scholarly journals Bona fide alveolar macrophage generation from long-term in vitro culture of fetal precursors provides a novel platform for gene therapy and functional studies

2021 ◽  
Author(s):  
Fengqi Li ◽  
Katarzyna Maria Okreglicka ◽  
Lea Maria Pohlmeier ◽  
Christoph Schneider ◽  
Manfred Kopf
Keyword(s):  
Gene Therapy ◽  
Alveolar Macrophage ◽  
Genetic Manipulation ◽  
Fetal Liver ◽  
Influenza Virus Infection ◽  
Gm Csf ◽  
Functional Studies ◽  
Bona Fide

Tissue-resident macrophage (MΦTR)-based immune therapies have been proposed for various diseases. However, it is still a challenge to generate a considerable amount of MΦTR that possess tissue-specific functions. Here we show that fetal liver monocytes (FLiMo) cultured with GM-CSF (also known as CSF2) rapidly differentiate into a long-lived, homogeneous alveolar macrophage (AM)-like population in vitro. CSF2-cultured FLiMo (CSF2-cFLiMo) remain the capacity to develop into bona fide AM upon transfer into Csf2ra-/- neonates and prevent development of alveolar proteinosis for at least 1 year in vivo. Compared to bone marrow-derived macrophages (BMM), CSF2-cFliMo more efficiently engraft empty AM niches in the lung and protect mice from influenza virus infection. Notably, to harness the potential of this approach for gene therapy, we restored a disrupted Csf2ra gene in FLiMo and their capacity to develop into AM in vivo, by retroviral expression of a wild-type gene. Taken together, we provide a novel platform for high-throughput genetic manipulation of AM that can be used for biological and therapeutic studies.

scholarly journals Glioblastoma epigenome profiling identifies SOX10 as a master regulator of molecular tumour subtype

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Yonghe Wu ◽  
Michael Fletcher ◽  
Zuguang Gu ◽  
Qi Wang ◽  
Barbara Costa ◽  
...  
Keyword(s):  
Regulatory Networks ◽  
Immune Cell ◽  
Sequencing Data ◽  
Master Regulator ◽  
Mesenchymal Transition ◽  
Functional Studies ◽  
Bona Fide ◽  
Oligodendrocyte Precursor

AbstractGlioblastoma frequently exhibits therapy-associated subtype transitions to mesenchymal phenotypes with adverse prognosis. Here, we perform multi-omic profiling of 60 glioblastoma primary tumours and use orthogonal analysis of chromatin and RNA-derived gene regulatory networks to identify 38 subtype master regulators, whose cell population-specific activities we further map in published single-cell RNA sequencing data. These analyses identify the oligodendrocyte precursor marker and chromatin modifier SOX10 as a master regulator in RTK I-subtype tumours. In vitro functional studies demonstrate that SOX10 loss causes a subtype switch analogous to the proneural–mesenchymal transition observed in patients at the transcriptomic, epigenetic and phenotypic levels. SOX10 repression in an in vivo syngeneic graft glioblastoma mouse model results in increased tumour invasion, immune cell infiltration and significantly reduced survival, reminiscent of progressive human glioblastoma. These results identify SOX10 as a bona fide master regulator of the RTK I subtype, with both tumour cell-intrinsic and microenvironmental effects.

scholarly journals Pluripotent stem cell-derived epithelium misidentified as brain microvascular endothelium requires ETS factors to acquire vascular fate

2021 ◽  
Vol 118 (8) ◽  
pp. e2016950118 ◽  
Author(s):  
Tyler M. Lu ◽  
Sean Houghton ◽  
Tarig Magdeldin ◽  
José Gabriel Barcia Durán ◽  
Andrew P. Minotti ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Neurological Diseases ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Microvascular Endothelium ◽  
Functional Studies ◽  
Bona Fide ◽  
Microvascular Endothelial

Cells derived from pluripotent sources in vitro must resemble those found in vivo as closely as possible at both transcriptional and functional levels in order to be a useful tool for studying diseases and developing therapeutics. Recently, differentiation of human pluripotent stem cells (hPSCs) into brain microvascular endothelial cells (ECs) with blood–brain barrier (BBB)-like properties has been reported. These cells have since been used as a robust in vitro BBB model for drug delivery and mechanistic understanding of neurological diseases. However, the precise cellular identity of these induced brain microvascular endothelial cells (iBMECs) has not been well described. Employing a comprehensive transcriptomic metaanalysis of previously published hPSC-derived cells validated by physiological assays, we demonstrate that iBMECs lack functional attributes of ECs since they are deficient in vascular lineage genes while expressing clusters of genes related to the neuroectodermal epithelial lineage (Epi-iBMEC). Overexpression of key endothelial ETS transcription factors (ETV2, ERG, and FLI1) reprograms Epi-iBMECs into authentic endothelial cells that are congruent with bona fide endothelium at both transcriptomic as well as some functional levels. This approach could eventually be used to develop a robust human BBB model in vitro that resembles the human brain EC in vivo for functional studies and drug discovery.

