In Vivo Marking of Rhesus Monkey Lymphocytes by Adeno-Associated Viral Vectors: Direct Comparison With Retroviral Vectors

Blood ◽  
1999 ◽  
Vol 94 (7) ◽  
pp. 2263-2270 ◽  
Author(s):  
Yutaka Hanazono ◽  
Kevin E. Brown ◽  
Atsushi Handa ◽  
Mark E. Metzger ◽  
Dominik Heim ◽  
...  
Keyword(s):  
Rhesus Monkey ◽  
Dna Sequences ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Retroviral Vectors ◽  
Peripheral Blood Lymphocytes ◽  
Transduction Efficiency ◽  
Monkey Model

We have compared adeno-associated virus (AAV)-based and retrovirus-based vectors for their ability to transduce primary T lymphocytes in vitro and then tracked the persistence of these genetically marked lymphocytes in vivo, using the rhesus monkey model. To avoid the complication of immune rejection of lymphocytes transduced with xenogeneic genes in tracking studies primarily designed to investigate transduction efficiency and in vivo kinetics, the vectors were designed without expressed genes. All vectors contained identically mutated β-galactosidase gene (β-gal) and neomycin resistance gene (neo) DNA sequences separated by different length polylinkers, allowing simple differentiation by polymerase chain reaction (PCR). Each of 2 aliquots of peripheral blood lymphocytes from 4 rhesus monkeys were transduced with either AAV or retroviral vectors. The in vitro transduction efficiency (mean vector copy number/cell) after the ex vivo culture was estimated by PCR at 0.015 to 3.0 for AAV, varying depending on the multiplicity of infection (MOI) used for transduction, and 0.13 to 0.19 for the retroviral transductions. Seven days after transduction, Southern blot analysis of AAV-transduced lymphocytes showed double-stranded and head-to-tail concatemer forms but failed to show integration of the AAV vector. AAV and retroviral aliquots were reinfused concurrently into each animal. Although the retrovirally marked lymphocytes could be detected for much longer after infusion, AAV transduction resulted in higher short-term in vivo marking efficiency compared with retroviral vectors, suggesting possible clinical applications of AAV vectors in lymphocyte gene therapy when long-term vector persistence is not required or desired.

scholarly journals Canine Adenovirus Vectors for Lung-Directed Gene Transfer: Efficacy, Immune Response, and Duration of Transgene Expression Using Helper-Dependent Vectors

2006 ◽  
Vol 80 (3) ◽  
pp. 1487-1496 ◽  
Author(s):  
Anne Keriel ◽  
Céline René ◽  
Chad Galer ◽  
Joseph Zabner ◽  
Eric J. Kremer
Keyword(s):  
Gene Transfer ◽  
Respiratory Tract ◽  
Neutralizing Antibodies ◽  
Transgene Expression ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Adenovirus Type ◽  
Transduction Efficiency

ABSTRACT A major hurdle to the successful clinical use of some viral vectors relates to the innate, adaptive, and memory immune responses that limit the efficiency and duration of transgene expression. Some of these drawbacks may be circumvented by using vectors derived from nonhuman viruses such as canine adenovirus type 2 (CAV-2). Here, we evaluated the potential of CAV-2 vectors for gene transfer to the respiratory tract. We found that CAV-2 transduction was efficient in vivo in the mouse respiratory tract, and ex vivo in well-differentiated human pulmonary epithelia. Notably, the in vivo and ex vivo efficiency was poorly inhibited by sera from mice immunized with a human adenovirus type 5 (HAd5, a ubiquitous human pathogen) vector or by human sera containing HAd5 neutralizing antibodies. Following intranasal instillation in mice, CAV-2 vectors also led to a lower level of inflammatory cytokine secretion and cellular infiltration compared to HAd5 vectors. Moreover, CAV-2 transduction efficiency was increased in vitro in human pulmonary cells and in vivo in the mouse respiratory tract by FK228, a histone deacetylase inhibitor. Finally, by using a helper-dependent CAV-2 vector, we increased the in vivo duration of transgene expression to at least 3 months in immunocompetent mice without immunosuppression. Our data suggest that CAV-2 vectors may be efficient and safe tools for long-term clinical gene transfer to the respiratory tract.

