Cellular barcoding tool for clonal analysis in the hematopoietic system

Blood ◽  
2010 ◽  
Vol 115 (13) ◽  
pp. 2610-2618 ◽  
Author(s):  
Alice Gerrits ◽  
Brad Dykstra ◽  
Olga J. Kalmykowa ◽  
Karin Klauke ◽  
Evgenia Verovskaya ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Integration Site ◽  
Retroviral Vectors ◽  
Clonal Analysis ◽  
Great Promise ◽  
Hematopoietic Stem ◽  
Site Analysis ◽  
Integration Site Analysis ◽  
Cellular Barcoding

Abstract Clonal analysis is important for many areas of hematopoietic stem cell research, including in vitro cell expansion, gene therapy, and cancer progression and treatment. A common approach to measure clonality of retrovirally transduced cells is to perform integration site analysis using Southern blotting or polymerase chain reaction–based methods. Although these methods are useful in principle, they generally provide a low-resolution, biased, and incomplete assessment of clonality. To overcome those limitations, we labeled retroviral vectors with random sequence tags or “barcodes.” On integration, each vector introduces a unique, identifiable, and heritable mark into the host cell genome, allowing the clonal progeny of each cell to be tracked over time. By coupling the barcoding method to a sequencing-based detection system, we could identify major and minor clones in 2 distinct cell culture systems in vitro and in a long-term transplantation setting. In addition, we demonstrate how clonal analysis can be complemented with transgene expression and integration site analysis. This cellular barcoding tool permits a simple, sensitive assessment of clonality and holds great promise for future gene therapy protocols in humans, and any other applications when clonal tracking is important.

Comprehensive and Unbiased Integration Site Analysis in Clinical Gene Therapy.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2351-2351
Author(s):  
Richard Gabriel ◽  
Anna Paruzynski ◽  
Ralph Eckenberg ◽  
Cynthia C. Bartholomae ◽  
Ali Nowrouzi ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Restriction Enzyme ◽  
Integration Site ◽  
Restriction Enzymes ◽  
Retroviral Vectors ◽  
Site Analysis ◽  
Cell Clones ◽  
Integration Site Analysis ◽  
Integration Sites ◽  
Clinical Gene Therapy

Abstract Retroviral vectors are commonly used gene delivery tools in clinical gene therapy providing stable integration and continuous gene expression of the transgene in the treated host cell. However, integration of the reverse transcribed vector DNA into the host genome is, by itself, a mutagenic eventthat may directly contribute to severe adverse events. The latter has dramatically been obbserved in individual cases in several, otherwise successful, gene therapy trials. Thus, a comprehensive analysis of the existing integration site pool in a transduced sample is indispensable to identify potential in vivo selection of affected cell clones and uncontrolled vector-induced cell proliferation. To date, there are several methods available to study the integration site distribution of retroviral vectors or other integrating elements as transposons. Each of these techniques makes use of restriction enzymes to digest the genomic DNA. To reveal particular vector integrations, a recognition motif of the used restriction enzyme has to be located in an appropriate distance to the integration locus in the host genome. Therefore, the genomic distribution of the recognition sequences directly impact the outcome of restriction enzyme dependent integration site analysis. We here report a validated genomic accessibility model which precisely determines the fraction of the human genome that can be analyzed with one reaction set up (i.e. restriction enzyme used). For our modeling, we used the clinically relevant linear amplification mediated PCR (LAM-PCR) as integration site analysis method of choice and the commonly used frequently cutting restriction enzymes (‘four-cutters’). We show that the most frequent four cutter motif (AATT) gives access to 54.5% of all possible integrations in the human genome, whereas the rarest distributed motif (CGCG) only identifies 2.9%. This restriction bias can be minimized by analyzing the same sample with different enzymes. A combination of the 5 most potent four cutter restriction enzymes gives access to 88.7% of the analyzable genome. Furthermore, we established an unbiased, non-restrictive integration site analysis technique based on (nr) LAM-PCR. Direct ligation of a single-stranded DNA sequence to the linear PCR product evades the need for restriction enzymes to recover integration sites. While standard LAM-PCR was done repeatedly with 3 different enzymes to detect integration sites present in lentivirally transduced single cell clones, nrLAM-PCR detected all integrations in these clones in one single reaction setup. This newly developed method comprehensively recovers genomic locations of integrating elements regardless of a restriction enzyme introduced bias. Our data show that the recovery rate of integration sites present in a transduced sample strongly depends on the restriction enzyme(s) used. However, we demonstrate that the genomic accessibility of viral integration sites indeed can be determined and minimized a priori, and that a non restrictive LAM-PCR approach circumvents the existing limitations. Analysis of the clonal inventory by these methods will allow determining the pharmacodynamics of insertional vectors with unprecedented precision, facilitating development and clinical testing of insertional vector systems.

