Frequency Of DNA Double-strand Breaks Research Articles

Transplastomic plants — new approaches to solving “old” problems

Transplastomic plants are capable to accumulate the significant amounts (up to 70% of TSP) of target recombinant proteins in tissues. However, the production of such forms is severely limited by the low yield of initial transformants. This problem requires the development and optimization of new approaches to the delivery of transgenes into chloroplasts and an increase in the frequency of their integration into the plastome. Transplastomic tobacco plants expressing thegfpreporter gene and theaadAselectable marker under the control of thePrrnG10Lpromoter and theTpsbAterminator were obtained in the laboratory of plant bioengineering. It is known that the selected promoter and insertion region (between the tRNA genes of isoleucine and alanine) are capable to provide a high yield of recombinant proteins in the leaves of transplastomic plants [1]. However, the content of recombinant GFP in the leaves of the obtained transplastomic plants was determined at the level of 0.12%, and the variability for this trait was minimal and ranged from 0.09 to 0.16% of TSP. Insufficient accumulation of the target protein in transformants is not associated with transcription disorders or the presence of non-transgenic copies of the plastome. Probably, the low frequency of transformation and the lack of variability between the transformants are the reasons that make it difficult to select highly productive forms. It is proposed to increase the efficiency of targeted delivery of genetic constructs to plastids using single-walled carbon nanotubes loaded with recombinant DNA. This process can also be facilitated by our proposed approach to increase the frequency of DNA double-strand breaks in target regions of the plastome through the use of the CRISPR-Cas9 genome editing system.
 This work was supported by the Russian Science Foundation grant No. 23-24-00545.

Follicular DNA Damage and Pesticide Exposure Among Latinx Children in Rural and Urban Communities

AbstractThe intersectional risks of children in United States immigrant communities include environmental exposures. Pesticide exposures and their biological outcomes are not well characterized in this population group. We assessed pesticide exposure and related these exposures to DNA double-strand breaks (DSBs) in Latinx children from rural, farmworker families (FW; N = 30) and from urban, non-farmworker families (NFW; N = 15) living in North Carolina. DSBs were quantified in hair follicular cells by immunostaining of 53BP1, and exposure to 72 pesticides and pesticide degradation products were determined using silicone wristbands. Cholinesterase activity was measured in blood samples. DSB frequencies were higher in FW compared to NFW children. Seasonal effects were detected in the FW group, with highest DNA damage levels in April–June and lowest levels in October–November. Acetylcholinesterase depression had the same seasonality and correlated with follicular DNA damage. Organophosphate pesticides were more frequently detected in FW than in NFW children. Participants with organophosphate detections had increased follicular DNA damage compared to participants without organophosphate detection. Follicular DNA damage did not correlate with organochlorine or pyrethroid detections and was not associated with the total number of pesticides detected in the wristbands. These results point to rural disparities in pesticide exposures and their outcomes in children from vulnerable immigrant communities. They suggest that among the different classes of pesticides, organophosphates have the strongest genotoxic effects. Assessing pesticide exposures and their consequences at the individual level is key to environmental surveillance programs. To this end, the minimally invasive combined approach used here is particularly well suited for children.

A Gradual Transition Toward Anaplasia in Wilms Tumor Through Tolerance to Genetic Damage

Patients with Wilms tumor (WT) in general have excellent survival, but the prognosis of patients belonging to the subgroup of WT with diffuse anaplasia (DA) is poor due to frequent resistance to chemotherapy. We hypothesized that DA WT cells might undergo changes, such as acquiring a persistent tolerance to DNA damage and copy number aberrations (CNAs), which could eventually lead to their resistance to chemotherapy treatment. Tissue sections from chemotherapy-treated DA WTs (n = 12) were compared with chemotherapy-treated nonanaplastic WTs (n = 15) in a tissue microarray system, enabling analysis of 769 tumor regions. All regions were scored for anaplastic features and immunohistochemistry was used to quantify p53 expression, proliferation index (Ki67), and DNA double-strand breaks (γH2AX). CNAs were assessed by array-based genotyping and TP53 mutations using targeted sequencing. Proliferation index and the frequency of DNA double-strand breaks (γH2AX dot expression) increased with higher anaplasia scores. Almost all (95.6%) areas with full-scale anaplasia had TP53 mutations or loss of heterozygosity, along with an increased amount of CNAs. Interestingly, areas with wild-type TP53 with loss of heterozygosity and only one feature of anaplasia (anaplasia score 1) also had significantly higher proliferation indices, more DNA double-strand breaks, and more CNAs than regions without any anaplastic features (score 0); such areas may be preanaplastic cell populations under selective pressure for TP53 mutations. In conclusion, we suggest that chemoresistance of DA WTs may be partly explained by a high proliferative capability of anaplastic cells, which also have a high burden of double-stranded DNA breaks and CNAs, and that there is a gradual emergence of anaplasia in WT.

Initial experience on abdominal photon-counting computed tomography in clinical routine: general image quality and dose exposure

ObjectivesPhoton-counting computed tomography has lately found its way into clinical routine. The new technique could offer substantial improvements regarding general image quality, image noise, and radiation dose reduction. This study evaluated the first abdominal examinations in clinical routine and compared the results to conventional computed tomography.MethodsIn this single-center retrospective study, 66 patients underwent photon-counting and conventional abdominal CT. Four radiologists assessed general image quality, image noise, and image artifacts. Signal-to-noise ratio and dose properties of both techniques within the clinical application were compared. An ex vivo phantom study revealed the radiobiological impact by means of DNA double-strand break foci in peripheral blood cells by enumerating γ-H2AX+53BP1 foci.ResultsGeneral image quality in accordance with the Likert scale was found superior for photon-counting CT (4.74 ± 0.46 vs. 4.25 ± 0.54; p < 0.001). Signal-to-noise ratio (p < 0.001) and also dose exposure were higher for photon-counting CT (DLP: 419.2 ± 162.2 vs. 372.3 ± 236.6 mGy*cm; p = 0.0435). CT exposure resulted in significantly increased DNA damage in comparison to sham control (p < 0.001). Investigation of the average foci per cell and radiation-induced foci numbers revealed significantly elevated numbers (p = 0.004 and p < 0.0001, respectively) after photon-counting CT.ConclusionPhoton-counting CT in abdominal examinations showed superior results regarding general image quality and signal-to-noise ratio in clinical routine. However, this seems to be traded for a significantly higher dose exposure and corresponding double-strand break frequency. Optimization of standard protocols in further clinical applications is required to find a compromise regarding picture quality and dose exposure.Key Points• Photon-counting computed tomography promises to enhance the diagnostic potential of medical imaging in clinical routine.• Retrospective single-center study showed superior general image quality accompanied by higher dose exposure in initial abdominal PCCT protocols compared to state-of-the-art conventional CT.• A simultaneous ex vivo phantom study revealed correspondingly increased frequencies of DNA double-strand breaks after PCCT.

