Biology subjects

Pekrun, K.

Publications and source records attributed to Pekrun, K..

6 recordsLinked to original sources

bioRxiv2024

AAV-mediated genome editing is influenced by the formation of R-loops

Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone, 3 end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop- deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.

molecular biology↗

bioRxiv2022

Capsid-mediated chromatin state of the AAV vector genome controls host species range

Recombinant adeno-associated viral vectors (rAAVs) are among the most commonly used vehicles for in vivo based gene therapies. However, it is hard to predict which AAV capsid will provide the most robust expression in human subjects due to the observed discordance in vector-mediated transduction between species. We used a primate specific capsid, AAV-LK03, and demonstrated that the limitation of this capsid towards transduction of mouse cells was unrelated to cell entry and nuclear transport but rather due to depleted histone H3 chemical modifications related to active transcription, namely H3K4me3 and H3K27ac, on the vector DNA itself. A single-amino acid insertion into the AAV-LK03 capsid enabled efficient transduction and the accumulation of active histone marks on the vector chromatin in mouse without compromising transduction efficiency in human cells. Our study suggests that the capsid protein itself is involved in determining the chromatin status of the vector genome, most likely during the process of uncoating. Programming viral chromatin states by capsid design may enable facile DNA transduction between vector and host species.

molecular biology↗

bioRxiv2022

Selection of rAAV Vectors that Cross the Human Blood-Brain Barrier 1 and Target the Central Nervous System Using a Transwell Model

A limitation for rAAV-mediated gene transfer into the central nervous system (CNS) is the low penetration of vectors across the human blood-brain barrier (BBB). High doses of intravenously-delivered vector are required to reach the CNS, which has resulted in varying adverse effects. Moreover, selective transduction of various cell types might be important depending on the disorder being treated. To enhance BBB penetration and improve CNS cell selectivity, we screened an AAV capsid-shuffled library using an in vitro transwell BBB system with separate layers of human endothelial cells, primary astrocytes and/or hiPSC-derived cortical neurons. After multiple passages through the transwell, we identified chimeric AAV capsids with enhanced penetration and improved transduction of astrocytes and/or neurons compared to wild-type capsids. We identified the amino acid (aa) from region 451-470 of AAV2 associated with the capsids selected for neurons, and a combination of aa from regions 413-496 of AAV-rh10 and 538-598 of AAV3B/LK03 associated with capsids selected for astrocytes. An siRNA screen identified several genes that affect transcytosis of AAV across the BBB. Our work supports the use of a human transwell system for selecting enhanced AAV capsids targeting the CNS and may allow for unraveling the underlying molecular mechanisms of BBB penetration.

molecular biology↗

bioRxiv2019

Using a bar-coded AAV capsid library to select for novel clinically relevant gene therapy vectors

ABSTRACTWhile gene transfer using recombinant adeno-associated viral (rAAV) vectors have shown success in some clinical trials, there remain many tissues that are not well transduced. Because of the recent success in reprogramming islet derived cells into functional {beta}-cells in animal models, we constructed two highly complex barcoded replication competent capsid shuffled libraries and selected for high transducing variants on primary human islets. We describe a chimeric capsid (AAV-KP1) that penetrated and transduced primary human islet cells and human embryonic stem cell derived {beta}-cells with up to 10-fold higher efficiency compared to previously studied best in class AAV vectors. Remarkably, this chimeric capsid was also able to transduce both mouse and human hepatocytes at very high levels in a humanized-chimeric mouse model, thus providing a versatile vector which has the potential to be used in both preclinical testing and human clinical trials for both liver-based diseases and diabetes.

genetics↗

bioRxiv2018

The repressive genome compartment is established early in the cell cycle before forming the lamina associated domains

Three-dimensional (3D) genome organization is thought to be important for regulation of gene expression. Chromosome conformation capture-based studies have uncovered ensemble organizational principles such as active (A) and inactive (B) compartmentalization. In addition, large inactive regions of the genome associate with the nuclear lamina, the Lamina Associated Domains (LADs). Here we investigate the dynamic relationship between A/B-compartment organization and the 3D organization of LADs. Using refined algorithms to identify active (A) and inactive (B) compartments from Hi-C data and to define LADs from DamID, we confirm that the LADs correspond to the B-compartment. Using specialized chromosome conformation paints, we show that LAD and A/B-compartment organization are dependent upon chromatin state and A-type lamins. By integrating single-cell Hi-C data with live cell imaging and chromosome conformation paints, we demonstrate that self-organization of the B-compartment within a chromosome is an early event post-mitosis and occurs prior to organization of these domains to the nuclear lamina.

genomics↗

bioRxiv2017

Chromosome Conformation Paints Reveal The Role Of Lamina Association In Genome Organization And Regulation

Non-random, dynamic three-dimensional organization of the nucleus is important for regulation of gene expression. Numerous studies using chromosome conformation capture strategies have uncovered ensemble organizational principles of individual chromosomes, including organization into active (A) and inactive (B) compartments. In addition, large inactive regions of the genome appear to be associated with the nuclear lamina, the so-called Lamina Associated Domains (LADs). However, the interrelationship between overall chromosome conformation and association of domains with the nuclear lamina remains unclear. In particular, the 3D organization of LADs within the context of the entire chromosome has not been investigated. In this study, we describe \"chromosome conformation paints\" to determine the relationship in situ between LAD and non-LAD regions of the genome in single cells. We find that LADs organize into constrained and compact regions at the nuclear lamina, and these findings are supported by an integrated analysis of both DamID and Hi-C data. Using a refined algorithm to identify active (A) and inactive (B) compartments from Hi-C data, we demonstrate that the LADs correspond to the B compartment. We demonstrate that in situ single cell chromosome organization is strikingly predicted by integrating both Hi-C and DamID data into a chromosome conformation model. In addition, using the chromosome conformation paints, we demonstrate that LAD (and B-compartment) organization is dependent upon both chromatin state and Lamin A/C. Finally, we demonstrate that small regions within LADs escape the repressive regime at the peripheral zone to interact with the A-compartment and are enriched for both transcription start sites (TSSs) and active enhancers.

cell biology↗