Improvement of effectiveness in maize breeding

2006 ◽  
Vol 54 (3) ◽  
pp. 351-358 ◽  
Author(s):  
P. Pepó
Keyword(s):  
Recurrent Selection ◽  
Genetic Manipulation ◽  
Field Testing ◽  
Tissue Cultures ◽  
Maize Breeding ◽  
Breeding Programmes ◽  
Speed Up ◽  
Selection For

Plant regeneration via tissue culture is becoming increasingly more common in monocots such as maize (Zea mays L.). Pollen (gametophytic) selection for resistance to aflatoxin in maize can greatly facilitate recurrent selection and the screening of germplasm for resistance at much less cost and in a shorter time than field testing. In vivo and in vitro techniques have been integrated in maize breeding programmes to obtain desirable agronomic attributes, enhance the genes responsible for them and speed up the breeding process. The efficiency of anther and tissue cultures in maize and wheat has reached the stage where they can be used in breeding programmes to some extent and many new cultivars produced by genetic manipulation have now reached the market.

SiRNA technology, the gene therapy of the future?

2008 ◽  
Vol 149 (4) ◽  
pp. 153-159 ◽  
Author(s):  
Zsuzsanna Rácz ◽  
Péter Hamar
Keyword(s):  
Gene Therapy ◽  
Short Interfering Rna ◽  
The Future ◽  
Interfering Rna

A genetikában új korszak kezdődött 17 éve, amikor a petúniában felfedezték a koszuppressziót. Később a koszuppressziót azonosították a növényekben és alacsonyabb rendű eukariótákban megfigyelt RNS-interferenciával (RNSi). Bár a növényekben ez ősi vírusellenes gazdaszervezeti védekezőmechanizmus, emlősökben az RNSi élettani szerepe még nincs teljesen tisztázva. Az RNSi-t rövid kettős szálú interferáló RNS-ek (short interfering RNA, siRNS) irányítják. A jelen cikkben összefoglaljuk az RNSi történetét és mechanizmusát, az siRNS-ek szerkezete és hatékonysága közötti összefüggéseket, a célsejtbe való bejuttatás virális és nem virális módjait. Az siRNS-ek klinikai alkalmazásának legfontosabb akadálya az in vivo alkalmazás. Bár a hidrodinamikus kezelés állatokban hatékony, embereknél nem alkalmazható. Lehetőséget jelent viszont a szervspecifikus katéterezés. A szintetizált siRNS-ek ismert mellékhatásait szintén tárgyaljuk. Bár a génterápia ezen új területén számos problémával kell szembenézni, a sikeres in vitro és in vivo kísérletek reményt jelentenek emberi betegségek siRNS-sel történő kezelésére.

Exosome as a Natural Gene Delivery Vector for Cancer Treatment

2020 ◽  
Vol 20 (11) ◽  
pp. 821-830
Author(s):  
Prasad Pofali ◽  
Adrita Mondal ◽  
Vaishali Londhe
Keyword(s):  
Gene Therapy ◽  
Gene Delivery ◽  
Nucleic Acids ◽  
Cancer Treatment ◽  
Intestinal Barrier ◽  
Gene Carrier ◽  
Gene Delivery Vector ◽  
Delivery Vector

Background: Current gene therapy vectors such as viral, non-viral, and bacterial vectors, which are used for cancer treatment, but there are certain safety concerns and stability issues of these conventional vectors. Exosomes are the vesicles of size 40-100 nm secreted from multivesicular bodies into the extracellular environment by most of the cell types in-vivo and in-vitro. As a natural nanocarrier, exosomes are immunologically inert, biocompatible, and can cross biological barriers like the blood-brain barrier, intestinal barrier, and placental barrier. Objective: This review focusses on the role of exosome as a carrier to efficiently deliver a gene for cancer treatment and diagnosis. The methods for loading of nucleic acids onto the exosomes, advantages of exosomes as a smart intercellular shuttle for gene delivery and therapeutic applications as a gene delivery vector for siRNA, miRNA and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and also the limitations of exosomes as a gene carrier are all reviewed in this article. Methods: Mostly, electroporation and chemical transfection are used to prepare gene loaded exosomes. Results: Exosome-mediated delivery is highly promising and advantageous in comparison to the current delivery methods for systemic gene therapy. Targeted exosomes, loaded with therapeutic nucleic acids, can efficiently promote the reduction of tumor proliferation without any adverse effects. Conclusion: In the near future, exosomes can become an efficient gene carrier for delivery and a biomarker for the diagnosis and treatment of cancer.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

scholarly journals POLRMT mutations impair mitochondrial transcription causing neurological disease

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Monika Oláhová ◽  
Bradley Peter ◽  
Zsolt Szilagyi ◽  
Hector Diaz-Maldonado ◽  
Meenakshi Singh ◽  
...  
Keyword(s):  
Progressive External Ophthalmoplegia ◽  
Global Developmental Delay ◽  
Disease Mechanism ◽  
Mitochondrial Transcription ◽  
Functional Studies ◽  
Mtdna Deletions ◽  
Molecular Nature ◽  
Important Disease