scholarly journals Adoptive cell therapy targeting common p53 neoantigens in human solid cancers

2021 ◽  
Author(s):  
Sanghyun Kim ◽  
Nolan Vale ◽  
Nikolaos Zacharakis ◽  
Sri Krishna ◽  
Zhiya Yu ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Tumor Regression ◽  
Ex Vivo ◽  
Adoptive Cell Therapy ◽  
Peripheral Blood Lymphocytes ◽  
Mutant P53 ◽  
Autologous Tumor ◽  
Clinical Responses

Abstract Adoptive cell therapy (ACT) targeting neoantigens can achieve durable clinical responses in patients with cancer. Most neoantigens arise from rare mutations, requiring highly individualized treatments. To broaden the applicability of ACT targeting neoantigens, we focused on TP53 mutations commonly shared across different cancer types. Here, we describe a library of T cell receptors (TCRs) that can target TP53 mutations shared among 7.3% of patients with solid cancers. These TCRs recognized tumor cells in a TP53 mutation- and human leucocyte antigen (HLA)-specific manner both in vitro and in vivo. Patients with chemorefractory epithelial cancers treated with ex vivo-expanded autologous tumor infiltrating lymphocytes (TILs) naturally reactive with mutant p53 experienced limited clinical responses (2 PRs/12 patients), and we detected low frequencies, exhausted phenotypes, and poor persistence of the infused mutant p53-reactive TILs. Alternatively, we treated one patient with a chemorefractory breast cancer with ACT by transducing autologous peripheral blood lymphocytes with an HLA-A*02-restricted anti-p53R175H TCR. The infused cells exhibited an improved immunophenotype and prolonged persistence compared to the TIL ACT and the patient experienced an objective tumor regression (-55%) that lasted 6 months. Collectively, these data demonstrate the feasibility of off-the-shelf TCR-engineered cell therapies targeting shared p53 neoantigens to treat human cancers.

DNA damage and susceptibility to oxidative damage in lymphocytes: effects of carotenoidsin vitroandin vivo

2004 ◽  
Vol 91 (1) ◽  
pp. 53-61 ◽  
Author(s):  
Siân B. Astley ◽  
David A. Hughes ◽  
Anthony J. A. Wright ◽  
Ruan M. Elliott ◽  
Susan Southon
Keyword(s):  
Oxidative Damage ◽  
Ex Vivo ◽  
Human Peripheral Blood ◽  
Plasma Concentrations ◽  
Peripheral Blood Lymphocytes ◽  
Repair Mechanisms ◽  
Oxidative Challenge ◽  
Β Carotene

Reports on the effects of carotenoids are conflicting. The present paper examines similarities and differences from contiguous studiesin vitroandin vivo. Single-cell gel electrophoresis was used to measure the frequency of single-strand breaks (SSB) in the cell line MOLT-17 (as a model system) and human peripheral blood lymphocytes (PBL). MOLT-17 cells were supplemented with β-carotene, lutein or lycopene at a range of concentrations (0·00–8·00 μmol/l) using a liposome delivery method. Uptake was dose-dependent. β-Carotene concentration in the media had no effect on SSB in control cells, but incubation with lycopene or lutein (>2·00 μmol/l) increased the numbers of SSB in control cells. MOLT-17 DNA was less susceptible to oxidative damage (100 μmol H2O2/l, 5 min, 4 °C) following incubation with carotenoids between 0·50 and 1·00 μmol/l; at >1·00 μmol/l the effects were ambiguous. Apparently healthy male volunteers supplemented their habitual diets with lutein, β-carotene or lycopene (natural isolate capsules, 15 mg/d, 4 weeks) in three independent studies, raising plasma concentrations to different extents. Lycopene and lutein had no effect on SSB in control PBL or following oxidative challenge. However, increased plasma β-carotene was associated with more SSB in control cells whilst PBL DNA resistance to oxidative damageex vivowas unaffected. These results suggest that the carotenoids are capable of exerting two overlapping but distinct effects: antioxidant protection by scavenging DNA-damaging free radicals and modulation of DNA repair mechanisms.

scholarly journals Single adeno-associated virus-based multiplexed CRISPR-Cas9 system to nullify core components of the mammalian molecular clock