DNA replication timing directly regulates the frequency of oncogenic chromosomal translocations.

Chromosomal translocations result from the joining of DNA double-strand breaks (DSBs) and frequently cause cancer. However, the steps linking DSB formation to DSB ligation remain undeciphered. We report that DNA replication timing (RT) directly regulates lymphomagenic Myc translocations during antibody maturation in B cells downstream of DSBs and independently of DSB frequency. Depletion of minichromosome maintenance complexes alters replication origin activity, decreases translocations, and deregulates global RT. Ablating a single origin at Myc causes an early-to-late RT switch, loss of translocations, and reduced proximity with the immunoglobulin heavy chain (Igh) gene, its major translocation partner. These phenotypes were reversed by restoring early RT. Disruption of early RT also reduced tumorigenic translocations in human leukemic cells. Thus, RT constitutes a general mechanism in translocation biogenesis linking DSB formation to DSB ligation.

Multi-color dSTORM microscopy in Hormad1-/- spermatocytes reveals alterations in meiotic recombination intermediates and synaptonemal complex structure.

Recombinases RAD51 and its meiosis-specific paralog DMC1 accumulate on single-stranded DNA (ssDNA) of programmed DNA double strand breaks (DSBs) in meiosis. Here we used three-color dSTORM microscopy, and a mouse model with severe defects in meiotic DSB formation and synapsis (Hormad1-/-) to obtain more insight in the recombinase accumulation patterns in relation to repair progression. First, we used the known reduction in meiotic DSB frequency in Hormad1-/- spermatocytes to be able to conclude that the RAD51/DMC1 nanofoci that preferentially localize at distances of ~300 nm form within a single DSB site, whereas a second preferred distance of ~900 nm, observed only in wild type, represents inter-DSB distance. Next, we asked whether the proposed role of HORMAD1 in repair inhibition affects the RAD51/DMC1 accumulation patterns. We observed that the two most frequent recombinase configurations (1 DMC1 and 1 RAD51 nanofocus (D1R1), and D2R1) display coupled frequency dynamics over time in wild type, but were constant in the Hormad1-/- model, indicating that the lifetime of these intermediates was altered. Recombinase nanofoci were also smaller in Hormad1-/- spermatocytes, consistent with changes in ssDNA length or protein accumulation. Furthermore, we established that upon synapsis, recombinase nanofoci localized closer to the synaptonemal complex (SYCP3), in both wild type and Hormad1-/- spermatocytes. Finally, the data also revealed a hitherto unknown function of HORMAD1 in inhibiting coil formation in the synaptonemal complex. SPO11 plays a similar but weaker role in coiling and SYCP1 had the opposite effect. Using this large super-resolution dataset, we propose models with the D1R1 configuration representing one DSB end containing recombinases, and the other end bound by other ssDNA binding proteins, or both ends loaded by the two recombinases, but in below-resolution proximity. This may then often evolve into D2R1, then D1R2, and finally back to D1R1, when DNA synthesis has commenced.

WASp modulates RPA function on single-stranded DNA in response to replication stress and DNA damage

Perturbation in the replication-stress response (RSR) and DNA-damage response (DDR) causes genomic instability. Genomic instability occurs in Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency disorder, yet the mechanism remains largely uncharacterized. Replication protein A (RPA), a single-strand DNA (ssDNA) binding protein, has key roles in the RSR and DDR. Here we show that human WAS-protein (WASp) modulates RPA functions at perturbed replication forks (RFs). Following genotoxic insult, WASp accumulates at RFs, associates with RPA, and promotes RPA:ssDNA complexation. WASp deficiency in human lymphocytes destabilizes RPA:ssDNA-complexes, impairs accumulation of RPA, ATR, ETAA1, and TOPBP1 at genotoxin-perturbed RFs, decreases CHK1 activation, and provokes global RF dysfunction. las17 (yeast WAS-homolog)-deficient S. cerevisiae also show decreased ScRPA accumulation at perturbed RFs, impaired DNA recombination, and increased frequency of DNA double-strand break (DSB)-induced single-strand annealing (SSA). Consequently, WASp (or Las17)-deficient cells show increased frequency of DSBs upon genotoxic insult. Our study reveals an evolutionarily conserved, essential role of WASp in the DNA stress-resolution pathway, such that WASp deficiency provokes RPA dysfunction-coupled genomic instability.

RNA-DNA hybrids regulate meiotic recombination.

RNA-DNA hybrids are often associated with genome instability and also function as a cellular regulator in many biological processes. In this study, we show that accumulated RNA-DNA hybrids cause multiple defects in budding yeast meiosis, including decreased sporulation efficiency and spore viability. Further analysis shows that these RNA-DNA hybrid foci colocalize with RPA/Rad51 foci on chromosomes. The efficient formation of RNA-DNA hybrid foci depends on Rad52 and ssDNA ends of meiotic DNA double-strand breaks (DSBs), and their number is correlated with DSB frequency. Interestingly, RNA-DNA hybrid foci and recombination foci show similar dynamics. The excessive accumulation of RNA-DNA hybrids around DSBs competes with Rad51/Dmc1, impairs homolog bias, and decreases crossover and noncrossover recombination. Furthermore, precocious removal of RNA-DNA hybrids by RNase H1 overexpression also impairs meiotic recombination similarly. Taken together, our results demonstrate that RNA-DNA hybrids form at ssDNA ends of DSBs to actively regulate meiotic recombination.

Gene expression changes and DNA damage after ex vivo exposure of peripheral blood cells to various CT photon spectra

Dual-energy CT provides enhanced diagnostic power with similar or even reduced radiation dose as compared to single-energy CT. Its principle is based on the distinct physical properties of low and high energetic photons, which, however, may also affect the biological effectiveness and hence the extent of CT-induced cellular damage. Therefore, a comparative analysis of biological effectiveness of dual- and single-energy CT scans with focus on early gene regulation and frequency of radiation-induced DNA double strand breaks (DSBs) was performed. Blood samples from three healthy individuals were irradiated ex vivo with single-energy (80 kV and 150 kV) and dual-energy tube voltages (80 kV/Sn150kV) employing a modern dual source CT scanner resulting in Volume Computed Tomography Dose Index (CTDIvol) of 15.79–18.26 mGy and dose length product (DLP) of 606.7–613.8 mGy*cm. Non-irradiated samples served as a control. Differential gene expression in peripheral blood mononuclear cells was analyzed 6 h after irradiation using whole transcriptome sequencing. DSB frequency was studied by 53BP1 + γH2AX co-immunostaining and microscopic evaluation of their focal accumulation at DSBs. Neither the analysis of gene expression nor DSB frequency provided any evidence for significantly increased biological effectiveness of dual-energy CT in comparison to samples irradiated with particular single-energy CT spectra. Relative to control, irradiated samples were characterized by a significantly higher rate of DSBs (p < 0.001) and the shared upregulation of five genes, AEN, BAX, DDB2, FDXR and EDA2R, which have already been suggested as radiation-induced biomarkers in previous studies. Despite steadily decreasing doses, CT diagnostics remain a genotoxic stressor with impact on gene regulation and DNA integrity. However, no evidence was found that varying X-ray spectra of CT impact the extent of cellular damage.