AbstractWhile >300 disease-causing variants have been identified in the mitochondrial DNA (mtDNA) polymerase γ, no mitochondrial phenotypes have been associated with POLRMT, the RNA polymerase responsible for transcription of the mitochondrial genome. Here, we characterise the clinical and molecular nature of POLRMT variants in eight individuals from seven unrelated families. Patients present with global developmental delay, hypotonia, short stature, and speech/intellectual disability in childhood; one subject displayed an indolent progressive external ophthalmoplegia phenotype. Massive parallel sequencing of all subjects identifies recessive and dominant variants in the POLRMT gene. Patient fibroblasts have a defect in mitochondrial mRNA synthesis, but no mtDNA deletions or copy number abnormalities. The in vitro characterisation of the recombinant POLRMT mutants reveals variable, but deleterious effects on mitochondrial transcription. Together, our in vivo and in vitro functional studies of POLRMT variants establish defective mitochondrial transcription as an important disease mechanism.

Neuropilin-1 on hematopoietic cells as a source of vascular development

Blood ◽  
2003 ◽  
Vol 101 (5) ◽  
pp. 1801-1809 ◽  
Author(s):  
Yoshihiro Yamada ◽  
Yuichi Oike ◽  
Hisao Ogawa ◽  
Yasuhiro Ito ◽  
Hajime Fujisawa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Fetal Liver ◽  
Vascular Development ◽  
Hematopoietic Cells ◽  
Vegf Receptor ◽  
Knock Out ◽  
Neuropilin 1 ◽  
Vasculogenesis And Angiogenesis

Neuropilin-1 (NP-1) is a receptor for vascular endothelial growth factor-165 (VEGF165) and acts as a coreceptor that enhances the function of VEGF165 through VEGF receptor-2 (VEGFR-2). Studies using transgenic and knock-out mice of NP-1 indicated that this molecule is important for vascular development as well as neuronal development. We recently reported that clustered soluble NP-1 phosphorylates VEGFR-2 on endothelial cells with a low dose of VEGF165 and rescues the defective vascularity of the NP-1−/− embryo in vitro and in vivo. Here we show that NP-1 is expressed by CD45+ hematopoietic cells in the fetal liver, can bind VEGF165, and phosphorylates VEGFR-2 on endothelial cells. CD45+NP-1+ cells rescued the defective vasculogenesis and angiogenesis in the NP-1−/− P-Sp (para-aortic splanchnopleural mesodermal region) culture, although CD45+NP-1− cells did not. Moreover, CD45+NP-1+ cells together with VEGF165 induced angiogenesis in an in vivo Matrigel assay and cornea neovascularization assay. The extracellular domain of NP-1 consists of “a,” “b,” and “c” domains, and it is known that the “a” and “c” domains are necessary for dimerization of NP-1. We found that both the “a” and “c” domains are essential for such rescue of defective vascularities in the NP-1 mutant. These results suggest that NP-1 enhances vasculogenesis and angiogenesis exogenously and that dimerization of NP-1 is important for enhancing vascular development. In NP-1−/− embryos, vascular sprouting is impaired at the central nervous system (CNS) and pericardium where VEGF is not abundant, indicating that NP-1–expressing cells are required for normal vascular development.

scholarly journals Preclinical Development of Autologous Hematopoietic Stem Cell-Based Gene Therapy for Immune Deficiencies: A Journey from Mouse Cage to Bed Side

Pharmaceutics ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 549
Author(s):  
Laura Garcia-Perez ◽  
Anita Ordas ◽  
Kirsten Canté-Barrett ◽  
Pauline Meij ◽  
Karin Pike-Overzet ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Severe Combined Immunodeficiency ◽  
Good Manufacturing Practice ◽  
Proof Of Concept ◽  
Preclinical Development ◽  
Hematopoietic Stem ◽  
Immune Deficiencies ◽  
Suitable Vector

Recent clinical trials using patient’s own corrected hematopoietic stem cells (HSCs), such as for primary immunodeficiencies (Adenosine deaminase (ADA) deficiency, X-linked Severe Combined Immunodeficiency (SCID), X-linked chronic granulomatous disease (CGD), Wiskott–Aldrich Syndrome (WAS)), have yielded promising results in the clinic; endorsing gene therapy to become standard therapy for a number of diseases. However, the journey to achieve such a successful therapy is not easy, and several challenges have to be overcome. In this review, we will address several different challenges in the development of gene therapy for immune deficiencies using our own experience with Recombinase-activating gene 1 (RAG1) SCID as an example. We will discuss product development (targeting of the therapeutic cells and choice of a suitable vector and delivery method), the proof-of-concept (in vitro and in vivo efficacy, toxicology, and safety), and the final release steps to the clinic (scaling up, good manufacturing practice (GMP) procedures/protocols and regulatory hurdles).

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