October 23, 2019
Unexpected CRISPR on-target effects.
Cas9 can induce extensive on-target damage, including large deletions, inversions, and insertions.
Auto Tag: Clinical
October 23, 2019
Bioengineered viral platform for intramuscular passive vaccine delivery to human skeletal muscle.
Skeletal muscle is ideal for passive vaccine administration as it is easily accessible by intramuscular injection. Recombinant adeno-associated virus (rAAV) vectors are in consideration for passive vaccination clinical trials for HIV and influenza. However, greater human skeletal muscle transduction is needed for therapeutic efficacy than is possible with existing serotypes. To bioengineer capsids with therapeutic levels of transduction, we utilized a directed evolution approach to screen libraries of shuffled AAV capsids in pools of surgically resected human skeletal muscle cells from five patients. Six rounds of evolution were performed in various muscle cell types, and evolved variants were validated against existing muscle-tropic serotypes rAAV1, 6, and 8. We found that evolved variants NP22 and NP66 had significantly increased primary human and rhesus skeletal muscle fiber transduction from surgical explants ex vivo and in various primary and immortalized myogenic lines in vitro. Importantly, we demonstrated reduced seroreactivity compared to existing serotypes against normal human serum from 50 adult donors. These capsids represent powerful tools for human skeletal muscle expression and secretion of antibodies from passive vaccines.
October 23, 2019
Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells.
CRISPR/Cas9 is an attractive platform to potentially correct dominant genetic diseases by gene editing with unprecedented precision. In the current proof-of-principle study, we explored the use of CRISPR/Cas9 for gene-editing in myotonic dystrophy type-1 (DM1), an autosomal-dominant muscle disorder, by excising the CTG-repeat expansion in the 3′-untranslated-region (UTR) of the human myotonic dystrophy protein kinase (DMPK) gene in DM1 patient-specific induced pluripotent stem cells (DM1-iPSC), DM1-iPSC-derived myogenic cells and DM1 patient-specific myoblasts. To eliminate the pathogenic gain-of-function mutant DMPK transcript, we designed a dual guide RNA based strategy that excises the CTG-repeat expansion with high efficiency, as confirmed by Southern blot and single molecule real-time (SMRT) sequencing. Correction efficiencies up to 90% could be attained in DM1-iPSC as confirmed at the clonal level, following ribonucleoprotein (RNP) transfection of CRISPR/Cas9 components without the need for selective enrichment. Expanded CTG repeat excision resulted in the disappearance of ribonuclear foci, a quintessential cellular phenotype of DM1, in the corrected DM1-iPSC, DM1-iPSC-derived myogenic cells and DM1 myoblasts. Consequently, the normal intracellular localization of the muscleblind-like splicing regulator 1 (MBNL1) was restored, resulting in the normalization of splicing pattern of SERCA1. This study validates the use of CRISPR/Cas9 for gene editing of repeat expansions.
October 23, 2019
Nuclease-mediated gene editing by homologous recombination of the human globin locus.
Tal-effector nucleases (TALENs) are engineered proteins that can stimulate precise genome editing through specific DNA double-strand breaks. Sickle cell disease and ß-thalassemia are common genetic disorders caused by mutations in ß-globin, and we engineered a pair of highly active TALENs that induce modification of 54% of human ß-globin alleles near the site of the sickle mutation. These TALENS stimulate targeted integration of therapeutic, full-length beta-globin cDNA to the endogenous ß-globin locus in 19% of cells prior to selection as quantified by single molecule real-time sequencing. We also developed highly active TALENs to human ?-globin, a pharmacologic target in sickle cell disease therapy. Using the ß-globin and ?-globin TALENs, we generated cell lines that express GFP under the control of the endogenous ß-globin promoter and tdTomato under the control of the endogenous ?-globin promoter. With these fluorescent reporter cell lines, we screened a library of small molecule compounds for their differential effect on the transcriptional activity of the endogenous ß- and ?-globin genes and identified several that preferentially upregulate ?-globin expression.
October 23, 2019
Adeno-associated virus type 2 wild-type and vector-mediated genomic integration profiles of human diploid fibroblasts analyzed by third-generation PacBio DNA sequencing.
