Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems

CRISPR/Cas9 technology is currently considered the most advanced tool for targeted genome engineering. Its sequence-dependent specificity has been explored for locus-directed transcriptional modulation. Such modulation, in particular transcriptional activation, has been proposed as key approach to overcome silencing of dormant HIV provirus in latently infected cellular reservoirs. Currently available agents for provirus activation, so-called latency reversing agents (LRAs), act indirectly through cellular pathways to induce viral transcription. However, their clinical performance remains suboptimal, possibly because reservoirs have diverse cellular identities and/or proviral DNA is intractable to the induced pathways. We have explored two CRISPR/Cas9-derived activator systems as targeted approaches to induce dormant HIV-1 proviral DNA. These systems recruit multiple transcriptional activation domains to the HIV 5'long terminal repeat (LTR), for which we have identified an optimal target region within the LTR U3 sequence. Using this target region, we demonstrate transcriptional activation of proviral genomes via the synergistic activation mediator complex in various in culture model systems for HIV latency. Observed levels of induction are comparable or indeed higher than treatment with established LRAs. Importantly, activation is complete, leading to production of infective viral particles. Our data demonstrate that CRISPR/Cas9-derived technologies can be applied to counteract HIV latency and may therefore represent promising novel approaches in the quest for HIV elimination

PRDM12 Is Required for Initiation of the Nociceptive Neuron Lineage during Neurogenesis

Summary: The sensation of pain is essential for the preservation of the functional integrity of the body. However, the key molecular regulators necessary for the initiation of the development of pain-sensing neurons have remained largely unknown. Here, we report that, in mice, inactivation of the transcriptional regulator PRDM12, which is essential for pain perception in humans, results in a complete absence of the nociceptive lineage, while proprioceptive and touch-sensitive neurons remain. Mechanistically, our data reveal that PRDM12 is required for initiation of neurogenesis and activation of a cascade of downstream pro-neuronal transcription factors, including NEUROD1, BRN3A, and ISL1, in the nociceptive lineage while it represses alternative fates other than nociceptors in progenitor cells. Our results thus demonstrate that PRDM12 is necessary for the generation of the entire lineage of pain-initiating neurons. : The sensation of pain, temperature, and itch by neurons of the nociceptive lineage is essential for animal survival. Bartesaghi et al. report that the transcriptional regulator PRDM12 is indispensable in neural crest cells (NCCs) for the initiation of the sensory neuronal differentiation program that generates the entire nociceptive lineage. Keywords: neurogenesis, pain, nociceptive neurons, Prdm12, neural crest cell

Highly significant antiviral activity of HIV-1 LTR-specific tre-recombinase in humanized mice

Stable integration of HIV proviral DNA into host cell chromosomes, a hallmark and essential feature of the retroviral life cycle, establishes the infection permanently. Current antiretroviral combination drug therapy cannot cure HIV infection. However, expressing an engineered HIV-1 long terminal repeat (LTR) site-specific recombinase (Tre), shown to excise integrated proviral DNA in vitro, may provide a novel and highly promising antiviral strategy. We report here the conditional expression of Tre-recombinase from an advanced lentiviral self-inactivation (SIN) vector in HIV-infected cells. We demonstrate faithful transgene expression, resulting in accurate provirus excision in the absence of cytopathic effects. Moreover, pronounced Tre-mediated antiviral effects are demonstrated in vivo, particularly in humanized Rag2−/−γc−/− mice engrafted with either Tre-transduced primary CD4+ T cells, or Tre-transduced CD34+ hematopoietic stem and progenitor cells (HSC). Taken together, our data support the use of Tre-recombinase in novel therapy strategies aiming to provide a cure for HIV

Genetic landscape of congenital insensitivity to pain and hereditary sensory and autonomic neuropathies

Congenital insensitivity to pain (CIP) and hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders exclusively or predominantly affecting the sensory and autonomic neurons. Due to the rarity of the diseases and findings based mainly on single case reports or small case series, knowledge about these disorders is limited. Here, we describe the molecular workup of a large international cohort of CIP/HSAN patients including patients from normally under-represented countries. We identify 80 previously unreported pathogenic or likely pathogenic variants in a total of 73 families in the >20 known CIP/HSAN-associated genes. The data expand the spectrum of disease-relevant alterations in CIP/HSAN, including novel variants in previously rarely recognized entities such as ATL3-, FLVCR1- and NGF-associated neuropathies and previously under-recognized mutation types such as larger deletions. In silico predictions, heterologous expression studies, segregation analyses and metabolic tests helped to overcome limitations of current variant classification schemes that often fail to categorize a variant as disease-related or benign. The study sheds light on the genetic causes and disease-relevant changes within individual genes in CIP/HSAN. This is becoming increasingly important with emerging clinical trials investigating subtype or gene-specific treatment strategies

