scholarly journals AAV ablates neurogenesis in the adult murine hippocampus

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Stephen Johnston ◽  
Sarah Parylak ◽  
Stacy Kim ◽  
Nolan Mac ◽  
Christina Lim ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Cell Death ◽  
Adult Neurogenesis ◽  
Granule Cells ◽  
Viral Vector ◽  
Cell Types ◽  
Early Gene ◽  
Post Injection

Recombinant adeno-associated virus (rAAV) has been widely used as a viral vector across mammalian biology and has been shown to be safe and effective in human gene therapy. We demonstrate that neural progenitor cells (NPCs) and immature dentate granule cells (DGCs) within the adult murine hippocampus are particularly sensitive to rAAV-induced cell death. Cell loss is dose dependent and nearly complete at experimentally relevant viral titers. rAAV-induced cell death is rapid and persistent, with loss of BrdU-labeled cells within 18 hours post-injection and no evidence of recovery of adult neurogenesis at 3 months post-injection. The remaining mature DGCs appear hyperactive 4 weeks post-injection based on immediate early gene expression, consistent with previous studies investigating the effects of attenuating adult neurogenesis. In vitro application of AAV or electroporation of AAV2 inverted terminal repeats (ITRs) is sufficient to induce cell death. Efficient transduction of the dentate gyrus (DG)-without ablating adult neurogenesis-can be achieved by injection of rAAV2-retro serotyped virus into CA3. rAAV2-retro results in efficient retrograde labeling of mature DGCs and permits in vivo 2-photon calcium imaging of dentate activity while leaving adult neurogenesis intact. These findings expand on recent reports implicating rAAV-linked toxicity in stem cells and other cell types and suggest that future work using rAAV as an experimental tool in the DG and as a gene therapy for diseases of the central nervous system (CNS) should be carefully evaluated.

scholarly journals AAV Ablates Neurogenesis in the Adult Murine Hippocampus

2020 ◽  
Author(s):  
ST Johnston ◽  
SL Parylak ◽  
S Kim ◽  
N Mac ◽  
CK Lim ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Cell Death ◽  
Adult Neurogenesis ◽  
Granule Cells ◽  
Viral Vector ◽  
Cell Types ◽  
Early Gene ◽  
Post Injection

ABSTRACTRecombinant adeno-associated virus (rAAV) has been widely used as a viral vector across mammalian biology and has been shown to be safe and effective in human gene therapy. We demonstrate that neural progenitor cells (NPCs) and immature dentate granule cells (DGCs) within the adult murine hippocampus are particularly sensitive to rAAV-induced cell death. Cell loss is dose dependent and nearly complete at experimentally relevant viral titers. rAAV-induced cell death is rapid and persistent, with loss of BrdU-labeled cells within 18 hours post-injection and no evidence of recovery of adult neurogenesis at 3 months post-injection. The remaining mature DGCs appear hyperactive 4 weeks post-injection based on immediate early gene expression, consistent with previous studies investigating the effects of attenuating adult neurogenesis. In vitro application of AAV or electroporation of AAV2 inverted terminal repeats (ITRs) is sufficient to induce cell death. Efficient transduction of the dentate gyrus (DG)—without ablating adult neurogenesis—can be achieved by injection of rAAV2-retro serotyped virus into CA3. rAAV2-retro results in efficient retrograde labeling of mature DGCs and permits in vivo 2-photon calcium imaging of dentate activity while leaving adult neurogenesis intact. These findings expand on recent reports implicating rAAV-linked toxicity in stem cells and other cell types and suggest that future work using rAAV as an experimental tool in the DG and as a gene therapy for diseases of the central nervous system (CNS) should be carefully evaluated.

scholarly journals In Vitro and In Vivo Evaluation of Human Adenovirus Type 49 as a Vector for Therapeutic Applications

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1483
Author(s):  
Emily A. Bates ◽  
John R. Counsell ◽  
Sophie Alizert ◽  
Alexander T. Baker ◽  
Natalie Suff ◽  
...  
Keyword(s):  
Viral Vector ◽  
Ex Vivo ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Cell Types ◽  
In Vivo Evaluation ◽  
In Vivo Biodistribution ◽  
Adenovirus Type 5

The human adenovirus phylogenetic tree is split across seven species (A–G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of pre-existing immunity detected across screened populations. However, many aspects of the basic virology of species D—such as their cellular tropism, receptor usage, and in vivo biodistribution profile—remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49)—a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry, but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting, whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells, and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen, whilst avoiding liver interactions, such as intravascular vaccine applications.