PON2 subverts metabolic gatekeeper functions in B cells to promote leukemogenesis

Unlike other cell types, developing B cells undergo multiple rounds of somatic recombination and hypermutation to evolve high-affinity antibodies. Reflecting the high frequency of DNA double-strand breaks, adaptive immune protection by B cells comes with an increased risk of malignant transformation. B lymphoid transcription factors (e.g., IKZF1 and PAX5) serve as metabolic gatekeepers by limiting glucose to levels insufficient to fuel transformation. We here identified aberrant expression of the lactonase PON2 in B cell acute lymphoblastic leukemia (B-ALL) as a mechanism to bypass metabolic gatekeeper functions. Compared to normal pre-B cells, PON2 expression was elevated in patient-derived B-ALL samples and correlated with poor clinical outcomes in pediatric and adult cohorts. Genetic deletion of Pon2 had no measurable impact on normal B cell development. However, in mouse models for BCR-ABL1 and NRASG12D-driven B-ALL, deletion of Pon2 compromised proliferation, colony formation, and leukemia initiation in transplant recipient mice. Compromised leukemogenesis resulted from defective glucose uptake and adenosine triphosphate (ATP) production in PON2-deficient murine and human B-ALL cells. Mechanistically, PON2 enabled glucose uptake by releasing the glucose-transporter GLUT1 from its inhibitor stomatin (STOM) and genetic deletion of STOM largely rescued PON2 deficiency. While not required for glucose transport, the PON2 lactonase moiety hydrolyzes the lactone-prodrug 3OC12 to form a cytotoxic intermediate. Mirroring PON2 expression levels in B-ALL, 3OC12 selectively killed patient-derived B-ALL cells but was well tolerated in transplant recipient mice. Hence, while B-ALL cells critically depend on aberrant PON2 expression to evade metabolic gatekeeper functions, PON2 lactonase activity can be leveraged as synthetic lethality to overcome drug resistance in refractory B-ALL.

A New Perspective on the Origin of DNA Double-Strand Breaks and Its Implications for Ageing.

It is estimated that 10–50 DNA double-strand breaks (DSBs) occur in a nucleated human cell per cell cycle. We reviewed the present state of knowledge and hypothesized that the currently accepted mechanisms cannot explain such high frequency of DSBs occurring daily under normal physiological conditions. We propose an alternative model that implicates illegitimate genomic integration into healthy cells of cell-free chromatin (cfCh) particles released from the billions of cells that die in the body every day. Repeated genomic integration of cfCh may have catastrophic consequences for the cell, such as DSBs, their faulty repair by nonhomologous end joining (NHEJ) followed by apoptosis with release of more cfCh which would integrate into genomes of surrounding cells. This can creates a vicious cycle of cfCh integration, DSBs, NHEJ, and more apoptosis, thereby providing a potential explanation as to why so many billions of cells die in the body on a daily basis. We also recount the recent observation that cfCh integration and the resulting DSBs activate inflammatory cytokines. This leads us to propose that concurrent DSBs and induction of inflammation occurring throughout life may be the underlying cause of ageing, degenerative disorders, and cancer. Finally, we discuss the prospect that agents that can inactivate/degrade cfCh may hold the key to making healthy ageing a realizable goal.

Age-associated deficient recruitment of 53BP1 in G1 cells directs DNA double-strand break repair to BRCA1/CtIP-mediated DNA-end resection.

DNA repair mechanisms play a crucial role in maintaining genome integrity. However, the increased frequency of DNA double-strand breaks (DSBs) and genome rearrangements in aged individuals suggests an age-associated DNA repair deficiency. Previous work from our group revealed a delayed firing of the DNA damage response in human mammary epithelial cells (HMECs) from aged donors. We now report a decreased activity of the main DSB repair pathways, the canonical non-homologous end-joining (c-NHEJ) and the homologous recombination (HR) in these HMECs from older individuals. We describe here a deficient recruitment of 53BP1 to DSB sites in G1 cells, probably influenced by an altered epigenetic regulation. 53BP1 absence at some DSBs is responsible for the age-associated DNA repair defect, as it permits the ectopic formation of BRCA1 foci while still in the G1 phase. CtIP and RPA foci are also formed in G1 cells from aged donors, but RAD51 is not recruited, thus indicating that extensive DNA-end resection occurs in these breaks although HR is not triggered. These results suggest an age-associated switch of DSB repair from canonical to highly mutagenic alternative mechanisms that promote the formation of genome rearrangements, a source of genome instability that might contribute to the aging process.

High-resolution, ultrasensitive and quantitative DNA double-strand break labeling in eukaryotic cells using i-BLESS.

DNA double-strand breaks (DSBs) are implicated in various physiological processes, such as class-switch recombination or crossing-over during meiosis, but also present a threat to genome stability. Extensive evidence shows that DSBs are a primary source of chromosome translocations or deletions, making them a major cause of genomic instability, a driving force of many diseases of civilization, such as cancer. Therefore, there is a great need for a precise, sensitive, and universal method for DSB detection, to enable both the study of their mechanisms of formation and repair as well as to explore their therapeutic potential. We provide a detailed protocol for our recently developed ultrasensitive and genome-wide DSB detection method: immobilized direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing (i-BLESS), which relies on the encapsulation of cells in agarose beads and labeling breaks directly and specifically with biotinylated linkers. i-BLESS labels DSBs with single-nucleotide resolution, allows detection of ultrarare breaks, takes 5 d to complete, and can be applied to samples from any organism, as long as a sufficient amount of starting material can be obtained. We also describe how to combine i-BLESS with our qDSB-Seq approach to enable the measurement of absolute DSB frequencies per cell and their precise genomic coordinates at the same time. Such normalization using qDSB-Seq is especially useful for the evaluation of spontaneous DSB levels and the estimation of DNA damage induced rather uniformly in the genome (e.g., by irradiation or radiomimetic chemotherapeutics).

Metabolic Gatekeepers of Pathological B Cell Activation.

Unlike other cell types, B cells undergo multiple rounds of V(D)J recombination and hypermutation to evolve high-affinity antibodies. Reflecting high frequencies of DNA double-strand breaks, adaptive immune protection by B cells comes with an increased risk of malignant transformation. In addition, the vast majority of newly generated B cells express an autoreactive B cell receptor (BCR). Thus, B cells are under intense selective pressure to remove autoreactive and premalignant clones. Despite stringent negative selection, B cells frequently give rise to autoimmune disease and B cell malignancies. In this review, we discuss mechanisms that we term metabolic gatekeepers to eliminate pathogenic B cell clones on the basis of energy depletion. Chronic activation signals from autoreactive BCRs or transforming oncogenes increase energy demands in autoreactive and premalignant B cells. Thus, metabolic gatekeepers limit energy supply to levels that are insufficient to fuel either a transforming oncogene or hyperactive signaling from an autoreactive BCR.