2020 ◽  
Author(s):  
Boil Kim ◽  
Jihoon Kim ◽  
Minjeong Chun ◽  
Inah Park ◽  
Mijung Choi ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Molecular Clock ◽  
Viral Vectors ◽  
Clock Genes ◽  
Ex Vivo ◽  
Physiological Role ◽  
Guide Rnas ◽  
Multiple Genes

ABSTRACTThe mammalian molecular clock is based on a transcription-translation feedback loop (TTFL) containing Period1, 2 (Per1, 2), Cryptochrome1, 2 (Cry1, 2), and Brain and Muscle ARNT-Like 1 (Bmal1). TTFL robustness is endowed by genetic complementation between these components; therefore, multiple genes must be knocked out to physiologically investigate the molecular clock, which requires extensive research resources. To facilitate molecular clock disruption, we developed a CRISPR-Cas9-based single adeno-associated viral (AAV) system targeting the circadian clock (CSAC) for Pers, Crys, or Bmal1. First, we designed single guide RNAs (sgRNAs) targeting individual clock genes using an in silico approach and validated their efficiency in Neuro2a cells. To target multiple genes, multiplex sgRNA plasmids were constructed using Golden Gate assembly and expressed in viral vectors. CSAC efficiency was demonstrated by decreased protein expression in vitro and ablated molecular oscillation ex vivo. We also measured locomotor activity and body temperature in Cas9-expressing mice injected with CSAC at the suprachiasmatic nucleus. Circadian rhythm disruption was observed under free-running conditions, indicating that CSAC can efficiently and robustly disrupt molecular circadian clock. Thus, CSAC is a simple and powerful tool for investigating the physiological role of the molecular clock in vivo.

scholarly journals Advances in siRNA delivery to T-cells: potential clinical applications for inflammatory disease, cancer and infection

2013 ◽  
Vol 455 (2) ◽  
pp. 133-147 ◽  
Author(s):  
Michael Freeley ◽  
Aideen Long
Keyword(s):  
T Cells ◽  
Gene Silencing ◽  
Inflammatory Disease ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Sirna Delivery ◽  
Attractive Potential ◽  
Therapeutic Benefits

The specificity of RNAi and its ability to silence ‘undruggable’ targets has made inhibition of gene expression in T-cells with siRNAs an attractive potential therapeutic strategy for the treatment of inflammatory disease, cancer and infection. However, delivery of siRNAs into primary T-cells represents a major hurdle to their use as potential therapeutic agents. Recent advances in siRNA delivery through the use of electroporation/nucleofection, viral vectors, peptides/proteins, nanoparticles, aptamers and other agents have now enabled efficient gene silencing in primary T-cells both in vitro and in vivo. Overcoming such barriers in siRNA delivery offers exciting new prospects for directly targeting T-cells systemically with siRNAs, or adoptively transferring T-cells back into patients following ex vivo manipulation with siRNAs. In the present review, we outline the challenges in delivering siRNAs into primary T-cells and discuss the mechanism and therapeutic opportunities of each delivery method. We emphasize studies that have exploited RNAi-mediated gene silencing in T-cells for the treatment of inflammatory disease, cancer and infection using mouse models. We also discuss the potential therapeutic benefits of manipulating T-cells using siRNAs for the treatment of human diseases.

scholarly journals Recent Advances in CRISPR/Cas9 Delivery Strategies

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 839 ◽  
Author(s):  
Bon Ham Yip
Keyword(s):  
Viral Vectors ◽  
Insertional Mutagenesis ◽  
Gene Editing ◽  
Ex Vivo ◽  
Extracellular Vesicle ◽  
Delivery Methods ◽  
Delivery Strategies ◽  
In Vivo Delivery

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has revolutionized the field of gene editing. Continuous efforts in developing this technology have enabled efficient in vitro, ex vivo, and in vivo gene editing through a variety of delivery strategies. Viral vectors are commonly used in in vitro, ex vivo, and in vivo delivery systems, but they can cause insertional mutagenesis, have limited cloning capacity, and/or elicit immunologic responses. Physical delivery methods are largely restricted to in vitro and ex vivo systems, whereas chemical delivery methods require extensive optimization to improve their efficiency for in vivo gene editing. Achieving a safe and efficient in vivo delivery system for CRISPR/Cas9 remains the most challenging aspect of gene editing. Recently, extracellular vesicle-based systems were reported in various studies to deliver Cas9 in vitro and in vivo. In comparison with other methods, extracellular vesicles offer a safe, transient, and cost-effective yet efficient platform for delivery, indicating their potential for Cas9 delivery in clinical trials. In this review, we first discuss the pros and cons of different Cas9 delivery strategies. We then specifically review the development of extracellular vesicle-mediated gene editing and highlight the strengths and weaknesses of this technology.

