scholarly journals Answered and Unanswered Questions in Early-Stage Viral Vector Transduction Biology and Innate Primary Cell Toxicity for Ex-Vivo Gene Editing

2021 ◽  
Vol 12 ◽  
Author(s):  
Amanda Mary Dudek ◽  
Matthew Hebden Porteus
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Gene Editing ◽  
Ex Vivo ◽  
Early Stage ◽  
Innate Immune ◽  
Hematopoietic Stem ◽  
Damage Response ◽  
Vector Transduction

Adeno-associated virus is a highly efficient DNA delivery vehicle for genome editing strategies that employ CRISPR/Cas9 and a DNA donor for homology-directed repair. Many groups have used this strategy in development of therapies for blood and immune disorders such as sickle-cell anemia and severe-combined immunodeficiency. However, recent events have called into question the immunogenicity of AAV as a gene therapy vector and the safety profile dictated by the immune response to this vector. The target cells dictating this response and the molecular mechanisms dictating cellular response to AAV are poorly understood. Here, we will investigate the current known AAV capsid and genome interactions with cellular proteins during early stage vector transduction and how these interactions may influence innate cellular responses. We will discuss the current understanding of innate immune activation and DNA damage response to AAV, and the limitations of what is currently known. In particular, we will focus on pathway differences in cell line verses primary cells, with a focus on hematopoietic stem and progenitor cells (HSPCs) in the context of ex-vivo gene editing, and what we can learn from HSPC infection by other parvoviruses. Finally, we will discuss how innate immune and DNA damage response pathway activation in these highly sensitive stem cell populations may impact long-term engraftment and clinical outcomes as these gene-editing strategies move towards the clinic, with the aim to propose pathways relevant for improved hematopoietic stem cell survival and long-term engraftment after AAV-mediated genome editing.

scholarly journals Precise Gene Editing Preserves Hematopoietic Stem Cell Function following Transient p53-Mediated DNA Damage Response

2019 ◽  
Vol 24 (4) ◽  
pp. 551-565.e8 ◽  
Author(s):  
Giulia Schiroli ◽  
Anastasia Conti ◽  
Samuele Ferrari ◽  
Lucrezia della Volpe ◽  
Aurelien Jacob ◽  
...  
Keyword(s):  
Dna Damage ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Dna Damage Response ◽  
Gene Editing ◽  
Cell Function ◽  
Hematopoietic Stem ◽  
Damage Response

Thrombopoietin-Increased DNA-PK-Dependent DNA Repair Limits Hematopoietic STEM and Progenitor CELL Mutagenesis in Response to Irradiation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3447-3447
Author(s):  
Bérengère de Laval ◽  
Patrycja Pawlikowska ◽  
Benoit Roch ◽  
Laurence Petit-Cocault ◽  
Chrystele Bilhou-Nabera ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Dna Damage Repair ◽  
Dsb Repair ◽  
Hematopoietic Stem ◽  
Damage Repair ◽  
Damage Response ◽  
Radiation Induced

Abstract Abstract 3447 Radiation-induced double-strand breaks (DSBs) represent a serious threat to the preservation of genetic information when it reaches hematopoietic stem cells (HSCs). Residual loss of HSC functions and increased risk of developing hematopoietic malignancies are two concerning complications of anti-cancer radiotherapy. Management of acute myelosuppression following radio- or chemotherapy has been significantly improved in recent years by the use of growth factors. However, how cytokine/environmental signals integrate the DNA damage responses in HSCs and regulate the long-term residual HSC defects following radio-or chemotherapy is unknown. Notably, the contribution of cytokines regulating HSC functions to HSC intrinsic DNA damage repair processes remains to be delineated. Thrombopoietin (TPO) and its receptor, Mpl, are critical factors supporting HSC self-renewal, survival and expansion posttransplantation. In this study, we uncover an unknown and unique function for TPO/Mpl in the regulation the DNA damage response. We show that DSB repair, measured by both γH2Ax foci resolution and neutral comet assays, following γ-irradiation (IR) or topoisomerase II inhibitor treatments, is defective in Mpl−/− and Mpl+/− HS and progenitor cells (HSPCs). Similar defects were found in wild-type cells treated in the absence of TPO. This indicates that the impaired DNA repair of Mpl−/− and Mpl+/− cells results from a specific loss of TPO-mediated DNA damage response signaling at the time of IR rather than from intrinsic constitutive differences. TPO stimulates DNA repair by increasing IR-induced DNA-PK phosphorylation at Ser2056 and Thr2609 and non-homologous end joining (NHEJ) efficiency in both HSPCs and the human UT7-Mpl cell line. This is to our knowledge the first demonstration that a cytokine involved in the homeostatic maintenance of HSCs may also regulate their response to external DNA damaging insults by controlling the DSB repair machinery. Short TPO treatment in vitro or single TPO injection to TPO/Mpl proficient mice prior to sublethal total body IR reduced IR-induced HSC genomic instability and loss of long-term reconstitution ability. This may open new avenues for administration of TPO agonists before radiotherapy to minimize radiation-induced HSC injury and mutagenesis. In addition, since Mpl is haploinsufficient in the regulation of DNA damage repair, these data suggest that Mpl might also act as a tumor suppressor in response to radiotherapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals DNA Damage Response Pathway and Replication Fork Stress During Oligonucleotide Directed Gene Editing