Deploying MMEJ using MENdel in precision gene editing applications for gene therapy and functional genomics.

Gene-editing experiments commonly elicit the error-prone non-homologous end joining for DNA double-strand break (DSB) repair. Microhomology-mediated end joining (MMEJ) can generate more predictable outcomes for functional genomic and somatic therapeutic applications. We compared three DSB repair prediction algorithms – MENTHU, inDelphi, and Lindel – in identifying MMEJ-repaired, homogeneous genotypes (PreMAs) in an independent dataset of 5,885 distinct Cas9-mediated mouse embryonic stem cell DSB repair events. MENTHU correctly identified 46% of all PreMAs available, a ∼2- and ∼60-fold sensitivity increase compared to inDelphi and Lindel, respectively. In contrast, only Lindel correctly predicted predominant single-base insertions. We report the new algorithm MENdel, a combination of MENTHU and Lindel, that achieves the most predictive coverage of homogeneous out-of-frame mutations in this large dataset. We then estimated the frequency of Cas9-targetable homogeneous frameshift-inducing DSBs in vertebrate coding regions for gene discovery using MENdel. 47 out of 54 genes (87%) contained at least one early frameshift-inducing DSB and 49 out of 54 (91%) did so when also considering Cas12a-mediated deletions. We suggest that the use of MENdel helps researchers use MMEJ at scale for reverse genetics screenings and with sufficient intra-gene density rates to be viable for nearly all loss-of-function based gene editing therapeutic applications.

MSH2 shapes the meiotic crossover landscape in relation to interhomolog polymorphism in Arabidopsis.

During meiosis, DNA double‐strand breaks undergo interhomolog repair to yield crossovers between homologous chromosomes. To investigate how interhomolog sequence polymorphism affects crossovers, we sequenced multiple recombinant populations of the model plant Arabidopsis thaliana. Crossovers were elevated in the diverse pericentromeric regions, showing a local preference for polymorphic regions. We provide evidence that crossover association with elevated diversity is mediated via the Class I crossover formation pathway, although very high levels of diversity suppress crossovers. Interhomolog polymorphism causes mismatches in recombining molecules, which can be detected by MutS homolog (MSH) mismatch repair protein heterodimers. Therefore, we mapped crossovers in a msh2 mutant, defective in mismatch recognition, using multiple hybrid backgrounds. Although total crossover numbers were unchanged in msh2 mutants, recombination was remodelled from the diverse pericentromeres towards the less‐polymorphic sub‐telomeric regions. Juxtaposition of megabase heterozygous and homozygous regions causes crossover remodelling towards the heterozygous regions in wild type Arabidopsis, but not in msh2 mutants. Immunostaining showed that MSH2 protein accumulates on meiotic chromosomes during prophase I, consistent with MSH2 regulating meiotic recombination. Our results reveal a pro‐crossover role for MSH2 in regions of higher sequence diversity in A. thaliana.

Nucleosides Rescue Replication-Mediated Genome Instability of Human Pluripotent Stem Cells.

SummaryHuman pluripotent stem cells (PSCs) are subject to the appearance of recurrent genetic variants on prolonged culture. We have now found that, compared with isogenic differentiated cells, PSCs exhibit evidence of considerably more DNA damage during the S phase of the cell cycle, apparently as a consequence of DNA replication stress marked by slower progression of DNA replication, activation of latent origins of replication, and collapse of replication forks. As in many cancers, which, like PSCs, exhibit a shortened G1 phase and DNA replication stress, the resulting DNA damage may underlie the higher incidence of abnormal and abortive mitoses in PSCs, resulting in chromosomal non-dysjunction or cell death. However, we have found that the extent of DNA replication stress, DNA damage, and consequent aberrant mitoses can be substantially reduced by culturing PSCs in the presence of exogenous nucleosides, resulting in improved survival, clonogenicity, and population growth.

Intragenic meiotic recombination in Schizosaccharomyces pombe is sensitive to environmental temperature changes

Changes in environmental temperature influence cellular processes and their dynamics, and thus affect the life cycle of organisms that are unable to control their cell/body temperature. Meiotic recombination is the cellular process essential for producing healthy haploid gametes by providing physical links (chiasmata) between homologous chromosomes to guide their accurate segregation. Additionally, meiotic recombination—initiated by programmed DNA double-strand breaks (DSBs)—can generate genetic diversity and, therefore, is a driving force of evolution. Environmental temperature influencing meiotic recombination outcome thus may be a crucial determinant of reproductive success and genetic diversity. Indeed, meiotic recombination frequency in fungi, plants and invertebrates changes with temperature. In most organisms, these temperature-induced changes in meiotic recombination seem to be mediated through the meiosis-specific chromosome axis organization, the synaptonemal complex in particular. The fission yeast Schizosaccharomyces pombe does not possess a synaptonemal complex. Thus, we tested how environmental temperature modulates meiotic recombination frequency in the absence of a fully-fledged synaptonemal complex. We show that intragenic recombination (gene conversion) positively correlates with temperature within a certain range, especially at meiotic recombination hotspots. In contrast, crossover recombination, which manifests itself as chiasmata, is less affected. Based on our observations, we suggest that, in addition to changes in DSB frequency, DSB processing could be another temperature-sensitive step causing temperature-induced recombination rate alterations.

ESGCT 27th Annual Congress In collaboration with SETGyc Barcelona, Spain October 22–25, 2019 Abstracts

