Genome-Edited T Cell Therapies

PURPOSE OF REVIEW: Alternative approaches to conventional drug-based cancer treatments have seen T cell therapies deployed more widely over the last decade. This is largely due to their ability to target and kill specific cell types based on receptor recognition. Introduction of recombinant T cell receptors (TCRs) using viral vectors and HLA-independent T cell therapies using chimeric antigen receptors (CARs) are discussed. This article reviews the tools used for genome editing, with particular emphasis on the applications of site-specific DNA nuclease mediated editing for T cell therapies. RECENT FINDINGS: Genetic engineering of T cells using TCRs and CARs with redirected antigen-targeting specificity has resulted in clinical success of several immunotherapies. In conjunction, the application of genome editing technologies has resulted in the generation of HLA-independent universal T cells for allogeneic transplantation, improved T cell sustainability through knockout of the checkpoint inhibitor, programmed cell death protein-1 (PD-1), and has shown efficacy as an antiviral therapy through direct targeting of viral genomic sequences and entry receptors. SUMMARY: The combined use of engineered antigen-targeting moieties and innovative genome editing technologies have recently shown success in a small number of clinical trials targeting HIV and hematological malignancies and are now being incorporated into existing strategies for other immunotherapies

The complete chloroplast genome sequence of the chlorophycean green alga Scenedesmus obliquus reveals a compact gene organization and a biased distribution of genes on the two DNA strands

BACKGROUND: The phylum Chlorophyta contains the majority of the green algae and is divided into four classes. While the basal position of the Prasinophyceae is well established, the divergence order of the Ulvophyceae, Trebouxiophyceae and Chlorophyceae (UTC) remains uncertain. The five complete chloroplast DNA (cpDNA) sequences currently available for representatives of these classes display considerable variability in overall structure, gene content, gene density, intron content and gene order. Among these genomes, that of the chlorophycean green alga Chlamydomonas reinhardtii has retained the least ancestral features. The two single-copy regions, which are separated from one another by the large inverted repeat (IR), have similar sizes, rather than unequal sizes, and differ radically in both gene contents and gene organizations relative to the single-copy regions of prasinophyte and ulvophyte cpDNAs. To gain insights into the various changes that underwent the chloroplast genome during the evolution of chlorophycean green algae, we have sequenced the cpDNA of Scenedesmus obliquus, a member of a distinct chlorophycean lineage. RESULTS: The 161,452 bp IR-containing genome of Scenedesmus features single-copy regions of similar sizes, encodes 96 genes, i.e. only two additional genes (infA and rpl12) relative to its Chlamydomonas homologue and contains seven group I and two group II introns. It is clearly more compact than the four UTC algal cpDNAs that have been examined so far, displays the lowest proportion of short repeats among these algae and shows a stronger bias in clustering of genes on the same DNA strand compared to Chlamydomonas cpDNA. Like the latter genome, Scenedesmus cpDNA displays only a few ancestral gene clusters. The two chlorophycean genomes share 11 gene clusters that are not found in previously sequenced trebouxiophyte and ulvophyte cpDNAs as well as a few genes that have an unusual structure; however, their single-copy regions differ considerably in gene content. CONCLUSION: Our results underscore the remarkable plasticity of the chlorophycean chloroplast genome. Owing to this plasticity, only a sketchy portrait could be drawn for the chloroplast genome of the last common ancestor of Scenedesmus and Chlamydomonas

Variable Copy Number, Intra-Genomic Heterogeneities and Lateral Transfers of the 16S rRNA Gene in Pseudomonas