2012 ◽  
Vol 1 ◽  
pp. e18 ◽  
Author(s):  
Melissa Bonner ◽  
Bryan Strouse ◽  
Mindy Applegate ◽  
Paula Livingston ◽  
Eric B Kmiec
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Replication Fork ◽  
Gene Editing ◽  
Damage Response ◽  
Dna Damage Response Pathway

scholarly journals Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1506 ◽  
Author(s):  
Cecilia Aquino Perez ◽  
Matous Palek ◽  
Lenka Stolarova ◽  
Patrick von Morgen ◽  
Libor Macurek
Keyword(s):  
Dna Damage ◽  
Stress Response ◽  
Osmotic Stress ◽  
Dna Damage Response ◽  
Protein Phosphatase ◽  
Gene Editing ◽  
Kinase Activity ◽  
Cell Cycle Control ◽  
Damage Response ◽  
Transformed Cell Lines

Polo-like kinases play essential roles in cell cycle control and mitosis. In contrast to other members of this kinase family, PLK3 has been reported to be activated upon cellular stress including DNA damage, hypoxia and osmotic stress. Here we knocked out PLK3 in human non-transformed RPE cells using CRISPR/Cas9-mediated gene editing. Surprisingly, we find that loss of PLK3 does not impair stabilization of HIF1α after hypoxia, phosphorylation of the c-Jun after osmotic stress and dynamics of DNA damage response after exposure to ionizing radiation. Similarly, RNAi-mediated depletion of PLK3 did not impair stress response in human transformed cell lines. Exposure of cells to various forms of stress also did not affect kinase activity of purified EGFP-PLK3. We conclude that PLK3 is largely dispensable for stress response in human cells. Using mass spectrometry, we identify protein phosphatase 6 as a new interacting partner of PLK3. Polo box domain of PLK3 mediates the interaction with the PP6 complex. Finally, we find that PLK3 is phosphorylated at Thr219 in the T-loop and that PP6 constantly dephosphorylates this residue. However, in contrast to PLK1, phosphorylation of Thr219 does not upregulate enzymatic activity of PLK3, suggesting that activation of both kinases is regulated by distinct mechanisms.

Viral Infection Sentinel Rig-I Maintains Hematopoietic Stem Cell Function Through Regulating DNA-Damage Response

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1166-1166
Author(s):  
Wu Zhang ◽  
Meng-Lei Ding ◽  
Xian-Yang Li ◽  
He-Zhou Guo ◽  
Hong-Xin Zhang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Dna Lesions ◽  
Type I ◽  
Hematopoietic Stem ◽  
Damage Response ◽  
Underlying Mechanisms ◽  
And Function

Abstract Throughout life hematopoietic stem cells (HSCs) have to cope with various kinds of insults from inflammation to DNA damage constantly to maintain the integrity of stemness. It is possible that certain core factors are commonly implicated in the maintenance of HSC pool and function under discrete physiological and pathological conditions. However, the underlying mechanisms remain largely unexplored. Previous works have demonstrated that retinoic acid inducible gene I (Rig-I) plays an essential role in recognizing viral RNA and activating type I IFN transcription, but whether Rig-I is involved in the core program governing HSCs’ behaviors is unclear. Here, we report that in the steady status Rig-I deficiency significantly increased HSC number by dysregulating the cell-cycling status of HSCs in mice. However, HSCs in Rig-I-/- mice were actually more sensitive to genotoxic treatments such as irradiation as compared to wild type HSCs, causing more Rig-I-/- mice to die of hematopoietic exhaustion. In accordance, HSC transplantation assays showed a significant impact of Rig-I loss on the hematopoietic regeneration capacity. Mechanistically, we found that Rig-I represented a pivotal component of the molecular pathways that mediate DNA-damage response and the repair of DNA lesions. Taken together, these data indicate a crucial role of innate immunity-regulatory factor Rig-I in the maintenance of HSCs. Disclosures: No relevant conflicts of interest to declare.