Human Gene TherapyVol. 30, No. 11 AbstractsFree AccessESGCT 27th Annual Congress In collaboration with SETGyc Barcelona, Spain October 22–25, 2019 AbstractsPublished Online:7 Nov 2019https://doi.org/10.1089/hum.2019.29095.abstractsAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Selected Oral PresentationsOR01Paired-nickase S.aureus Cas9 system is an efficient and potentially safer in vivo treatment for Primary Hyperoxaluria Type 1L Torella1 I Raimondi1 A Vales1 C Olague1 A Abad1 J R Rodriguez-Madoz3 M Huarte1 E Salido2 G Gonzalez-Aseguinolaza1 N Zabaleta141: Gene Therapy and Regulation of Gene Expression Program, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain. Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona, Spain. 2: Hospital Universitario de Canarias, Universidad La Laguna, Tenerife, Spain. Centre for Biomedical Research on Rare Diseases (CIBERER). 3: Cell Therapy Program, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain. Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona, Spain. 4: Harvard Medical School, Grousbeck Gene Therapy Center, Schepens Eye Research Institute, Mass. Eye and Ear, Boston (MA)Primary hyperoxaluria type 1 (PH1) is a rare genetic metabolic disorder associated with mutations in AGXT gene, causing hepatic alanine-glyoxylate aminotransferase (AGT) deficiency. Consequently, oxalate is overproduced in the liver and accumulated in kidneys causing life-threatening renal damage. The only curative treatment is liver transplantation, thus, new therapies are required. The inhibition of glycolate oxidase (GO), the enzyme implicated in the synthesis of glyoxylate (precursor of oxalate), has been proven to be an efficient substrate reduction therapy (SRT) to treat PH1. Recently, AAV8-CRISPR/Cas9-mediated in vivo SRT was shown to greatly diminish GO expression, resulting in urine oxalate reduction and prevention of kidney damage. Nevertheless, concerns regarding CRISPR/Cas9 off-target effects should not be underestimated. Our approach to decrease off-target modifications was to use a nickase Cas9 combined with two gRNAs targeting nearby regions on the opposite strand. PH1 mice were treated with D10A-SaCas9 nickase mutants and two gRNAs previously tested efficient for the WT SaCas9. Simultaneous nicks greatly interfered with transcription and translation of Hao1, showing no significant differences with WT Cas9, while individual nicks did not interfere with the target gene expression. As expected, GO inhibition decreased urine oxalate levels and crystal accumulation in kidney parenchyma. Moreover, potential off-target sites for each gRNA were identified by CIRCLE-seq and NGS analysis is being performed in mice treated with WT or nickase SaCas9. In conclusion, dual nickase Cas9 should be preferred over nuclease strategy for more efficient and safer in vivo treatment of this monogenic disease.OR02HDR-CRISPR: a novel system to promote Cas9-mediated homology-directed DNA repairA Carusillo1 T Cathomen1 C Mussolino11: Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of FreiburgPrecise genome editing requires the harnessing of homologous recombination-based homology-directed repair (HDR) pathway and a proper donor template to precisely seal a designer nuclease (DN)-induced DNA double strand break (DSB). However, competing DNA repair pathways, such as non-homologous end-joining (NHEJ), predominate in mammalian cells, often resulting in HDR frequencies far below the thresholds required for clinical translation. While chemical compounds have been used to synchronize the cells in cell cycle phases when the HDR pathway is most active or, alternatively, to inhibit NHEJ, their potential side effects limit the use of such drugs in clinical settings. To overcome this limitation, we sought to increase at the DSB the local concentration of factors critical for either engaging HDR or inhibiting NHEJ, and generated 13 different Cas9-fusion proteins (referred to as HDR-CRISPRs). We used a traffic light reporter (TLR) system to assess the frequency of DSBs that are either repaired by NHEJ or HDR, respectively, and achieved up to 3-fold increase in HDR-mediated repair events with selected HDR-CRISPRs. Importantly, the simultaneous inhibition of the NHEJ pathway further improved the HDR to NHEJ ratio, leading to an almost even distribution of HDR to NHEJ. These results support our hypothesis that the local recruitment of factors to the DSB to either promote HDR or inhibit NHEJ can modulate the DNA repair choice without altering the physiology of the target cells. We envision that this strategy is readily translatable to clinically relevant applications.OR03Targeted gene correction of human hematopoietic stem cells for the treatment of Wiskott-Aldrich SyndromeABSTRACT WITHDRAWNOR04Adeno-associated virus in human liver: natural history and consequences in tumor developmentT La Bella1 S Imbeaud1 C Peneau1 I Mami1 S Datta1 Q Bayard1 S Caruso1 T Z Hirsch1 J Calderaro2 G Morcrette13 C Guettier3 V Paradis4 G Amaddeo2 A Laurent2 L Possenti5 L Chiche6 P Bioulac-Sage7 J F Blanc7 E Letouzé1 J C Nault18 J Zucman-Rossi191: Functional Genomics of Solid Tumors, Centre de Recherche des Cordeliers, Inserm U1138, Paris, France. 2: Groupe Hospitalier Henri Mondor, AP-HP, Inserm U955, Université Paris-Est, Créteil, France. 3: Hôpitaux Paul Brousse et Bicêtre, Inserm U1193, Le Kremlin Bicêtre, France. 4: Hôpital Beaujon, Clichy, France. 5: CHU Bordeaux, Hôpital Haut-Lévêque, Bordeaux, France. 6: CHU Bordeaux, Centre Médico Chirurgical Magellan, Haut-Lévêque Hospital, Pessac, France. 7: BaRITOn, Inserm U1053, Bordeaux, France 8: Hôpitaux Universitaires Paris–Seine Saint-Denis, Site Jean Verdier, Bondy, France. 9: European Hospital Georges Pompidou, AP-HP, Paris, France.Adeno-associated virus (AAV) is a defective mono-stranded DNA virus, endemic in human population (35–80%). Recurrent clonal AAV2 insertions are associated with the pathogenesis of rare human hepatocellular carcinoma (HCC) developed on normal liver. This study aimed to characterize the natural history of AAV infection in the liver and its consequence in tumor development. Viral DNA was quantified in tumor and non-tumor liver tissues of 1461 patients and in silico analyses using viral capture data explored viral variants and new clonal insertions. AAV DNA was detected in 21% of the patients, more frequently in the non-tumor counterpart (18%) than in tumor (8%). The full-length viral sequences were reconstructed in 57 patients leading to identify two distinct AAV subtypes: one similar to AAV2, the other hybrid between AAV2 and AAV13 sequences. Episomal viral forms were found in 4% of the non-tumor tissues, frequently associated with viral RNA expression and human herpesvirus type 6 (HHV6), the candidate natural AAV helper virus. In 30 HCC, clonal AAV insertions were recurrently identified in CCNA2, CCNE1, TERT, TNFSF10, KMT2B and GLI1/INHBE. AAV insertion triggered oncogenic overexpression through multiple mechanisms that differ according to the localization of the integration site. Clonal AAV insertions were positively selected during HCC development on non-cirrhotic liver challenging the notion of AAV as a non-pathogenic virus. In conclusion, this is the first large scale study that provides an integrated analysis of wild type AAV infection in the liver with the identification of viral genotypes, molecular forms, helper virus relationship and viral integrations.OR05Paracrine delivery of therapeutic biologics for cancerS N Smith1 R Schubert2 B Simic1 D Brücher1 M Schmid1 V Gradinaru2 A Plückthun11: University of Zurich 2: California Institute of TechnologyA fundamental goal of cancer drug delivery is to achieve sufficient levels within the tumour without leading to high systemic concentrations that might cause off-target toxicities. In situ production of protein-based therapeutics by tumour cells provides an attractive alternative to treatment with repeated high bolus injections, as secretion by the tumour itself could provide high local concentrations that act in a paracrine fashion over an extended duration. For this purpose, we have developed a non-oncolytic adenoviral delivery system that allows for targeting of Ad5 to discrete cell types by redirecting viral tropism to cell surface biomarkers through the use of interchangeable adapters. Furthermore, we recently described the engineering of a protein-based ‘shield’ that is coated on the Ad5 capsid, which, together with the retargeting adapters, allows for improved tumour specificity and prevention of viral clearance. To test this delivery strategy in vivo, SCID-beige mice bearing orthotopic BT474 xenografts were treated with three doses of either a cancer-specific, non-replicative Ad5 that encodes a secreted anti-HER2 antibody, trastuzumab, in its genome, or with the protein therapeutic itself (Herceptin®). We have employed state-of-the-art whole tumour clearing and imaging with confocal microscopy at high spatial resolution in 3D to assess biodistribution, and large volumetric imaging has revealed that the secreted therapeutic diffuses significantly throughout the tumour leading to a therapeutic effect and delayed tumour outgrowth. Moreover, the systemic concentration of antibody is significantly reduced with viral delivery, suggesting that paracrine delivery may be a promising strategy for delivery of biologics with narrow therapeutic indices.OR06Base editor-mediated CD33 engineering to improve safety and efficacy of CD33-targeted cancer therapyO Humbert1 S Cook1 M Llevellyn1 G Laszlo1 J K Joung23 B Kleinstiver23 R B Walter14 H P Kiem141: Fred Hutchinson Cancer Research Center 2: Massachusetts General Hospital 3: Harvard Medical School 4: University of WashingtonAntigen-specific immunotherapies for myeloid malignancies, including acute myeloid leukemia (AML), have largely focused