Even though the 16S rRNA gene is the most commonly used taxonomic marker in microbial ecology, its poor resolution is still not fully understood at the intra-genus level. In this work, the number of rRNA gene operons, intra-genomic heterogeneities and lateral transfers were investigated at a fine-scale resolution, throughout the Pseudomonas genus. In addition to nineteen sequenced Pseudomonas strains, we determined the 16S rRNA copy number in four other Pseudomonas strains by Southern hybridization and Pulsed-Field Gel Electrophoresis, and studied the intra-genomic heterogeneities by Denaturing Gradient Gel Electrophoresis and sequencing. Although the variable copy number (from four to seven) seems to be correlated with the evolutionary distance, some close strains in the P. fluorescens lineage showed a different number of 16S rRNA genes, whereas all the strains in the P. aeruginosa lineage displayed the same number of genes (four copies). Further study of the intra-genomic heterogeneities revealed that most of the Pseudomonas strains (15 out of 19 strains) had at least two different 16S rRNA alleles. A great difference (5 or 19 nucleotides, essentially grouped near the V1 hypervariable region) was observed only in two sequenced strains. In one of our strains studied (MFY30 strain), we found a difference of 12 nucleotides (grouped in the V3 hypervariable region) between copies of the 16S rRNA gene. Finally, occurrence of partial lateral transfers of the 16S rRNA gene was further investigated in 1803 full-length sequences of Pseudomonas available in the databases. Remarkably, we found that the two most variable regions (the V1 and V3 hypervariable regions) had probably been laterally transferred from another evolutionary distant Pseudomonas strain for at least 48.3 and 41.6% of the 16S rRNA sequences, respectively. In conclusion, we strongly recommend removing these regions of the 16S rRNA gene during the intra-genus diversity studies

Desarrollo de nuevos marcadores genómicos para estudios de biodiversidad en fotobiontes liquénicos. Microalgas eucarióticas como fuente de recursos de utilidad biotecnológica

Las microalgas constituyen un grupo heterogéneo de microorganismos fotosintéticos resultado de los procesos endosimbióticos que han sucedido en la historia evolutiva de los plastidios. Uno de los grupos más importantes y diversos de microalgas es el que constituyen las algas verdes. Dentro de ellas cabe destacar a los fotobiontes liquénicos, un grupo que es capaz de establecer asociaciones simbióticas con hongos para constituir los líquenes. La complejidad de formas y características dificulta enormemente la clasificación taxonómica basada en caracteres morfológicos, tanto de algas verdes en general como fotobiontes en particular. Sin embargo, la disponibilidad de un mayor número de secuencias genéticas y el desarrollo de marcadores moleculares ha permitido construir una nueva taxonomía y sistemática de estos organismos, que está en continua remodelación. Además, la correcta clasificación de las algas verdes es un paso crucial si se desea trabajar en biotecnología. Actualmente, diversos géneros son empleados en industria con diferentes propósitos. Entre estos cabe destacar el interés biomédico que suscitan unas enzimas (llamadas Homing Endonucleasas), codificadas en intrones de tipo I insertos en diversas regiones del genoma de estos organismos. La presente memoria de Tesis Doctoral describe los experimentos llevados a cabo en diferentes especies de fotobiontes liquénicos y otras microalgas con el objetivo de determinar su biodiversidad y posible aplicabilidad biotecnológica. Para ello, se han desarrollado herramientas moleculares destinadas a mejorar la clasificación y el estudio de las relaciones filogenéticas de estos organismos. Además, se ha explorado una región del genoma cloroplástico con elevada frecuencia de intrones de tipo I que codifican para Homing Endonucleasas. Las herramientas desarrolladas fueron utilizadas para caracterizar la diversidad genética de fotobiontes aislados de dos especies muy diferentes de líquenes recolectados en su hábitat natural. En conjunto, los resultados obtenidos sugieren que la generación y revisión constante de nuevas herramientas moleculares en microalgas puede contribuir, por una parte, a aumentar el conocimiento de las características genéticas de estos organismos y las interacciones que establecen con su entorno; y por otra parte, a sustentar su aplicabilidad como fuente de recursos de utilidad biotecnológica

Desarrollo de nuevos marcadores genómicos para estudios de biodiversidad en fotobiontes liquénicos. Microalgas eucarióticas como fuente de recursos de utilidad biotecnológica