The fdp1 and cif1 mutations are caused by different single nucleotide changes in the yeast CIF1 gene

The allelism between the mutations cif1 and fdp1 from Saccharomyces cerevisiae has been demonstrated using PCR techniques and complementation of function. The cif1 mutation results in a shortened version of the protein while the fdp1 mutation introduces a charged residue in a highly hydrophobic stretch.This work was supported by grants from the DGICYT (PB91-0056) to C. Gancedo and the Fonds der Chemischen Industrie and Deutsche Forschungsgemeinschaft (SFB190, Miinchen) to H. Feldmann. M.A.B. was a Fellow of the Universidad Autónoma de Madrid.Peer Reviewe

A newly identified chromosomal microdeletion and an N‐box mutation of the AChRϵ gene cause a congenital myasthenic syndrome

Congenital myasthenic syndromes (CMSs) are frequently caused by mutations of the coding region of the acetylcholine receptor epsilon subunit (AChRϵ) gene leading to a reduced expression of the acetylcholine receptor (AChR) at the postsynaptic membrane. Two recent observations have linked two different N‐box mutations of the human AChRϵ promoter to a clinical CMS phenotype. N‐boxes are regulatory sequence elements of mammalian promoters that confer synapse‐specific expression of several genes, including the AChR subunit genes. Here, we report on a novel point mutation (ϵ‐154G→A) in the N‐box of the AChRϵ promoter in a German CMS pedigree. Semiquantitative analysis of AChRϵ mRNA levels in the patient's muscle indicated significantly impaired AChRϵ expression. We provide additional evidence of a pathogenic role for this mutation using the mutated promoter (ϵ‐154G→A) driving a heterologous gene (luciferase) in rat skeletal muscle. We show that agrin‐induced gene expression is significantly reduced by the N‐box mutant (mt) compared with the wild‐type (wt) promoter. Refined haplotype analysis and direct sequencing revealed maternal inheritance of the mutant AChRϵ promoter (ϵ‐154G→A) together with paternal inheritance of a chromosomal microdeletion (Δ1290 bp) encompassing the promoter and the first two exons of the AChRϵ gene in the index patient. In conclusion, we provide genetic and functional evidence that a mutation of the AChRϵ subunit promoter (ϵ‐154G→A) causes CMS due to the reduction of gene expression in skeletal muscle. Moreover, this is the first report of a chromosomal microdeletion affecting an AChR gene. This type of mutation may be missed in standard screening techniques of CMS patient

Intragenic deletion of TRIM32 in compound heterozygotes with sarcotubular myopathy/LGMD2H

In 2005 the commonality of sarcotubular myopathy (STM) and limb girdle muscular dystrophy type 2H (LGMD2H) was demonstrated, as both are caused by the p D487N missense mutation in TRIM32 originally found in the Manitoba Hutterite population. Recently, three novel homozygous TRIM32 mutations have been described in LGMD patients. Here we describe a three generation Swedish family clinically presenting with limb girdle muscular weakness and histological features of a microvacuolar myopathy. The two index patients were compound heterozygotes for a frameshift mutation in TRIM32 (c.1560delC ) and a 30 kb intragenic deletion, encompassing parts of intron 1 and the entire exon 2 of TRIM32. In these patients, no full-length or truncated TRIM32 could be detected. Interestingly, heterozygous family members carrying only one mutation showed mild clinical symptoms and vacuolar changes in muscle. In our family, the phenotype encompasses additionally a mild demyelinating polyneuropathic syndrome. Thus STM and LGMD2H are the result of loss of function mutations that can be either deletions or missense mutations

Conservation of optimal gRNAs and the effect of sequence variation on SAM-mediated LTR activation.

<p>(<b>A</b>) Sequence conservation of HXB2-derived gRNA3, -4, -5 and -6 based on the Los Alamos HIV Sequence Database considering all HIV subtypes (upper panel), subtype B only (middle panel) or subtype C only (lower panel). Percentages of sequence hits are shown, allowing for 0 to 9 point mutations in the indicated gRNA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158294#sec008" target="_blank">Materials and Methods</a> for details). (<b>B</b>) Frequency distribution of gRNA5 mutations observed in database hits according to nucleotide (nt) position. Different colors indicate the number of mutations (1–9). (<b>C</b>) The effect of sequence variations in gRNA5 on SAM-mediated LTR activation was tested by transient expression of gRNA mutants (gRNA *X, where X is the mutated nucleotide position) with SAM system components in TZM-bl cells. Activation levels were measured at 48 h post transfection by luciferase assays performed on whole cell lysates. Activation is shown as fold increase (light units/transfected cell) over negative control (no gRNA expression). Shown are results of two independent experiments.</p