Generation of a tyrosine hydroxylase-2A-Cre knockin non-human primate model by homology-directed-repair-biased CRISPR genome editing.

Non-human primates (NHPs) are the closest animal model to humans; thus, gene engineering technology in these species holds great promise for the elucidation of higher brain functions and human disease models. Knockin (KI) gene targeting is a versatile approach to modify gene(s) of interest; however, it generally suffers from the low efficiency of homology-directed repair (HDR) in mammalian cells, especially in non-expressed gene loci. In the current study, we generated a tyrosine hydroxylase (TH)-2A-Cre KI model of the common marmoset monkey (marmoset; Callithrix jacchus) using an HDR-biased CRISPR-Cas9 genome editing approach using Cas9-DN1S and RAD51. This model should enable labeling and modification of a specific neuronal lineage using the Cre-loxP system. Collectively, the current study paves the way for versatile gene engineering in NHPs, which may be a significant step toward further biomedical and preclinical applications.

Differential CXCR4 expression on hematopoietic progenitor cells versus stem cells directs homing and engraftment.

Gene therapy involves a substantial loss of hematopoietic stem and progenitor cells (HSPC) during processing and homing. Intra-BM (i.b.m.) transplantation can reduce homing losses, but prior studies have not yielded promising results. We studied the mechanisms involved in homing and engraftment of i.b.m. transplanted and i.v. transplanted genetically modified (GM) human HSPC. We found that i.b.m. HSPC transplantation improved engraftment of hematopoietic progenitor cells (HPC) but not of long-term repopulating hematopoietic stem cells (HSC). Mechanistically, HPC expressed higher functional levels of CXCR4 than HSC, conferring them a retention and homing advantage when transplanted i.b.m. Removing HPC and transplanting an HSC-enriched population i.b.m. significantly increased long-term engraftment over i.v. transplantation. Transient upregulation of CXCR4 on GM HSC-enriched cells, using a noncytotoxic portion of viral protein R (VPR) fused to CXCR4 delivered as a protein in lentiviral particles, resulted in higher homing and long-term engraftment of GM HSC transplanted either i.v. or i.b.m. compared with standard i.v. transplants. Overall, we show a mechanism for why i.b.m. transplants do not significantly improve long-term engraftment over i.v. transplants. I.b.m. transplantation becomes relevant when an HSC-enriched population is delivered. Alternatively, CXCR4 expression on HSC, when transiently increased using a protein delivery method, improves homing and engraftment specifically of GM HSC.

NRASQ61R mutation in human endothelial cells causes vascular malformations.

Somatic mutations in NRAS drive the pathogenesis of melanoma and other cancers but their role in vascular anomalies and specifically human endothelial cells is unclear. The goals of this study were to determine whether the somatic-activating NRASQ61R mutation in human endothelial cells induces abnormal angiogenesis and to develop in vitro and in vivo models to identify disease-causing pathways and test inhibitors. Here, we used mutant NRASQ61R and wild-type NRAS (NRASWT) expressing human endothelial cells in in vitro and in vivo angiogenesis models. These studies demonstrated that expression of NRASQ61R in human endothelial cells caused a shift to an abnormal spindle-shaped morphology, increased proliferation, and migration. NRASQ61R endothelial cells had increased phosphorylation of ERK compared to NRASWT cells indicating hyperactivation of MAPK/ERK pathways. NRASQ61R mutant endothelial cells generated abnormal enlarged vascular channels in a 3D fibrin gel model and in vivo, in xenografts in nude mice. These studies demonstrate that NRASQ61R can drive abnormal angiogenesis in human endothelial cells. Treatment with MAP kinase inhibitor U0126 prevented the change to a spindle-shaped morphology in NRASQ61R endothelial cells, whereas mTOR inhibitor rapamycin did not.

CRISPR-Cas9 fusion to dominant-negative 53BP1 enhances HDR and inhibits NHEJ specifically at Cas9 target sites.

Precise genome editing/correction of DNA double-strand breaks (DSBs) induced by CRISPR-Cas9 by homology-dependent repair (HDR) is limited by the competing error-prone non-homologous end-joining (NHEJ) DNA repair pathway. Here, we define a safer and efficient system that promotes HDR-based precise genome editing, while reducing NHEJ locally, only at CRISPR-Cas9-induced DSBs. We fused a dominant-negative mutant of 53BP1, DN1S, to Cas9 nucleases, and the resulting Cas9-DN1S fusion proteins significantly block NHEJ events specifically at Cas9 cut sites and improve HDR frequency; HDR frequency reached 86% in K562 cells. Cas9-DN1S protein maintains this effect in different human cell types, including leukocyte adhesion deficiency (LAD) patient-derived immortalized B lymphocytes, where nearly 70% of alleles were repaired by HDR and 7% by NHEJ. Our CRISPR-Cas9-DN1S system is clinically relevant to improve the efficiencies of precise gene correction/insertion, significantly reducing error-prone NHEJ events at the nuclease cleavage site, while avoiding the unwanted effects of global NHEJ inhibition.

A Versatile Tool for the Quantification of CRISPR/Cas9-Induced Genome Editing Events in Human Hematopoietic Cell Lines and Hematopoietic Stem/Progenitor Cells.

The efficient site-specific DNA double-strand breaks (DSB) created by CRISPR/Cas9 has revolutionized genome engineering and has great potential for editing hematopoietic stem/progenitor cells (HSPCs). However, detailed understanding of the variables that influence choice of DNA-DSB repair (DDR) pathways by HSPC is required for therapeutic levels of editing in these clinically relevant cells. We developed a hematopoietic-reporter system that rapidly quantifies the three major DDR pathways utilized at the individual DSB created by CRISPR/Cas9-NHEJ, MMEJ, and HDR-and show its applicability in evaluating the different DDR outcomes utilized by human hematopoietic cell lines and primary human HSPC.