Las microalgas constituyen un grupo heterogéneo de microorganismos fotosintéticos resultado de los procesos endosimbióticos que han sucedido en la historia evolutiva de los plastidios. Uno de los grupos más importantes y diversos de microalgas es el que constituyen las algas verdes. Dentro de ellas cabe destacar a los fotobiontes liquénicos, un grupo que es capaz de establecer asociaciones simbióticas con hongos para constituir los líquenes. La complejidad de formas y características dificulta enormemente la clasificación taxonómica basada en caracteres morfológicos, tanto de algas verdes en general como fotobiontes en particular. Sin embargo, la disponibilidad de un mayor número de secuencias genéticas y el desarrollo de marcadores moleculares ha permitido construir una nueva taxonomía y sistemática de estos organismos, que está en continua remodelación. Además, la correcta clasificación de las algas verdes es un paso crucial si se desea trabajar en biotecnología. Actualmente, diversos géneros son empleados en industria con diferentes propósitos. Entre estos cabe destacar el interés biomédico que suscitan unas enzimas (llamadas Homing Endonucleasas), codificadas en intrones de tipo I insertos en diversas regiones del genoma de estos organismos. La presente memoria de Tesis Doctoral describe los experimentos llevados a cabo en diferentes especies de fotobiontes liquénicos y otras microalgas con el objetivo de determinar su biodiversidad y posible aplicabilidad biotecnológica. Para ello, se han desarrollado herramientas moleculares destinadas a mejorar la clasificación y el estudio de las relaciones filogenéticas de estos organismos. Además, se ha explorado una región del genoma cloroplástico con elevada frecuencia de intrones de tipo I que codifican para Homing Endonucleasas. Las herramientas desarrolladas fueron utilizadas para caracterizar la diversidad genética de fotobiontes aislados de dos especies muy diferentes de líquenes recolectados en su hábitat natural. En conjunto, los resultados obtenidos sugieren que la generación y revisión constante de nuevas herramientas moleculares en microalgas puede contribuir, por una parte, a aumentar el conocimiento de las características genéticas de estos organismos y las interacciones que establecen con su entorno; y por otra parte, a sustentar su aplicabilidad como fuente de recursos de utilidad biotecnológica

RNA Exosome Regulated Antisense and Divergent Noncoding RNA Facilitate AID Targeting Throughout the B Cell Genome

Vertebrate immune systems are armed with the ability to generate highly specific immune responses capable of responding to nearly any foreign molecular threat. One of the major mediators of this response is immunoglobulins (Igs) produced by B lymphocytes. The specificity of individual Igs is created through a tightly orchestrated series of somatic DNA manipulations at Ig encoding loci resulting in functional gene rearrangements and nucleotide substitutions. These events serve to create a pool of naive B cells expressing Igs with distinct specificities, capable of expansion in response to antigen specific selection. Affinity of Ig towards antigen is enhanced through nucleotide substitutions introduced at the antigen binding variable region gene segments through the enzyme activation induced cytidine deaminase (AID) during the process of somatic hypermutation (SHM). AID also generates point mutations within noncoding DNA segments of the Ig heavy chain locus that are processed into double strand breaks leading to constant region isotype switching during class switch recombination (CSR). The Ig diversification processes of SHM and CSR critically depend upon transcriptional activation of the relevant DNA segments. Transcription is thought to facilitate single strand DNA substrate recognition by AID during unwinding of the DNA duplex. The 3'-5' exoribonuclease RNA exosome serves as a transcription dependent cofactor of AID. RNA exosome is comprised of multiple structurally integral core subunits and associated nuclease subunits. In this work, RNA exosome core subunit Exosc3 and nuclease Exosc10 have been targeted for conditional mutagenesis and loss of function analysis in mouse cells. RNA exosome deficient B cells were significantly impaired in AID dependent SHM and CSR Ig diversification processes. Transcriptome analyses revealed a striking accumulation of promoter proximal antisense divergent noncoding transcripts (xTSS-RNA) at a subset of genes upon loss of RNA exosome function. xTSS-RNAs mark regions of chromatin containing RNA exosome activity. Multiple known AID target sites including IgH and Myc were observed to express xTSS-RNA. Furthermore, genomic sites of recurrent AID dependent chromosomal translocations were enriched for xTSS-RNA. In addition to promoter proximal xTSS-RNA, cryptic intragenic antisense noncoding transcripts were found to accumulate at many genomic loci. In fact, multiple translocation hotspots precisely overlap regions of RNA exosome sensitive antisense transcription. AID targeted divergently transcribed promoters containing RNA exosome substrates possessed greater amounts of RNA:DNA hybrids, indicative of frequent transcriptional arrest. Lastly, RNA exosome deficient transcriptomes have revealed a substantial number of novel long intergenic noncoding RNAs and enhancer RNAs, indicating a hidden layer of cellular transcriptional activity. A model of AID targeting utilizing transcriptional arrest is becoming increasingly apparent. Transcribed chromatin prone to undergo transcriptional arrest, such as Ig loci or xTSS-RNA expressing regions, frequently undergoes premature transcription termination coupled to RNA exosome mediated degradation of the nascent transcript. This process results in the creation of AID substrates and serves to stabilize its association with chromatin through multiple interactions involving RNA exosome and transcription complex subunits