scholarly journals Baculoviruses as Vectors for Gene Therapy against Human Prostate Cancer

2003 ◽  
Vol 2003 (2) ◽  
pp. 79-91 ◽  
Author(s):  
Lindsay J. Stanbridge ◽  
Vincent Dussupt ◽  
Norman J. Maitland
Keyword(s):  
Prostate Cancer ◽  
Gene Therapy ◽  
Viral Entry ◽  
Mammalian Cells ◽  
Primary Tumour ◽  
Genetic Material ◽  
Cell Types ◽  
Attractive Alternative

Current curative strategies for prostate cancer are restricted to the primary tumour, and the effect of treatments to control metastatic disease is not sustained. Therefore, the application of gene therapy to prostate cancer is an attractive alternative. Baculoviruses are highly restricted insect viruses, which can enter, but not replicate in mammalian cells. Baculoviruses can incorporate large amounts of extra genetic material, and will express transgenes in mammalian cells when under the control of a mammalian or strong viral promoter. Successful gene delivery has been achieved both in vitro and in vivo and into both dividing and nondividing cells, which is important since prostate cancers divide relatively slowly. In addition, the envelope protein gp64 is sufficiently mutable to allow targeted transduction of particular cell types. In this review, the advantages of using baculoviruses for prostate cancer gene therapy are explored, and the mechanisms of viral entry and transgene expression are described.

scholarly journals In Vitro and In Vivo Evaluation of Human Adenovirus Type 49 as a Vector for Therapeutic Applications

2021 ◽  
Author(s):  
Emily A. Bates ◽  
John R. Counsell ◽  
Sophie Alizert ◽  
Alexander T. Baker ◽  
Natalie Suff ◽  
...  
Keyword(s):  
Viral Vector ◽  
Ex Vivo ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Cell Types ◽  
In Vivo Evaluation ◽  
In Vivo Biodistribution ◽  
Adenovirus Type 5

The human adenovirus phylogenetic tree is split across seven species (A-G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of preexisting immunity detected across screened populations. However, many aspects of the basic virology of species D, such as their cellular tropism, receptor usage and in vivo biodistribution profile, remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49), a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen whilst avoiding liver interactions, such as intravascular vaccine applications.

Simian virus 40 small t antigen cooperates with mitogen-activated kinases to stimulate AP-1 activity

1994 ◽  
Vol 14 (9) ◽  
pp. 6244-6252
Author(s):  
J A Frost ◽  
A S Alberts ◽  
E Sontag ◽  
K Guan ◽  
M C Mumby ◽  
...  
Keyword(s):  
Simian Virus 40 ◽  
Cell Types ◽  
Simian Virus ◽  
T Antigen ◽  
Mitogen Activated Protein Kinase ◽  
Early Gene ◽  
Mitogen Activated Protein ◽  
Small T Antigen

The simian virus 40 small tumor antigen (small t) specifically interacts with protein phosphatase type 2A (PP2A) in vivo and alters its catalytic activity in vitro. Among the substrates for PP2A in vitro are the activated forms of MEK and ERK kinases. Dephosphorylation of the activating phosphorylation sites on MEK and ERKs by PP2A in vitro results in a decrease in their respective kinase activities. Recently, it has been shown that overexpression of small t in CV-1 cells results in an inhibition of PP2A activity toward MEK and ERK2 and a constitutive upregulation of MEK and ERK2 activity. Previously, we have observed that overexpression of either ERK1, MEK1, or a constitutively active truncated form of c-Raf-1 (BXB) is insufficient to activate AP-1 in REF52 fibroblasts. We therefore examined whether overexpression of small t either alone or in conjunction with ERK1, MEK1, or BXB could activate AP-1. We found that coexpression of small t and either ERK1, MEK1, or BXB resulted in an increase in AP-1 activity, whereas expression of either small t or any of the kinases alone did not have any effect. Similarly, coexpression of small t and ERK1 activated serum response element-regulated promoters. Coexpression of kinase-deficient mutants of ERK1 and ERK2 inhibited the activation of AP-1 caused by expression of small t and either MEK1 or BXB. Coexpression of an interfering MEK, which inhibited AP-1 activation by small t and BXB, did not inhibit the activation of AP-1 caused by small t and ERK1. In contrast to REF52 cells, we observed that overexpression of either small or ERK1 alone in CV-1 cells was sufficient to stimulate AP-1 activity and that this stimulation was not enhanced by expression of small t and ERK1 together. These results show that the effects of small t on immediate-early gene expression depend on the cell type examined and suggest that the mitogen-activated protein kinase activation pathway is distinctly regulated in different cell types.