Foamy Virus Vector Carries a Strong Insulator in Its Long Terminal Repeat Which Reduces Its Genotoxic Potential.

Strong viral enhancers in gammaretrovirus vectors have caused cellular proto-oncogene activation and leukemia, necessitating the use of cellular promoters in "enhancerless" self-inactivating integrating vectors. However, cellular promoters result in relatively low transgene expression, often leading to inadequate disease phenotype correction. Vectors derived from foamy virus, a nonpathogenic retrovirus, show higher preference for nongenic integrations than gammaretroviruses/lentiviruses and preferential integration near transcriptional start sites, like gammaretroviruses. We found that strong viral enhancers/promoters placed in foamy viral vectors caused extremely low immortalization of primary mouse hematopoietic stem/progenitor cells compared to analogous gammaretrovirus/lentivirus vectors carrying the same enhancers/promoters, an effect not explained solely by foamy virus' modest insertional site preference for nongenic regions compared to gammaretrovirus/lentivirus vectors. Using CRISPR/Cas9-mediated targeted insertion of analogous proviral sequences into the LMO2 gene and then measuring LMO2 expression, we demonstrate a sequence-specific effect of foamy virus, independent of insertional bias, contributing to reduced genotoxicity. We show that this effect is mediated by a 36-bp insulator located in the foamy virus long terminal repeat (LTR) that has high-affinity binding to the CCCTC-binding factor. Using our LMO2 activation assay, LMO2 expression was significantly increased when this insulator was removed from foamy virus and significantly reduced when the insulator was inserted into the lentiviral LTR. Our results elucidate a mechanism underlying the low genotoxicity of foamy virus, identify a novel insulator, and support the use of foamy virus as a vector for gene therapy, especially when strong enhancers/promoters are required.IMPORTANCE Understanding the genotoxic potential of viral vectors is important in designing safe and efficacious vectors for gene therapy. Self-inactivating vectors devoid of viral long-terminal-repeat enhancers have proven safe; however, transgene expression from cellular promoters is often insufficient for full phenotypic correction. Foamy virus is an attractive vector for gene therapy. We found foamy virus vectors to be remarkably less genotoxic, well below what was expected from their integration site preferences. We demonstrate that the foamy virus long terminal repeats contain an insulator element that binds CCCTC-binding factor and reduces its insertional genotoxicity. Our study elucidates a mechanism behind the low genotoxic potential of foamy virus, identifies a unique insulator, and supports the use of foamy virus as a vector for gene therapy.

Placenta growth factor augments airway hyperresponsiveness via leukotrienes and IL-13.

Airway hyperresponsiveness (AHR) affects 55%-77% of children with sickle cell disease (SCD) and occurs even in the absence of asthma. While asthma increases SCD morbidity and mortality, the mechanisms underlying the high AHR prevalence in a hemoglobinopathy remain unknown. We hypothesized that placenta growth factor (PlGF), an erythroblast-secreted factor that is elevated in SCD, mediates AHR. In allergen-exposed mice, loss of Plgf dampened AHR, reduced inflammation and eosinophilia, and decreased expression of the Th2 cytokine IL-13 and the leukotriene-synthesizing enzymes 5-lipoxygenase and leukotriene-C4-synthase. Plgf-/- mice treated with leukotrienes phenocopied the WT response to allergen exposure; conversely, anti-PlGF Ab administration in WT animals blunted the AHR. Notably, Th2-mediated STAT6 activation further increased PlGF expression from lung epithelium, eosinophils, and macrophages, creating a PlGF/leukotriene/Th2-response positive feedback loop. Similarly, we found that the Th2 response in asthma patients is associated with increased expression of PlGF and its downstream genes in respiratory epithelial cells. In an SCD mouse model, we observed increased AHR and higher leukotriene levels that were abrogated by anti-PlGF Ab or the 5-lipoxygenase inhibitor zileuton. Overall, our findings indicate that PlGF exacerbates AHR and uniquely links the leukotriene and Th2 pathways in asthma. These data also suggest that zileuton and anti-PlGF Ab could be promising therapies to reduce pulmonary morbidity in SCD.

Impact of HIV-1 backbone on neutralization sensitivity: neutralization profiles of heterologous envelope glycoproteins expressed in native subtype C and CRF01_AE backbone.

Standardized assays to assess vaccine and antiviral drug efficacy are critical for the development of protective HIV-1 vaccines and drugs. These immune assays will be advanced by the development of standardized viral stocks, such as HIV-1 infectious molecular clones (IMC), that i) express a reporter gene, ii) are representative of globally diverse subtypes and iii) are engineered to easily exchange envelope (env) genes for expression of sequences of interest. Thus far, a subtype B IMC backbone expressing Renilla luciferase (LucR), and into which the ectodomain of heterologous env coding sequences can be expressed has been successfully developed but as execution of HIV-1 vaccine efficacy trials shifts increasingly to non-subtype B epidemics (Southern African and Southeast Asia), non-subtype B HIV-1 reagents are needed to support vaccine development. Here we describe two IMCs derived from subtypes C and CRF01_AE HIV-1 primary isolates expressing LucR (IMC.LucR) that were engineered to express heterologous gp160 Envs. 18 constructs expressing various subtypes C and CRF01_AE Envs, mostly acute, in subtype-matched and -unmatched HIV backbones were tested for functionality and neutralization sensitivity. Our results suggest a possible effect of non-env HIV-1 genes on the interaction of Env and neutralizing antibodies and highlight the need to generate a library of IMCs representative of the HIV-1 subtype spectrum to be used as standardized neutralization assay reagents for assessing HIV-1 vaccine efficacy.