on CD33, a myeloid differentiation antigen displayed on AML blasts and possibly on leukemia stem cells. Improved survival with the CD33 antibody-drug conjugate gemtuzumab ozogamicin (GO) in AML patients has validated CD33 as immunotherapeutic target. An important limitation of this approach is however the significant on-target/off-leukemia effects attributed to the expression of CD33 on normal myeloid cells, causing severe cytopenia in treated patients. Recent studies, including by our group, have demonstrated that CRISPR/Cas9 nuclease-based editing of CD34+ hematopoietic stem and progenitor cells (HSPCs) conferred protection from CD33-directed drugs. While promising, this CRISPR-based strategy suffers from off-target activity due to cleavage of a nearby CD33 homolog pseudogene and from activation of endogenous TP53-mediated DNA damage responses. To address these limitations, we have explored the use of cytosine base editors (CBE) that introduce precise nucleotide substitutions and circumvent the need for DNA double strand breaks. We investigated 2 different strategies for introducing non-sense and splicing mutations in CD33. CBE-treatment of human CD34+ HSPCs did not impair engraftment and differentiation in a mouse model, while reducing CD33 expression and protecting cells from in vivo GO administration. Next-generation sequencing analysis of blood nucleated cells confirmed the persistence and specificity of CBE-induced mutations in vivo. Together, these results validate the use of CBE for the generation of CD33 engineered hematopoiesis to improve safety and efficacy of CD33-targeted therapies.OR07De novo generation of functional human thymus organoids from induced pluripotent stem cellsA Chhatta1 M Cordes1 S Vloemans1 M Hanegraaf1 T Cupedo2 F Carlotti1 J Cornelissen2 D Salvatori1 R Hoeben1 M Mikkers1 W E Fibbe1F JT Staal11: Leiden University Medical Center 2: Erasmus Medical CenterA proper functional thymus is required for generation of T cell mediated immunity. This is dramatically illustrated by patients lacking a thymus, such as children with complete diGeorge Syndrome which is fatal if left untreated. Therapeutic options for such patients are limited and confined to transplantation of small fragments from allogeneic neonatal thymi. Following the concept that an autologous medical product would be advantageous for any condition in which thymic function is impaired, we set out to develop a preclinical strategy to generate functional human thymi from induced pluripotent stem cells (iPSC), as potential autologous stem cell source. Here we describe that human iPSC can be differentiated into induced thymic epithelial cells (iTEPC) following developmental stages that mimic normal development. This protocol is most robust when combined with directed differentiation enforced by lentiviral expression of FoxN1, the master regulator for thymic epithelial cells. When aggregated in organoids and transplanted in nude mice (that lack a thymus), these organoids supported the development of functional T cells with a broad TCR repertoire capable of cytokine production when stimulated via the T cell receptor. Thus, we provide proof-of-principle evidence that a combination of stem cell technology nad gene therapy can restore thymic function.OR08Self-assembly of human stem/progenitor cells creates neo-vascularized skin and skin organoidsP Peking1 A Hochreiter1 M Wolf1 C Scharler1 B Vári1 E Russe2 L Krisch3 K Schallmoser13 D Strunk11: Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University, Salzburg, Austria. 2: Department of Plastic, Aesthetic and Reconstructive Surgery, Hospital Barmherzige Brueder, Salzburg, Austria 3: Department of Transfusion Medicine, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University, Salzburg, AustriaStem/progenitor cells bear the potential to self-organize, creating organoids that resemble the organ functions in vitro. Here we established a humanized skin regeneration mouse model, based on self-assembly of adult as compared to iPSC-derived skin cell lineages forming neo-vascularized human skin. Adult endothelial cells (EC), skin fibroblasts (FB) and epidermal keratinocytes (KC) were propagated in 2D under xeno-free conditions. In addition, umbilical cord blood-derived iPSC were differentiated into iPS-EC,-FB, and–KC. Cell identity and purity were confirmed by flow cytometry and clonogenicity indicating their stem/progenitor potential. Skin organoid formation was performed to investigate cell self-organisation supported by human platelet-derived growth factors. Via life cell tracking sequential organoid assembly starting from stromal-vascular aggregation and followed by superficial anchorage of KC was revealed. Xeno-free human cell grafts, containing a mixture of KC, FB and EC in human platelet lysate (HPL) were transplanted onto full-thickness wounds of NSG mice using a transplant chamber to circumvent murine skin contraction. Two weeks after transplantation, histological analysis demonstrated appropriate cell organization into layered skin and a regular distribution of collagen fibers and ground substance. Immunohistochemistry confirmed the human origin of the grafts and a combination of murine and human neo-vasculature. Quantification showed significantly increased vessel numbers upon co-transplantation of EC compared to limited murine in-sprouting angiogenesis after transplantation of KC+FB only. The data show that self-assembly of human KC+FB combined with co-transplanted EC and HPL can create complex organoids in vitro and human neo-vascularized skin in vivo, building the basis for novel skin regeneration strategies.OR09Cell- and vector-engineering approaches for manufacturing high-titer GaLV pseudotyped lentiviral vectors from stable and constitutive producer cell linesA F Rodrigues12 R Nogueira1 T A Vaz1 M JT Carrondo1 A S Coroadinha1231: iBET – Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal 2: Instituto de Tecnologia Química e Biológica António Xavier, NOVA University Lisbon, Oeiras, Portugal 3: The Discoveries Centre for Regenerative and Precision Medicine, NOVA University Lisbon, Lisboa, PortugalLentiviral vectors (LV) are currently the first choice for cell and gene therapy applications when long-term expression is required and have consolidated as a preferential vector in the context of hematopoietic cells transduction. To cope with current and future demand of LV manufacturing, we have developed LentiPro26, stable and constitutive cell lines to support continuous production of LVs. LentiPro26 cells deliver competitive titers for amphotropic-pseudotyped vectors, but for Gibbon Ape Leukemia Virus (GaLV) envelope, preferable for hematopoietic cells transduction, obtaining higher titers is more challenging. Herein, we present two approaches to enable the manufacturing of high-titer LV GaLV-pseudotypes from stable and continuous producer cells lines. On the vector side, we created a panel of chimeric envelopes based on genetic modification of the cytoplasmic tail. The best envelope from our panel delivers transient titers near 10^7 TU/mL but also induced a strong cytotoxic phenotype. Thus, on the producer cell side, we abolished this phenotype by CRISPR-CAS knock-out of key cellular proteins. Genome-edited cells are capable of stably expressing the highest titer GaLV envelope with no evidence of envelope-induced cytotoxity. This work enables the use of constitutive packaging cell lines of GaLV LV pseudotypes, simultaneously featuring the competitive titres of transient production with the scalability, standardization and versatility of operation modes of stable and continuous production.OR10Generation of an automated GMP-grade protocol in a closed system for the expansion of polyclonal memory γδ-T cells for a “third party” cell bankV A Polito1 R Cristantielli1 G Weber1 T Belardinilli1 L Antonucci1 C M Arnone1 A Petretto2 F Ferrandino1 A Pitisci1 B De Angelis1 C Quintarelli1 F Del Bufalo1 F Locatelli13 I Caruana11: Department of Paediatric Haematology and Oncology, Cellular and Gene Therapy, Bambino Gesù Children's Hospital, Rome - Italy 2: Core Facilities-Proteomics Laboratory, Istituto Giannina Gaslini, Genoa, Italy 3: Department of Gynaecology/Obstetrics and Paediatrics, Sapienza University of Rome, Italyγδ-T cells are cells of the immune system with properties of both innate and adaptive compartment. Their powerful cytotoxic activity against bacteria, virus, tumours, together with their ability to recognize antigens in an HLA-independent manner and their negligible alloreactivity, make them attractive for clinical translation, especially in the perspective of a “third-party” T-cell bank. Unfortunately, γδ-T cells represent only a small lymphocyte population and therefore require in vitro expansion for clinical application. In this study, we developed a protocol to manually and automatically expand large numbers of polyclonal γδ-T memory cells, with the possibility of genetic modification to improve their anti-tumour activity. Artificial antigen presenting cells (aAPC) expressing CD86/41BBL/CD40L and the cytomegalovirus-pp65 antigen were used to induce expansion of γδ-T cells. To implement safety, aAPCs have been further modified with the inducible Caspase-9 suicide gene. γδ-T cells expanded 240 ± 109 times (day +21), expressing activation and memory markers maintaining a polyclonal phenotype (predominantly Vδ1). The extensive anti-tumour activity of this population was demonstrated in vitro with different tumour cell lines and leukaemic blasts (p < 0,01) and in vivo in a xenograft leukemia mouse model when compared to polyclonal αβ-T cells. In none of the cases, alloreactivity was observed. Phosphoproteomic and gene-expression studies reveal features between expanded and activated αβ and γδ-T (ability to operate as APCs, metabolism, pathways