scholarly journals Comprehensive Integration Site Analysis of Human Immunodeficiency Virus during In Vivo Infections Reveals Genomic Regions of Enrichment and Clonal Expansion

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2518-2518
Author(s):  
Kevin G Haworth ◽  
Lauren E Schefter ◽  
Zachary K. Norgaard ◽  
Jennifer E Adair ◽  
Hans-Peter Kiem
Keyword(s):  
Integration Site ◽  
Site Analysis ◽  
Genome Wide ◽  
Integration Site Analysis ◽  
Integration Sites ◽  
Infected Cells ◽  
Genomic Regions ◽  
Hiv Integration

Abstract BACKGROUND A key event in the lifecycle of Human Immunodeficiency Virus (HIV) is permanent integration into the infected cells genome. In addition to allowing long-term persistence of the virus, this results in a trackable mark carried in all infected cells. Active HIV replication represses cellular pathways, preventing further cell division. This would imply that any specific integration site (IS) which is clonally expanded either during active or repressed viral infection arises from either a dormant/inactive virus, or is perturbing local gene expression, leading to increased cell proliferation. Alternatively, a cell carrying HIV provirus could proliferate due to T-cell specific antigen stimulation. By analyzing the patterns of integration sites detected in cell cultures and tissue samples from animal models of HIV infection, we can better understand the basic virology of integration site selection and determine what may potentially drive infected cells to persist despite effective treatment regimens. METHODS Jurkat reporter cell lines or primary human CD4+ cells were cultured and infected with various strains of HIV including both CCR5 and CXCR4 tropic viruses. Infected cells were cultured up to 21 days post infection, then analyzed for HIV proviral integration sites by next-generation sequencing. For in vivo studies, NSG mice were infused with human CD34+ hematopoietic stem/progenitor cells, resulting in a reconstituted human immune system including high levels of CD4+ T cells capable of sustaining HIV infection. After 16 weeks post-challenge, tissues were collected and subjected to integration site analysis for HIV proviral DNA. Identified integration sites were mapped and compared across multiple parameters to identify chromosomal regions and associated genes enriched for integration events, as well as clonally expanded cells in vivo. RESULTS Genome-wide analysis of HIV integration sites reveals a remarkably similar chromosomal landscape both in tissue culture infection of Jurkat cells and in vivo infection data (Figure 1), as well as across multiple HIV strains. As previously observed, the majority of integrations occur near or within gene coding regions thought to be actively transcribed at time of infection. However, certain areas of the genome, and even unique genes, are enriched for IS in individual samples. In addition to these genomic regions of enrichment, we also observe specific clonal outgrowth of unique integration events in genes previously unidentified in the literature. Three genes in particular exhibit a significant increase of integration events during acute infection which are 3x higher than predicted by random chance alone. We also observe integration events in genes that have been documented by other labs in HIV+ clinical patient samples, however in our active infection models, we do not see those specific genes enriched or expanded. This could indicate that these genes play a role in persistence that is only present during anti-retroviral therapy which suppresses active replication. CONCLUSIONS We have cataloged the most extensive HIV IS library to date in both relevant tissue culture models and in vivo infection studies, including over 245,000 unique integration events and three different HIV strains commonly used in research. Genome-wide correlation studies reveal regions significantly enriched for HIV integrations and genes which repeatedly exhibit clonal outgrowth in multiple animals. These types of studies are now being applied to human patient samples to determine if latency and persistence of infection can be mapped to unique integration events or genes of interest. Such information may indicate when and how the latent HIV reservoir is seeded and what types of therapy or treatments are most effective at targeting and eliminating these populations. Circos plot comparing HIV integrations sites (IS) identified either during in vitro cell culture infections (black bars), or in vivo infection studies using humanized mice (red bars). The outer ring is composed of human chromosomes each of which are divided into 25kB fragment bins. Total number of unique integration sites identified in each bin is represented by the height of the histogram bars. The in vitro IS concentric ring scale represents increments of 25 outwards up to 250 while the in vivo IS scales inwards in increments of 2 up to 16. Figure 1 Comparison of in vitro vs in vivo HIV Integration Sites. Figure 1. Comparison of in vitro vs in vivo HIV Integration Sites. Disclosures Adair: Rocket Pharmaceuticals: Consultancy, Equity Ownership.