Genome-wide analysis of adeno-associated virus (AAV) type 2 integration in HeLa cells has shown that wild-type AAV integrates at numerous genomic sites, including AAVS1 on chromosome 19q13.42. Multiple GAGY/C repeats, resembling consensus AAV Rep-binding sites are preferred, whereas rep-deficient AAV vectors (rAAV) regularly show a random integration profile. This study is the first study to analyze wild-type AAV integration in diploid human fibroblasts. Applying high-throughput third-generation PacBio-based DNA sequencing, integration profiles of wild-type AAV and rAAV are compared side by side. Bioinformatic analysis reveals that both wild-type AAV and rAAV prefer open chromatin regions. Although genomic features of AAV integration largely reproduce previous findings, the pattern of integration hot spots differs from that described in HeLa cells before. DNase-Seq data for human fibroblasts and for HeLa cells reveal variant chromatin accessibility at preferred AAV integration hot spots that correlates with variant hot spot preferences. DNase-Seq patterns of these sites in human tissues, including liver, muscle, heart, brain, skin, and embryonic stem cells further underline variant chromatin accessibility. In summary, AAV integration is dependent on cell-type-specific, variant chromatin accessibility leading to random integration profiles for rAAV, whereas wild-type AAV integration sites cluster near GAGY/C repeats.Adeno-associated virus type 2 (AAV) is assumed to establish latency by chromosomal integration of its DNA. This is the first genome-wide analysis of wild-type AAV2 integration in diploid human cells and the first to compare wild-type to recombinant AAV vector integration side by side under identical experimental conditions. Major determinants of wild-type AAV integration represent open chromatin regions with accessible consensus AAV Rep-binding sites. The variant chromatin accessibility of different human tissues or cell types will have impact on vector targeting to be considered during gene therapy. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
October 23, 2019
Targeted gene addition in human CD34(+) hematopoietic cells for correction of X-linked chronic granulomatous disease.
Gene therapy with genetically modified human CD34(+) hematopoietic stem and progenitor cells (HSPCs) may be safer using targeted integration (TI) of transgenes into a genomic ‘safe harbor’ site rather than random viral integration. We demonstrate that temporally optimized delivery of zinc finger nuclease mRNA via electroporation and adeno-associated virus (AAV) 6 delivery of donor constructs in human HSPCs approaches clinically relevant levels of TI into the AAVS1 safe harbor locus. Up to 58% Venus(+) HSPCs with 6-16% human cell marking were observed following engraftment into mice. In HSPCs from patients with X-linked chronic granulomatous disease (X-CGD), caused by mutations in the gp91phox subunit of the NADPH oxidase, TI of a gp91phox transgene into AAVS1 resulted in ~15% gp91phox expression and increased NADPH oxidase activity in ex vivo-derived neutrophils. In mice transplanted with corrected HSPCs, 4-11% of human cells in the bone marrow expressed gp91phox. This method for TI into AAVS1 may be broadly applicable to correction of other monogenic diseases.
October 23, 2019
Bioengineered AAV capsids with combined high human liver transduction in vivo and unique humoral seroreactivity.
Existing recombinant adeno-associated virus (rAAV) serotypes for delivering in vivo gene therapy treatments for human liver diseases have not yielded combined high-level human hepatocyte transduction and favorable humoral neutralization properties in diverse patient groups. Yet, these combined properties are important for therapeutic efficacy. To bioengineer capsids that exhibit both unique seroreactivity profiles and functionally transduce human hepatocytes at therapeutically relevant levels, we performed multiplexed sequential directed evolution screens using diverse capsid libraries in both primary human hepatocytes in vivo and with pooled human sera from thousands of patients. AAV libraries were subjected to five rounds of in vivo selection in xenografted mice with human livers to isolate an enriched human-hepatotropic library that was then used as input for a sequential on-bead screen against pooled human immunoglobulins. Evolved variants were vectorized and validated against existing hepatotropic serotypes. Two of the evolved AAV serotypes, NP40 and NP59, exhibited dramatically improved functional human hepatocyte transduction in vivo in xenografted mice with human livers, along with favorable human seroreactivity profiles, compared with existing serotypes. These novel capsids represent enhanced vector delivery systems for future human liver gene therapy applications. Copyright © 2017. Published by Elsevier Inc.