activated upon stimulation, phenotype, migration). The automated protocol for the isolation and expansion has been optimized and validated in the Clinimacs Prodigy closed-system, which maintains all the features obtained in the manual process.OR11Modelling skeletal muscle laminopathies with human iPS cells and bio-engineered skeletal muscles: Prospects for genetic therapiesL Pinton126D Moore16 H Steele-Stallard126 S Sarcar1 T Ozdemir12 S M Maffioletti1 J M Cuisset3 G Bonne4 P S Zammit2 F S Tedesco151: Department of Cell and Developmental Biology, University College London, London, UK 2: Randall Centre for Cell and Molecular Biophysics, King's College London, London, UK 3: Centre de Référence des maladies neuromusculaires Nord/Est/Ile de France, Service de Neuropédiatrie, Hôpital Roger Salengro, CHRU Lille, Lille, France 4: Sorbonne Université, INSERM UMRS_974, Centre de Recherche en Myologie, Institut de Myologie, G.H. Pitié-Salpêtrière, Paris, France 5: Great Ormond Street Institute of Child Health, University College London, London, UK 6: Equally ContributingLaminopathies are severe genetic diseases caused by mutations in the LMNA gene, which encodes A-type lamins. Together with B-type lamins, they assemble into a mesh-like structure located beneath the nuclear membrane, providing structural stability and regulating gene expression. Laminopathies affect various cell types in a systemic or tissue specific manner, with the latter including striated muscle laminopathies. Although different mechanisms have been proposed, the precise pathophysiology of laminopathies remains unknown; additionally, therapy development is hindered by their rarity and lack of easily accessible cell types for ex vivo studies. To overcome these hurdles, we used induced pluripotent stem (iPS) cells from patients with skeletal muscle laminopathies such as LMNA-related congenital muscular dystrophy, limb-girdle muscular dystrophy 1B and Emery-Dreifuss muscular dystrophy (type 2 and 3), to model disease-associated phenotypes in vitro. iPS cells from four patients were differentiated into skeletal myogenic cells and myotubes. Characteristic pathological hallmarks, including nuclear shape abnormalities and mislocalisation of nuclear lamina proteins, were observed in LMNA-mutant iPS cell derivatives in proliferation and in differentiation. Notably, modelling in three-dimensional (3D) artificial muscle constructs resulted in higher fidelity recapitulation of nuclear shape abnormalities than in standard monolayer cultures, and identified nuclear length as a reproducible, mutation-specific phenotypic readout. Finally, we will present and discuss current efforts and future applications of this novel iPS cell-based platform to develop genetic therapies for laminopathies and other severe muscle disorders, including viral and non-viral strategies such as LMNA exon-skipping and gene editing.OR12Decoy-based gene therapy for Myotonic DystrophyM Matloka1234 L Arandel1234 F Rau1234 J Marie1234 M Ney1234 A Klein1234 A Sureau1234 N Naouar1234 A Ferry1234 N Sergeant567 D Furling12341: Center of Research in Myology 2: Sorbonne Université 3: Inserm UMRS974 4: Institute of Myology 5: Inserm UMRS1172 6: Alzheimer et Tauopathies, Université Lille Nord de France 7: Centre Jean Pierre AubertMyotonic dystrophy (DM) is an autosomal neuromuscular disease encompassing two distinct forms, DM1 and DM2, caused by abnormal microsatellite expansions of C(C)TG repeats in the non-coding regions of DMPK and ZNF9 genes, respectively. Mutant RNAs carrying expanded repeats are retained in the nucleus that abnormally sequester MBNL RNA-binding factors hampering their normal function in the regulation of alternative splicing events. Thus, several splicing changes in DM patients have been associated with clinical symptoms such as myotonia, muscle weakness and cognitive defects. Although various therapeutic approaches for DM are under development, to date there is no effective therapy available. Herein, we report a novel gene therapy strategy with the use of an engineered MBNLΔ RNA-binding protein that acts as a CUGexp-decoy to release sequestered endogenous MBNL factors and restore their proper functions. In vitro, the decoy interferes with CUG-expanded transcripts and normalizes splicing abnormalities. An RNAseq analysis confirmed that the vast majority of missplicing events are no more significantly altered in DM1-treated cells, indicating an almost complete recovery of MBNL1 activity. Further in vivo analysis showed that a single intramuscular injection of AAV-MBNLΔ vectors in a mouse model of the disease results in a complete and durable normalization of splicing misregulation up to one year. Moreover, systemic delivery of the decoy using AAV9 vectors improves hallmarks of the disease including abolition of the myotonia and correction of splicing defects. In conclusion, our results support this innovative decoy-based gene therapy approach as an alternate or complementary therapeutic intervention for DM treatment.OR13Shortened ex vivo transduction for hematopoietic stem cell gene therapy of Hurler disease: impact on hematopoietic reconstitution potentialE Zonari1 M E Bernardo1 M M Naldini12 I Galasso12 M Volpin1 G Desantis1 E Montini1 A Aiuti12 B Gentner11: San Raffaele Telethon Insitute for Gene Therapy (HSR-TIGET) 2: Universita Vita-Salute San RaffaeleSan Raffaele Telethon Institute for Gene Therapy (SR-TIGET) has recently started a phase I/II study on transplantation of ex vivo genetically-modified autologous CD34+ hematopoietic stem and progenitor cells (HSPC) in patients with Hurler disease (NCT03488394), implementing for the first time a shortened lentiviral transduction protocol containing prostaglandin E2. Preliminary data from the first five patients treated show rapid hematologic recovery following fludarabin/busulfan-based myeloablative conditioning: the median duration of grade 4 neutropenia and thrombocytopenia was 17 and 0 days, respectively. Median duration of neutropenia (<100 cells/mcl) was 6 days. The drug product (DP) and non-cultured (NC) CD34+ cells from the first 3 patients were xenografted into mice. With the exception of the 4 week time-point where human CD45+ engraftment in peripheral blood was reduced by 50% in DP compared to NC, there were no significant differences between groups during the follow up until 24 weeks, including secondary transplantation. This data-set confirms that the shortened ex vivo transduction protocol preserves HSPC function. Vector integration site analysis on the first patient's DP graft in primary and secondary mice is ongoing and will allow to compare graft clonality with the reference database. To further characterize the impact of ex vivo culture, we performed longitudinal single cell RNA sequencing on CD34+CD90+ cell fractions. Cells were cultured for up to 1 week, showing metabolic changes and stress responses after prolonged culture, which may impact engraftment potential. These results are anticipated to help improve the ex vivo manufacturing process.OR14The SUNRISE-PD Study, a clinical trial of AXO-LENTI-PD: a CNS-directed gene therapy for the treatment of Parkinson's DiseaseG Corcoran1 P Korner1 Y Mo1 J Benoit1 K Binley2 Y Lad2 J E Miskin2 N J Tuckwell2 D Zamoryakhin2 T Foltynie3 R A Barker4 K A Mitrophanous2 S Palfi51: Axovant Sciences 2: Oxford Biomedica (UK) Ltd 3: Department of Clinical and Movement Neurosciences, UCL Institute of Neurology 4: John van Geest Centre for Brain Repair, Department of Clinical Neuroscience, Addenbrooke's Hospital Cambridge 5: AP-HP, Groupe Hospitalier Henri-Mondor, DHU PePsy, Neurochirurgie, Psychiatrie CréteilParkinson's disease (PD) is caused in part, by the progressive degeneration of dopaminergic neurons in the substantia nigra. The standard of care, L-dopa, is highly efficacious but long-term use is complicated by motor fluctuations from intermittent stimulation of dopamine receptors and off-target effects. Therefore, a therapy that provides a continuous supply of dopamine to the area of greatest loss in PD, namely the putamen, offers the potential for reduced motor fluctuations and off-target effects. AXO-Lenti-PD is a novel gene therapy that delivers three genes critical for de novo dopamine biosynthesis, to the putamen, using a high-capacity lentiviral vector. Transduced neuronal cells produce continuous dopamine from endogenous tyrosine in the striatum. The first-generation construct of this product (ProSavin®) was found to be well-tolerated with all patients displaying some improvement in the UPDRS part III OFF score, which was sustained in some patients up to six years. To further increase the potency of this construct, the second-generation product, AXO-Lenti-PD, was developed, utilizing the same genes but in a different configuration allowing for increased dopamine production per genetically modified cell. AXO-Lenti-PD is being investigated in the two-part SUNRISE-PD study, comprised of a dose-ranging phase to confirm the optimal therapeutic dose, followed by a sham-controlled trial to assess the safety and efficacy of the optimal dose from the first part of the study. Based on data from the lowest dose cohort, AXO-Lenti-PD was observed to be generally well-tolerated, and the data suggests it may have greater efficacy compared to the highest dose of ProSavin®.OR15In vivo generated human CAR T cells eradicate B cell leukemia in preclinical mouse modelsF B Thalheimer1 S Agarwal1 T Weidner1 A Frank1 C J Buchholz11: Paul-Ehrlich-InstitutChimeric antigen receptors (CAR) T cells have shown significant clinical benefits to patients with B-cell malignancies. However, production of CAR T cells requires extensive and time-consuming procedures of cell isolation, sorting, transduction and in vitro expansion of T cells. We investigate if CAR T cell production can be radical