Identification and Validation of Novel Hematopoietic Stem Cell Regulatory microRNAs

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3872-3872
Author(s):  
Elias Simon Peter Eckert ◽  
Peer Wünsche ◽  
Manfred Schmidt ◽  
Claudia Ball ◽  
Friederike Herbst ◽  
...  
Keyword(s):  
Gene Therapy ◽  
High Throughput Sequencing ◽  
Conflicts Of Interest ◽  
Selection Process ◽  
Integration Site ◽  
Selection Strategy ◽  
Hematopoietic Stem ◽  
Mirna Genes ◽  
Clinical Gene Therapy

Abstract Within this study we have developed a novel approach to identify hematopoietic regulatory genes by re-analyzing genome-wide viral integration site (IS) data from a clinical gene therapy trial of 10 Wiskott-Aldrich syndrome patients. We observed a statistically enriched contribution of individual cell clones marked by IS to benign human long-term hematopoiesis within a dataset comprising 130,699 unique IS in the proximity of 12,437 genes. Furthermore, when we assessed the genomic distribution of the IS, we found that clusters of IS (CIS) are significantly over-represented at certain coding and non-coding genetic loci, indicating a viral marking of actively transcribed genes or a proliferation advantage of specific clones due to viral induced deregulation of adjacent genes. Notably, 42 % of the 50 most enriched regions encode for human HSC regulators such as MECOM, LMO2 and HMGA2. Based on a stepwise and stringent selection process evaluating IS number, location and CIS size, we chose the top 12 ranked miRNA genes for further analysis. Of those, three miRNAs (miR-132, miR-142, miR-146a) have described roles in hematopoiesis, strongly validating our selection strategy. In order to functionally investigate the influence of selected candidate genes on hematopoiesis we developed a lentiviral (LV) based pooled overexpression (OE) approach using uniquely barcoded (BC) vectors encoding for the top 8 candidate miRNA HSC regulators in lin- sca1+ ckit+ (LSK) cells. The LV based library consists of two vectors per gene including corresponding GFP control vectors, and harbor a unique BC cassette serving as a molecular tag to allow its unbiased amplification and high-throughput sequencing of up to 96 indexed samples simultaneously. To assess the influence of the selected miRNA candidates on hematopoietic differentiation in vitro, we performed serial CFU assays using BC library transduced LSK-SLAM cells as a starting population. Low infection rates were used to ensure one vector copy per cell. We detected a 1.58±0.03 [SEM] fold increase (p=0.003, n=5) in myeloid colony forming potential of miRNA library OE cells compared to GFP control cells without a shift in colony phenotypes. After three rounds of serial replating, colonies expressing 2/8 candidates were significantly overrepresented (1.6±0.23 to 2.0±0.20 fold; p=0.049 and p=0.005; n =5) while BC vectors encoding for two other candidate miRNAs were significantly reduced (0.4±0.11 to 0.5±0.14 fold, p=0.006 and p=0.027) compared to GFP control (fc=1.0±0.12). To address the influence of candidate miRNA OE on multi-lineage engraftment and self-renewal in vivo, we serially transplanted miRNA or GFP control library transduced LSK cells in a cohort of 18 recipient mice. We revealed efficient candidate BC representation in all mice with a median read count of 7.1E4 (min=2451, max=8.4E5) 20 weeks post-transplant. In line with our results in vitro, we observed a 1.23±0.23 (p=0.044) fold enrichment of the same two miRNA candidates and a depletion of the two other miRNA genes (0.63±0.09 to 0.68±0.13fold; p=0.002 and p=0.033) in PB samples already eight weeks after transplantation. Interestingly, BC sequencing of lineage sorted fractions suggests that one of the overrepresented candidates has a strong influence on T-cell differentiation (fc=1.5±0.2, p=0.029, 20 weeks post TP) whereas the other candidate impacts on myelopoiesis (fc=1.6±0.35, p=0.114). In summary, our systematic stepwise approach combining the comprehensive analysis of the complete integration site repertoire in a clinical gene therapy study with subsequent functional validation using a pooled lentiviral overexpression approach is a useful strategy to identify and characterize novel HSC regulators. Disclosures No relevant conflicts of interest to declare.