Association of a DNA damage response deficiency (DDRD) assay and prognosis in early-stage esophageal adenocarcinoma.

2014 ◽  
Vol 32 (15_suppl) ◽  
pp. 4015-4015 ◽  
Author(s):  
Richard C. Turkington ◽  
Laura A. Hill ◽  
Damian McManus ◽  
Stephen McQuaid ◽  
Ken Arthur ◽  
...  
Keyword(s):  
Dna Damage ◽  
Esophageal Adenocarcinoma ◽  
Dna Damage Response ◽  
Early Stage ◽  
Damage Response

scholarly journals The heterogeneity of the DNA damage response landscape determines patient outcomes in ovarian cancer

2021 ◽  
Author(s):  
Thomas Walker ◽  
Zahra Faraahi ◽  
marcus price ◽  
Amy Hawarden ◽  
Catlin Waddell ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Ex Vivo ◽  
Single Strand ◽  
Cellular Growth ◽  
Response To Chemotherapy ◽  
Damage Response ◽  
Repair Pathways ◽  
Non Homologous End Joining

Defective DNA damage response (DDR) pathways allow cancer cells to accrue genomic aberrations and evade normal cellular growth checkpoints. Defective DDR also determines response to chemotherapy. However, the interaction and overlap between the two double strand repair pathways and the three single strand repair pathways is complex, and has remained poorly understood. Here we show that, in ovarian cancer, a disease hallmarked by chromosomal instability, explant cultures show a range of DDR abrogation patterns. Defective homologous recombination (HR) and non-homologous end joining (NHEJ) are near mutually exclusive with HR deficient (HRD) cells showing increased abrogation of the single strand repair pathways compared to NHEJ defective cells. When combined with global markers of DNA damage, including mitochondrial membrane functionality and reactive oxygen species burden, the pattern of DDR abrogation allows the construction of DDR signatures which are predictive of both ex vivo cytotoxicity, and more importantly, patient outcome.

scholarly journals Optimized cell culture conditions promote ex-vivo manipulation and expansion of primitive hematopoietic stem cells for therapeutic gene editing.

2022 ◽  
Author(s):  
Rajeev Rai ◽  
Winston Vetharoy ◽  
Asma Naseem ◽  
Zohar Steinberg ◽  
Adrian James Thrasher ◽  
...  
Keyword(s):  
Gene Editing ◽  
Culture Media ◽  
Ex Vivo ◽  
Culture Conditions ◽  
Fine Tuning ◽  
Great Promise ◽  
Hematopoietic Stem ◽  
Monogenic Diseases

During the last few years, gene editing has emerged as a powerful tool for the therapeutic correction of monogenic diseases. CRISPR/Cas9 applied to hematopoietic stem and progenitor cells (HSPCs) has shown great promise in proof-of-principle preclinical studies to treat haematological disorders, and clinical trials using these tools are now underway. Nonetheless, there remain important challenges that need to be addressed, such as the efficiency of targeting primitive, long-term repopulating HSPCs and expand them in vitro for clinical purposes. Here we have tested the effect exerted by different culture media compositions on the ability of HSPCs to proliferate and undergo homology directed repair-mediated knock-in of a reporter gene, while preserving their stemness features during ex-vivo culture. We tested different combinations of compounds and demonstrated that by supplementing the culture media with inhibitors of histone deacetylases, and/or by fine-tuning its cytokine composition it is possible to achieve high levels of gene targeting in long-term repopulating HSPCs both in vitro and in vivo, with a beneficial balance between preservation of stemness and cell expansion, thus allowing to obtain a significant amount of edited, primitive HSPCs compared to established, state-of-the-art culture conditions. Overall, the implantation of this optimized ex vivo HSPC culture protocol will improve the efficacy, feasibility and applicability of gene editing and will likely provide one step further to unlock the full therapeutic potential of such powerful technology.

Sign in / Sign up

Export Citation Format

Share Document