scholarly journals Failure of Poly(ADP-Ribose) Polymerase Cleavage by Caspases Leads to Induction of Necrosis and Enhanced Apoptosis

1999 ◽  
Vol 19 (7) ◽  
pp. 5124-5133 ◽  
Author(s):  
Zdenko Herceg ◽  
Zhao-Qi Wang
Keyword(s):  
Cell Death ◽  
Parp Inhibitor ◽  
Cell Types ◽  
Parp Cleavage ◽  
Dna Breaks ◽  
Necrosis Factor Alpha ◽  
Caspase Cleavage ◽  
Protein Resistant

ABSTRACT Activation of poly(ADP-ribose) polymerase (PARP) by DNA breaks catalyzes poly(ADP-ribosyl)ation and results in depletion of NAD+ and ATP, which is thought to induce necrosis. Proteolytic cleavage of PARP by caspases is a hallmark of apoptosis. To investigate whether PARP cleavage plays a role in apoptosis and in the decision of cells to undergo apoptosis or necrosis, we introduced a point mutation into the cleavage site (DEVD) of PARP that renders the protein resistant to caspase cleavage in vitro and in vivo. Here, we show that after treatment with tumor necrosis factor alpha, fibroblasts expressing this caspase-resistant PARP exhibited an accelerated cell death. This enhanced cell death is attributable to the induction of necrosis and an increased apoptosis and was coupled with depletion of NAD+ and ATP that occurred only in cells expressing caspase-resistant PARP. The PARP inhibitor 3-aminobenzamide prevented the NAD+ drop and concomitantly inhibited necrosis and the elevated apoptosis. These data indicate that this accelerated cell death is due to NAD+ depletion, a mechanism known to kill various cell types, caused by activation of uncleaved PARP after DNA fragmentation. The present study demonstrates that PARP cleavage prevents induction of necrosis during apoptosis and ensures appropriate execution of caspase-mediated programmed cell death.

scholarly journals Mechanizmy śmierci od komórki do całego organizmu

2019 ◽  
Author(s):  
Sylwia Ciesielska
Keyword(s):  
Cell Death ◽  
Living Organism ◽  
Cell Types ◽  
In Vivo Studies ◽  
Whole Body ◽  
Natural Phenomenon ◽  
Human Organism ◽  
Living Organisms

In vitro studies are alternative for in vivo studies carried on living organisms. They involve cell populations for both normal and cancer cells. The processes inside cells might be base for defining whole body processes. Starting with fundamental unit of every living organism which is cell, we can distinguish two main types of cell death – apoptosis and necrosis. Human organism is built from 1013–1014 cells of 300 different cell types. During cell division new cells are created and their number is strictly controlled in programmed cell death – apoptosis. Mainly old or damaged cells commit suicide and are removed from organism. This is natural phenomenon and every change in mechanisms of proliferation or apoptosis cause changes and damage in whole organism. Homeostasis in organism depends on correct action of death and survival system. The patterns of equilibrium in nature relies on similar regulation profiles, in which it is similar to death of singular organisms in population or species. It implicates death as natural phenomenon maintaining balance in the world.

scholarly journals Polyinosinic acid enhances delivery of adenovirus vectors in vivo by preventing sequestration in liver macrophages

2008 ◽  
Vol 89 (5) ◽  
pp. 1097-1105 ◽  
Author(s):  
Hidde J. Haisma ◽  
Jan A. A. M. Kamps ◽  
Gera K. Kamps ◽  
Josee A. Plantinga ◽  
Marianne G. Rots ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Kupffer Cells ◽  
Transgene Expression ◽  
Liver Toxicity ◽  
Viral Vector ◽  
Adenovirus Vector ◽  
Liver Macrophages ◽  
Polyinosinic Acid

