activation, whereas pathological IFN-γ induced prolonged STAT1 activation, which primed the pathway to be more responsive to a subsequent IFN-γ challenge. This is an IFN-γ specific response, as other IFNs and cytokines did not elicit such STAT1 activation nor priming in neurons. Additionally, we did not see the same effect in microglia or astrocytes, suggesting this non-canonical IFN-γ/STAT1 signaling is unique to neurons. Prolonged STAT1 activation was facilitated by continuous janus kinase (JAK) activity, even in the absence of IFN-γ. Finally, although IFN-γ initially induced a canonical IFN-γ transcriptional response in neurons, pathological levels of IFN-γ caused long-term changes in synaptic pathway transcripts. Overall, these findings suggest that IFN-γ signaling occurs via non-canonical mechanisms in neurons, and differential STAT1 activation may explain how neurons have both homeostatic and pathological responses to IFN-γ signaling.

Gene delivery using AAV8 in vivo for disease stabilization in a bimodal gene therapy approach for the treatment of ADA-deficient SCID.

Adenosine deaminase (ADA) deficiency is an inborn error of metabolism affecting multiple systems and causing severe combined immunodeficiency. We tested intravenous administration of recombinant adeno-associated virus (AAV) 2/8-ADA vector in ADA-deficient neonate and adult mice or as part of a bimodal approach comprised of rAAV treatment at birth followed by infusion of lentiviral vector (LV)-modified lineage-depleted bone marrow cells at 8 weeks. ADA-/- mice treated with rAAV and enzyme replacement therapy (ERT) for 30 days were rescued from the lethal pulmonary insufficiency, surviving out to 180 days without further treatment. rAAV vector copy number (VCN) was highest in liver, lung, and heart and was associated with near-normal ADA activity and thymocyte development. In the bimodal approach, rAAV-mediated ADA expression supported survival during the 4 weeks before infusion of the LV-modified bone marrow cells and during the engraftment period. Conditioning prior to infusion may have resulted in the replacement of rAAV marked cells in marrow and liver, with LV VCN 100- to 1,000-fold higher in hematopoietic tissue compared with rAAV VCN, and was associated with immune cell reconstitution. In conclusion, a bimodal approach may be an alternative for patients without reliable access to ERT before receiving a stem cell transplant or gene therapy.

Global and Local Manipulation of DNA Repair Mechanisms to Alter Site-Specific Gene Editing Outcomes in Hematopoietic Stem Cells.

Monogenic disorders of the blood system have the potential to be treated by autologous stem cell transplantation of ex vivo genetically modified hematopoietic stem and progenitor cells (HSPCs). The sgRNA/Cas9 system allows for precise modification of the genome at single nucleotide resolution. However, the system is reliant on endogenous cellular DNA repair mechanisms to mend a Cas9-induced double stranded break (DSB), either by the non-homologous end joining (NHEJ) pathway or by the cell-cycle regulated homology-directed repair (HDR) pathway. Here, we describe a panel of ectopically expressed DNA repair factors and Cas9 variants assessed for their ability to promote gene correction by HDR or inhibit gene disruption by NHEJ at the HBB locus. Although transient global overexpression of DNA repair factors did not improve the frequency of gene correction in primary HSPCs, localization of factors to the DSB by fusion to the Cas9 protein did alter repair outcomes toward microhomology-mediated end joining (MMEJ) repair, an HDR event. This strategy may be useful when predictable gene editing outcomes are imperative for therapeutic success.

Dosing and Re-Administration of Lentiviral Vector for In Vivo Gene Therapy in Rhesus Monkeys and ADA-Deficient Mice.

Adenosine deaminase (ADA)-deficient mice and healthy rhesus monkeys were studied to determine the impact of age at treatment, vector dosage, dosing schedule, repeat administration, biodistribution, and immunogenicity after systemic delivery of lentiviral vectors (LVs). In Ada -/- mice, neonatal treatment resulted in broad vector marking across all tissues analyzed, whereas adult treatment resulted in marking restricted to the liver, spleen, and bone marrow. Intravenous administration to infant rhesus monkeys also resulted in dose-dependent marking in the liver, spleen, and bone marrow. Using an ELISA to monitor anti-vector antibody development, Ada -/- neonatal mice did not produce an antibody response, whereas Ada -/- adult mice produced a strong antibody response to vector administration. In mice and monkeys with repeat administration of LV, a strong anti-vector antibody response was shown in response to the second LV administration, which resulted in LV inactivation. Three separate doses administered to immune competent mice resulted in acute toxicity. Pegylation of the vesicular stomatitis virus G protein (VSV-G)-enveloped LVs showed a less robust anti-vector response but did not prevent the inactivation of the second LV administration. These studies identify important factors to consider related to age and timing of administration when implementing systemic delivery of LVs as a potential therapeutic agent.