October 23, 2019
Adeno-associated virus genome population sequencing achieves full vector genome resolution and reveals human-vector chimeras
Recombinant adeno-associated virus (rAAV)-based gene therapy has entered a phase of clinical translation and commercialization. Despite this progress, vector integrity following production is often overlooked. Compromised vectors may negatively impact therapeutic efficacy and safety. Using single molecule, real-time (SMRT) sequencing, we can comprehensively profile packaged genomes as a single intact molecule and directly assess vector integrity without extensive preparation. We have exploited this methodology to profile all heterogeneic populations of self-complementary AAV genomes via bioinformatics pipelines and have coined this approach AAV-genome population sequencing (AAV-GPseq). The approach can reveal the relative distribution of truncated genomes versus full-length genomes in vector preparations. Preparations that seemingly show high genome homogeneity by gel electrophoresis are revealed to consist of less than 50% full-length species. With AAV-GPseq, we can also detect many reverse-packaged genomes that encompass sequences originating from plasmid backbone, as well as sequences from packaging and helper plasmids. Finally, we detect host-cell genomic sequences that are chimeric with inverted terminal repeat (ITR)-containing vector sequences. We show that vector populations can contain between 1.3% and 2.3% of this type of undesirable genome. These discoveries redefine quality control standards for viral vector preparations and highlight the degree of foreign products in rAAV-based therapeutic vectors.
September 22, 2019
Meet some code-breakers of noncoding RNAs.
The regulome—the part of the genome that regulates function—includes noncoding RNAs with varied functions yet to be deciphered.
September 22, 2019
Searching for convergent pathways in autism spectrum disorders: insights from human brain transcriptome studies.
Autism spectrum disorder (ASD) is one of the most heritable neuropsychiatric conditions. The complex genetic landscape of the disorder includes both common and rare variants at hundreds of genetic loci. This marked heterogeneity has thus far hampered efforts to develop genetic diagnostic panels and targeted pharmacological therapies. Here, we give an overview of the current literature on the genetic basis of ASD, and review recent human brain transcriptome studies and their role in identifying convergent pathways downstream of the heterogeneous genetic variants. We also discuss emerging evidence on the involvement of non-coding genomic regions and non-coding RNAs in ASD.
September 22, 2019
Transcriptional adaptations during long-term persistence of Staphylococcus aureus in the airways of a cystic fibrosis patient.
The lungs of Cystic fibrosis (CF) patients are often colonized and/or infected by Staphylococcus aureus for years, mostly by one predominant clone. For long-term survival in this environment, S. aureus needs to adapt during its interactions with host factors, antibiotics, and other pathogens. Here, we study long-term transcriptional as well as genomic adaptations of an isogenic pair of S. aureus isolates from a single patient using RNA sequencing (RNA-Seq) and whole genome sequencing (WGS). Mimicking in vivo conditions, we cultivated the S. aureus isolates using artificial sputum medium before harvesting RNA for subsequent analysis. We confirmed our RNA-Seq data using quantitative real-time (qRT)-PCR and additionally investigated intermediate isolates from the same patient representing in total 13.2 years of persistence in the CF airways. Comparative RNA-Seq analysis of the first and the last (“late”) isolate revealed significant differences in the late isolate after 13.2 years of persistence. Of the 2545 genes expressed in both isolates that were cultivated aerobically, 256 genes were up- and 161 were down-regulated with a minimum 2-fold change (2f). Focusing on 25 highly (=8f) up- (n=9) or down- (n=16) regulated genes, we identified several genes encoding for virulence factors involved in immune evasion, bacterial spread or secretion (e.g. spa, sak, and esxA). Moreover, these genes displayed similar expression trends under aerobic, microaerophilic and anaerobic conditions. Further qRT-PCR-experiments of highly up- or down-regulated genes within intermediate S. aureus isolates resulted in different gene expression patterns over the years. Using sequencing analysis of the differently expressed genes and their upstream regions in the late S. aureus isolate resulted in only few genomic alterations. Comparative transcriptomic analysis revealed adaptive changes affecting mainly genes involved in host-pathogen interaction. Although the underlying mechanisms were not known, our results suggest adaptive processes beyond genomic mutations triggered by local factors rather than by activation of global regulators. Copyright © 2014 The Authors. Published by Elsevier GmbH.. All rights reserved.