scholarly journals In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors

2002 ◽  
Vol 76 (3) ◽  
pp. 1496-1504 ◽  
Author(s):  
Ulrich Kuhn ◽  
Atsushi Terunuma ◽  
Wolfgang Pfutzner ◽  
Ruth Ann Foster ◽  
Jonathan C. Vogel
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Human Skin ◽  
Ex Vivo ◽  
Lentiviral Vectors ◽  
Retroviral Vectors ◽  
Immunocompromised Mice ◽  
In Vivo Assessment

ABSTRACT For skin gene therapy, introduction of a desired gene into keratinocyte progenitor or stem cells could overcome the problem of achieving persistent gene expression in a significant percentage of keratinocytes. Although keratinocyte stem cells have not yet been completely characterized and purified for gene targeting purposes, lentiviral vectors may be superior to retroviral vectors at gene introduction into these stem cells, which are believed to divide and cycle slowly. Our initial in vitro studies demonstrate that lentiviral vectors are able to efficiently transduce nondividing keratinocytes, unlike retroviral vectors, and do not require the lentiviral accessory genes for keratinocyte transduction. When lentiviral vectors expressing green fluorescent protein (GFP) were directly injected into the dermis of human skin grafted onto immunocompromised mice, transduction of dividing basal and nondividing suprabasal keratinocytes could be demonstrated, which was not the case when control retroviral vectors were used. However, flow cytometry analysis demonstrated low transduction efficiency, and histological analysis at later time points provided no evidence for progenitor cell targeting. In an alternative in vivo method, human keratinocytes were transduced in tissue culture (ex vivo) with either lentiviral or retroviral vectors and grafted as skin equivalents onto immunocompromised mice. GFP expression was analyzed in these human skin grafts after several cycles of epidermal turnover, and both the lentiviral and retroviral vector-transduced grafts had similar percentages of GFP-expressing keratinocytes. This ex vivo grafting study provides a good in vivo assessment of gene introduction into progenitor cells and suggests that lentiviral vectors are not necessarily superior to retroviral vectors at introducing genes into keratinocyte progenitor cells during in vitro culture.

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

Significance of Platelet β-Adrenoceptors for Platelet Responses In Vivo and In Vitro

1992 ◽  
Vol 68 (06) ◽  
pp. 687-693 ◽  
Author(s):  
P T Larsson ◽  
N H Wallén ◽  
A Martinsson ◽  
N Egberg ◽  
P Hjemdahl
Keyword(s):  
Ex Vivo ◽  
High Dose ◽  
Platelet Aggregability ◽  
Adrenoceptor Agonist ◽  
Dose Infusion ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Factor Antigen

SummaryThe significance of platelet β-adrenoceptors for platelet responses to adrenergic stimuli in vivo and in vitro was studied in healthy volunteers. Low dose infusion of the β-adrenoceptor agonist isoprenaline decreased platelet aggregability in vivo as measured by ex vivo filtragometry. Infusion of adrenaline, a mixed α- and β-adrenoceptor agonist, increased platelet aggregability in vivo markedly, as measured by ex vivo filtragometry and plasma β-thromboglobulin levels. Adrenaline levels were 3–4 nM in venous plasma during infusion. Both adrenaline and high dose isoprenaline elevated plasma von Willebrand factor antigen levels β-Blockade by propranolol did not alter our measures of platelet aggregability at rest or during adrenaline infusions, but inhibited adrenaline-induced increases in vWf:ag. In a model using filtragometry to assess platelet aggregability in whole blood in vitro, propranolol enhanced the proaggregatory actions of 5 nM, but not of 10 nM adrenaline. The present data suggest that β-adrenoceptor stimulation can inhibit platelet function in vivo but that effects of adrenaline at high physiological concentrations are dominated by an α-adrenoceptor mediated proaggregatory action.

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