DNA Double-Strand Breaks in Arctic Char (Salvelinus alpinus) from Bjørnøya in the Norwegian Arctic.

High levels of organochlorine contaminants (OCs) have been found in arctic char (Salvelinus alpinus) from Lake Ellasjøen, Bjørnøya (Norwegian Arctic). The aim of the present study was to investigate the potential genotoxic effect of environmental organochlorine contaminant exposure in arctic char from Ellasjøen compared with arctic char from the low-contaminated Lake Laksvatn nearby. Blood was analyzed using agarose gel electrophoresis and image data analysis to quantify the fraction of total DNA that migrated into the gel (DNA-FTM) as a relative measure of DNA double-strand breaks (DSBs). Analysis by GC-MS of muscle samples showed an average 43 times higher concentration of ΣOCs in arctic char from Ellasjøen (n = 18) compared with Laksvatn char (n = 21). Char from Lake Ellasjøen had a much higher frequency of DSBs, as measured by DNA-FTM, than char from Lake Laksvatn. Principal component analysis and multiple linear regressions show that there was a significant positive relationship between DSBs and levels of organochlorine contaminants in the char. In addition, DSBs were less frequent in reproductively mature char than in immature char. The results suggest that organochlorine contaminants are genotoxic to arctic char. Environ Toxicol Chem 2019;38:2405-2413. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.