New insights into the functional organization of xyloglucan biosynthetic enzymes and their catalytic mechanism

Xyloglucan (XyG) is the major hemicellulosic polysaccharide in the primary cell walls of most vascular dicotyledonous plants, and has important structural and physiological functions in plant growth and development. In Arabidopsis thaliana, a glucan synthase CSLC4, three xylosyltransferases, XXT1, XXT2, and XXT5, two galactosyltransferase, MUR3 and XLT2 and a fucosyltransferase, FUT1 synthesize xyloglucan in Golgi. The functional organization of these enzymes is not clear. To study the functional organization of these enzymes, Bimolecular Fluorescence Complementation (BiFC), in vitro pull-down assay and co-immunoprecipitation were used to elucidate the interactions among CSLC4, XXTs, MUR3, XLT2 and FUT1 proteins both in vivo and in vitro. Obtained results show agreement with each other and indicate the physical interactions and/or close proximity among these proteins. To further understand the stoichiometry and exact composition of the complex, the proteomics analysis of the protein complexes immunoprecipitated using different tagged glycosyltransferases as bait proteins is being performed. The other part of this dissertation is to understand the molecular mechanism of XXTs catalytic activity. Currently, not much information is known regarding the catalytic mechanism of cell wall related glycosyltransferases due to the lack of crystal structures. We conducted homology modeling and molecular simulation to predict structure of substrate binding domain (DXD motif) of XXT2 and XXT5. Using this information, we are performing now the site-directed mutagenesis to understand the importance of XXT2 and XXT5 DXD motifs for their functionality both in vitro and in vivo

Evolutionarily conserved and functionally important residues in the I-CeuI homing endonuclease.

Two approaches were used to discern critical amino acid residues for the function of the I- Ceu I homing endonuclease: sequence comparison of subfamilies of homologous proteins and genetic selection. The first approach revealed residues potentially involved in catalysis and DNA recognition. Because I- Ceu I is lethal in Escherichia coli , enzyme variants not perturbing cell viability were readily selected from an expression library. A collection of 49 variants with single amino acid substitutions at 37 positions was assembled. Most of these positions are clustered within or around the LAGLI-DADG dodecapeptide and the TQH sequence, two motifs found in all protein subfamilies examined. The Km and kcat values of the wild-type and nine variant enzymes synthesized in vitro were determined. Three variants, including one showing a substitution of the glutamine residue in the TQH motif, revealed no detectable endonuclease activity; five others showed reduced activity compared to the wild-type enzyme; whereas the remaining variant cleaved the top strand about three times more efficiently than the wild-type. Our results not only confirm recent reports indicating that amino acids in the LAGLI-DADG dodecapeptide are functionally critical, but they also suggest that some residues outside this motif directly participate in catalysis

The role of epigenetics in the maintenance of plant genome stability

xv, 280 leaves : ill. ; 29 cmSignificant alterations in the environmental conditions can have pronounced effects on plant genome stability. Recent evidence argues for a global involvement of the components of epigenetic modules in the regulation of genome homeostasis both immediately after stress exposure and long after environmental cues were acquired. The last observation is of particular interest as the memory of imposing stress can be maintained at the molecular level throughout plant ontogenesis and may be faithfully propagated into the following generation. Our study provides evidence that epigenetic repercussions exerted by stress exposure of parental plants manifest themselves in untreated progeny at all three levels of the epigenetic module: DNA methylation, histone posttranslational modifications and small RNA metabolism. Additionally, the results of our study shed new light on the engagement of the epigenetic machinery in the maintenance of plant genome integrity by counteracting the activity of invading nucleic acids