September 22, 2019
HIV-1 infection of primary CD4(+) T cells regulates the expression of specific HERV-K (HML-2) elements.
Endogenous retroviruses (ERVs) occupy extensive regions of the human genome. Although many of these retroviral elements have lost their ability to replicate, those whose insertion took place more recently, such as the HML-2 group of HERV-K elements, still retain intact open reading frames and the capacity to produce certain viral RNA and/or proteins. Transcription of these ERVs is, however, tightly regulated by dedicated epigenetic control mechanisms. Nonetheless, it has been reported that some pathologic states, such as viral infections and certain cancers, coincide with ERV expression suggesting transcriptional reawakening is possible. HML-2 elements are reportedly induced during HIV-1 infection, but the conserved nature of these elements has, until recently, rendered their expression profiling problematic.Here, we provide comprehensive HERV-K HML-2 expression profiles specific for productively HIV-1 infected primary human CD4(+) T cells. We combined enrichment of HIV-1 infected cells using a reporter virus expressing a surface reporter for gentle and efficient purification with long-read Single Molecule Real-Time sequencing. We show that three HML-2 proviruses, 6q25.1, 8q24.3, and 19q13.42 are up-regulated on average between 3- and 5-fold in HIV-1 infected CD4(+) T cells. One provirus, HML-2 12q24.33, in contrast, was repressed in the presence of active HIV replication.In conclusion, this report identifies the HERV-K HML-2 loci whose expression profiles differ upon HIV-1 infection in primary human CD4(+) T cells. These data will help pave the way for further studies on the influence of endogenous retroviruses on HIV-1 replication.Importance Endogenous retroviruses inhabit big portions of our genome. And although they are mainly inert some of the evolutionarily younger members maintain the ability to express both RNA as well as proteins. We have developed an approach using long-read SMRT sequencing that produces long reads, that provides us with ability to obtain detailed and accurate HERV-K HML-2 expression profiles. We have now applied this approach to study HERV-K expression in the presence and absence of productive HIV-1 infection of primary human CD4(+) T cells. In addition to using SMRT sequencing, our strategy also includes the magnetic selection of the infected cells so that levels of background expression due to uninfected cells are kept at a minimum. The results in this manuscript provide the blueprint for in-depth studies of the interactions of the authentic upregulated HERV-K HML-2 elements and HIV-1. Copyright © 2017 American Society for Microbiology.
September 22, 2019
neoantigenR: An annotation based pipeline for tumor neoantigen identification from sequencing data
Studies indicate that more than 90% of human genes are alternatively spliced, suggesting the complexity of the transcriptome assembly and analysis. The splicing process is often disrupted, resulting in both functional and non-functional end-products (Sveen et al. 2016) in many cancers. Harnessing the immune system to fight against malignant cancers carrying aberrantly mutated or spliced products is becoming a promising approach to cancer therapy. Advances in immune checkpoint blockade have elicited adaptive immune responses with promising clinical responses to treatments against human malignancies (Tumor Neoantigens in Personalized Cancer Immunotherapy 2017). Emerging data suggest that recognition of patient-specific mutation-associated cancer antigens (i.e. from alternative splicing isoforms) may allow scientists to dissect the immune response in the activity of clinical immunotherapies (Schumacher and Schreiber 2015). The advent of high-throughput sequencing technology has provided a comprehensive view of both splicing aberrations and somatic mutations across a range of human malignancies, allowing for a deeper understanding of the interplay of various disease mechanisms. Meanwhile, studies show that the number of transcript isoforms reported to date may be limited by the short-read sequencing due to the inherit limitation of transcriptome reconstruction algorithms, whereas long-read sequencing is able to significantly improve the detection of alternative splicing variants since there is no need to assemble full-length transcripts from short reads. The analysis of these high-throughput long-read sequencing data may permit a systematic view of tumor specific peptide epitopes (also known as neoantigens) that could serve as targets for immunotherapy (Tumor Neoantigens in Personalized Cancer Immunotherapy 2017). Currently, there is no software pipeline available that can efficiently produce mutation-associated cancer antigens from raw high-throughput sequencing data on patient tumor DNA (The Problem with Neoantigen Prediction 2017). In addressing this issue, we introduce a R package that allows the discoveries of peptide epitope candidates, which are the tumor-specific peptide fragments containing potential functional neoantigens. These peptide epitopes consist of structure variants including insertion, deletions, alternative sequences, and peptides from nonsynonymous mutations. Analysis of these precursor candidates with widely used tools such as netMHC allows for the accurate in-silico prediction of neoantigens. The pipeline named neoantigeR is currently hosted in https://github.com/ICBI/neoantigeR.