Adenovirus is among the preferred vectors for gene therapy because of its superior in vivo gene-transfer efficiency. However, upon systemic administration, adenovirus is preferentially sequestered by the liver, resulting in reduced adenovirus-mediated transgene expression in targeted tissues. In the liver, Kupffer cells are responsible for adenovirus degradation and contribute to the inflammatory response. As scavenger receptors present on Kupffer cells are responsible for the elimination of blood-borne pathogens, we investigated the possible implication of these receptors in the clearance of the adenovirus vector. Polyinosinic acid [poly(I)], a scavenger receptor A ligand, was analysed for its capability to inhibit adenovirus uptake specifically in macrophages. In in vitro studies, the addition of poly(I) before virus infection resulted in a specific inhibition of adenovirus-induced gene expression in a J774 macrophage cell line and in primary Kupffer cells. In in vivo experiments, pre-administration of poly(I) caused a 10-fold transient increase in the number of adenovirus particles circulating in the blood. As a consequence, transgene expression levels measured in different tissues were enhanced (by 5- to 15-fold) compared with those in animals that did not receive poly(I). Finally, necrosis of Kupffer cells, which normally occurs as a consequence of systemic adenovirus administration, was prevented by the use of poly(I). No toxicity, as measured by liver-enzyme levels, was observed after poly(I) treatment. From our data, we conclude that poly(I) can prevent adenovirus sequestration by liver macrophages. These results imply that, by inhibiting adenovirus uptake by Kupffer cells, it is possible to reduce the dose of the viral vector to diminish the liver-toxicity effect and to improve the level of transgene expression in target tissues. In systemic gene-therapy applications, this will have great impact on the development of targeted adenoviral vectors.

THE PROSPECTS FOR APPLICATION OF LACTOFERRIN AND ITS DERIVATIVES IN THE TREATMENT OF CANCER

2019 ◽  
Vol 65 (6) ◽  
pp. 785-790
Author(s):  
Veronika Zorina
Keyword(s):  
Gene Therapy ◽  
Synthetic Peptides ◽  
Cell Types ◽  
In Vitro Models ◽  
The World ◽  
Specific Receptors ◽  
Methods Of Treatment ◽  
Universal Protein

Annually more than 250 thousand deaths from malignant diseases are registered in Russia and 8 million - in the world. The effectiveness of traditional methods of treatment, as well as of monoclonal antibodies and gene therapy is limited in the case of resistant forms of cancer and hard to reach tumors. Lactoferrin (LF) is a universal protein with anticancer activity and cross-species biocompatibility, which modulates the immune response and the redox/antioxidant system. Due to the ability to interact not only with specific receptors, but also with signaling and endocytosis receptors, TLR on various cell types, LF can overcome tissue barriers. The therapeutic efficacy of lactoferrin, its cleavage products, and synthetic peptides against various types of malignant proliferation was demonstrated with in vivo and in vitro models. The prospects of using LF as an adjuvant before chemotherapy and radiotherapy and as a carrier for gene therapy of cancer were experimentally demonstrated. However, the results of clinical studies are few and require further study.

scholarly journals Intramyocardial Injection of Recombinant Adeno-Associated Viral Vector Coexpressing PR39/Adrenomedullin Enhances Angiogenesis and Reduces Apoptosis in a Rat Myocardial Infarction Model

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
Rui An ◽  
Cong Xi ◽  
Jian Xu ◽  
Ying Liu ◽  
Shumiao Zhang ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Gene Therapy ◽  
Myocardial Injury ◽  
Viral Vector ◽  
Saline Group ◽  
Intramyocardial Injection ◽  
New Gene ◽  
Therapy Strategies

Cotransfer of angiogenic and antiapoptotic genes could be the basis of new gene therapy strategies for myocardial infarction. In this study, rAAV-PR39-ADM, coexpressing antimicrobial peptide (PR39) and adrenomedullin (ADM), was designed with the mediation of recombinant adeno-associated virus. In vitro, CRL-1730 cells were divided into four groups, namely, the sham group, the AAV-null group, the NS (normal saline) group, and the PR39-ADM group. Immunocytochemistry analysis, CCK-8 assays, Matrigel assays, and apoptotic analysis were performed; in vivo, myocardial infarction model was established through ligation of the left coronary artery on rats, and treatment groups corresponded to those used in vitro. Myocardial injury, cardiac performance, and the extent of myocardial apoptosis were assessed. Results suggested that rAAV-PR39-ADM administration after myocardial infarction improved cell viability and cardiac function, attenuated apoptosis and myocardial injury, and promoted angiogenesis. Subsequently, levels of 6×His, HIF-1α, VEGF, p-Akt, Akt, ADM, Bcl-2, and Bax were measured by western blot. rAAV-PR39-ADM increased p-Akt, HIF-1α, and VEGF levels and induced higher Bcl-2 expression and lower Bax expression. In conclusion, our results demonstrate that rAAV-PR39-ADM mitigates myocardial injury by promoting angiogenesis and reducing apoptosis. This study suggests a potential novel gene therapy-based method that could be used clinically for myocardial infarction.

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