scholarly journals Multiple Displacement Amplification Enables Large-Scale Clonal Analysis following Retroviral Gene Therapy

2007 ◽  
Vol 82 (5) ◽  
pp. 2448-2455 ◽  
Author(s):  
S. Bleier ◽  
P. Maier ◽  
H. Allgayer ◽  
F. Wenz ◽  
W. J. Zeller ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Genomic Dna ◽  
Large Scale ◽  
Integration Site ◽  
Multiple Displacement Amplification ◽  
Site Analysis ◽  
Integration Site Analysis ◽  
Reliable Quantification ◽  
Therapy Studies ◽  
First Time

ABSTRACT Analysis of the fate of retrovirally transduced cells after transplantation is often hampered by the scarcity of available DNA. We evaluated a promising method for whole-genome amplification, called multiple displacement amplification (MDA), with respect to even and accurate representation of retrovirally transduced genomic DNA. We proved that MDA is a suitable method to subsequently quantify engraftment efficiencies by quantitative real-time PCR by analyzing retrovirally transduced DNA in a background of untransduced DNA and retroviral integrations found in primary material from a retroviral transplantation model. The portion of these retroviral integrations in the amplified samples was 1.02-fold (range 0.2, to 2.1-fold) the portion determined in the original genomic DNA. Integration site analysis by ligation-mediated PCR (LM-PCR) is essential for the detection of retroviral integrations. The combination of MDA and LM-PCR showed an increase in the sensitivity of integration site analysis, as a specific integration site could be detected in a background of untransduced DNA, while the transduced DNA made up only 0.001%. These results show for the first time that MDA enables large-scale sensitive detection and reliable quantification of retrovirally transduced human genomic DNA and therefore facilitates follow-up analysis in gene therapy studies even from the smallest amounts of starting material.

scholarly journals Gene Therapy for β-Thalassemia

Hematology ◽  
2005 ◽  
Vol 2005 (1) ◽  
pp. 45-50 ◽  
Author(s):  
Punam Malik ◽  
Paritha I. Arumugam
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Globin Gene ◽  
Thalassemia Major ◽  
Retroviral Vectors ◽  
Thalassemia Intermedia ◽  
Hematopoietic Stem ◽  
Variable Expression

AbstractGene transfer for β-thalassemia requires gene transfer into hematopoietic stem cells using integrating vectors that direct regulated expression of β globin at therapeutic levels. Among integrating vectors, oncoretroviral vectors carrying the human β-globin gene and portions of the locus control region (LCR) have suffered from problems of vector instability, low titers and variable expression. In recent studies, human immunodeficiency virus–based lentiviral (LV) vectors were shown to stably transmit the human β-globin gene and a large LCR element, resulting in correction of β-thalassemia intermedia in mice. Several groups have since demonstrated correction of the mouse thalassemia intermedia phenotype, with variable levels of β-globin expression. These levels of expression were insufficient to fully correct the anemia in thalassemia major mouse model. Insertion of a chicken hypersensitive site-4 chicken insulator element (cHS4) in self-inactivating (SIN) LV vectors resulted in higher and less variable expression of human β-globin, similar to the observations with cHS4-containing retroviral vectors carrying the human γ-globin gene. The levels of β-globin expression achieved from insulated SIN-LV vectors were sufficient to phenotypically correct the thalassemia phenotype from 4 patients with human thalassemia major in vitro, and this correction persisted long term for up to 4 months, in xeno-transplanted mice in vivo. In summary, LV vectors have paved the way for clinical gene therapy trials for Cooley’s anemia and other β-globin disorders. SIN-LV vectors address several safety concerns of randomly integrating viral vectors by removing viral transcriptional elements and providing lineage-restricted expression. Flanking the proviral cassette with chromatin insulator elements, which additionally have enhancer-blocking properties, may further improve SIN-LV vector safety.

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