September 22, 2019
A novel lactobacilli-based teat disinfectant for improving bacterial communities in the milks of cow teats with subclinical mastitis.
Teat disinfection pre- and post-milking is important for the overall health and hygiene of dairy cows. The objective of this study was to evaluate the efficacy of a novel probiotic lactobacilli-based teat disinfectant based on changes in somatic cell count (SCC) and profiling of the bacterial community. A total of 69 raw milk samples were obtained from eleven Holstein-Friesian dairy cows over 12 days of teat dipping in China. Single molecule, real-time sequencing technology (SMRT) was employed to profile changes in the bacterial community during the cleaning protocol and to compare the efficacy of probiotic lactic acid bacteria (LAB) and commercial teat disinfectants. The SCC gradually decreased following the cleaning protocol and the SCC of the LAB group was slightly lower than that of the commercial disinfectant (CD) group. Our SMRT sequencing results indicate that raw milk from both the LAB and CD groups contained diverse microbial populations that changed over the course of the cleaning protocol. The relative abundances of some species were significantly changed during the cleaning process, which may explain the observed bacterial community differences. Collectively, these results suggest that the LAB disinfectant could reduce mastitis-associated bacteria and improve the microbial environment of the cow teat. It could be used as an alternative to chemical pre- and post-milking teat disinfectants to maintain healthy teats and udders. In addition, the Pacific Biosciences SMRT sequencing with the full-length 16S ribosomal RNA gene was shown to be a powerful tool for monitoring changes in the bacterial population during the cleaning protocol.
September 22, 2019
Rapid infectious disease identification by next-generation DNA sequencing.
Currently, there is a critical need to rapidly identify infectious organisms in clinical samples. Next-Generation Sequencing (NGS) could surmount the deficiencies of culture-based methods; however, there are no standardized, automated programs to process NGS data. To address this deficiency, we developed the Rapid Infectious Disease Identification (RIDI™) system. The system requires minimal guidance, which reduces operator errors. The system is compatible with the three major NGS platforms. It automatically interfaces with the sequencing system, detects their data format, configures the analysis type, applies appropriate quality control, and analyzes the results. Sequence information is characterized using both the NCBI database and RIDI™ specific databases. RIDI™ was designed to identify high probability sequence matches and more divergent matches that could represent different or novel species. We challenged the system using defined American Type Culture Collection (ATCC) reference standards of 27 species, both individually and in varying combinations. The system was able to rapidly detect known organisms in <12h with multi-sample throughput. The system accurately identifies 99.5% of the DNA sequence reads at the genus-level and 75.3% at the species-level in reference standards. It has a limit of detection of 146cells/ml in simulated clinical samples, and is also able to identify the components of polymicrobial samples with 16.9% discrepancy at the genus-level and 31.2% at the species-level. Thus, the system's effectiveness may exceed current methods, especially in situations where culture methods could produce false negatives or where rapid results would influence patient outcomes. Copyright © 2016 Elsevier B.V. All rights reserved.