scholarly journals Directed Evolution of AAV Targeting Primate Retina by Intravitreal Injection Identifies R100, a Variant Demonstrating Robust Gene Delivery and Therapeutic Efficacy in Non-Human Primates

2021 ◽  
Author(s):  
Melissa Ann Kotterman ◽  
Ghezal Beliakoff ◽  
Roxanne Croze ◽  
Tandis Vazin ◽  
Christopher Schmitt ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Nonhuman Primates ◽  
Target Cells ◽  
Retinal Cells ◽  
Efficient Delivery ◽  
Monogenic Diseases ◽  
Retinal Angiogenesis ◽  
Intravitreal Treatment ◽  
Specific Tissue ◽  
Tissue Target

Targeted AAV vectors are needed for safe and efficient delivery to and transduction of specific tissue target(s) in patients. Effective intravitreal delivery for retina gene therapy is not feasible with wildtype AAV. We employed directed evolution in nonhuman primates (NHP) to discover an AAV variant (R100) for intravitreal treatment of multiple target cells in the primate retina. R100 demonstrated superior transduction of human retinal cells compared to wildtype AAV. Furthermore, three R100-based gene therapeutics demonstrated safety, delivery, and durable pan-retinal expression of intracellular or secreted transgenes throughout the NHP retina following intravitreal administration. Finally, efficacy of R100 mediated delivery of therapeutic transgenes was demonstrated in patient-derived retinal cells (monogenic diseases) and in an NHP model of pathogenic retinal angiogenesis.

scholarly journals Characterisation of Ex Vivo Liver Thermal Properties for Electromagnetic-Based Hyperthermic Therapies

Sensors ◽  
2020 ◽  
Vol 20 (10) ◽  
pp. 3004 ◽  
Author(s):  
Nuno P. Silva ◽  
Anna Bottiglieri ◽  
Raquel C. Conceição ◽  
Martin O’Halloran ◽  
Laura Farina
Keyword(s):  
Thermal Properties ◽  
Ex Vivo ◽  
Tissue Perfusion ◽  
Heating Process ◽  
Volumetric Heat Capacity ◽  
Accurate Knowledge ◽  
Different Temperatures ◽  
Treatment Plans ◽  
Specific Tissue ◽  
Tissue Target

Electromagnetic-based hyperthermic therapies induce a controlled increase of temperature in a specific tissue target in order to increase the tissue perfusion or metabolism, or even to induce cell necrosis. These therapies require accurate knowledge of dielectric and thermal properties to optimise treatment plans. While dielectric properties have been well investigated, only a few studies have been conducted with the aim of understanding the changes of thermal properties as a function of temperature; i.e., thermal conductivity, volumetric heat capacity and thermal diffusivity. In this study, we experimentally investigate the thermal properties of ex vivo ovine liver in the hyperthermic temperature range, from 25 °C to 97 °C. A significant increase in thermal properties is observed only above 90 °C. An analytical model is developed to model the thermal properties as a function of temperature. Thermal properties are also investigated during the natural cooling of the heated tissue. A reversible phenomenon of the thermal properties is observed; during the cooling, thermal properties followed the same behaviour observed in the heating process. Additionally, tissue density and water content are evaluated at different temperatures. Density does not change with temperature; mass and volume losses change proportionally due to water vaporisation. A 30% water loss was observed above 90 °C.

scholarly journals Immunoadsorption enables successful rAAV5-mediated repeated hepatic gene delivery in nonhuman primates

2019 ◽  
Vol 3 (17) ◽  
pp. 2632-2641 ◽  
Author(s):  
David Salas ◽  
Karin L. Kwikkers ◽  
Nerea Zabaleta ◽  
Andrea Bazo ◽  
Harald Petry ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Neutralizing Antibodies ◽  
Nonhuman Primates ◽  
Human Subjects ◽  
Factor Ix ◽  
Target Cells ◽  
Targeted Gene Delivery ◽  
Human Factor Ix ◽  
Relevant Animal Model ◽  
Liver Gene

Abstract Adeno-associated virus (AAV)–based liver gene therapy has been shown to be clinically successful. However, the presence of circulating neutralizing antibodies (NABs) against AAV vector capsids remains a major challenge as it may prevent successful transduction of the target cells. Therefore, there is a need to develop strategies that would enable AAV-mediated gene delivery to patients with preexisting anti-AAV NABs. In the current study, the feasibility of using an immunoadsorption (IA) procedure for repeated, liver-targeted gene delivery in nonhuman primates was explored. The animals were administered IV with recombinant AAV5 (rAAV5) carrying the reporter gene human secreted embryonic alkaline phosphatase (hSEAP). Seven weeks after the first rAAV treatment, all of the animals were readministered with rAAV5 carrying the therapeutic hemophilia B gene human factor IX (hFIX). Half of the animals administered with rAAV5-hSEAP underwent IA prior to the second rAAV5 exposure. The transduction efficacies of rAAV5-hSEAP and rAAV5-hFIX were assessed by measuring the levels of hSEAP and hFIX proteins. Although no hFIX was detected after rAAV5-hFIX readministration without prior IA, all animals submitted to IA showed therapeutic levels of hFIX expression, and a threshold of anti-AAV5 NAB levels compatible with successful readministration was demonstrated. In summary, our data demonstrate that the use of a clinically applicable IA procedure enables successful readministration of an rAAV5-based gene transfer in a clinically relevant animal model. Finally, the analysis of anti-AAV NAB levels in human subjects submitted to IA confirmed the safety and efficacy of the procedure to reduce anti-AAV NABs. Furthermore, clinical translation was assessed using an immunoglobulin G assay as surrogate.

scholarly journals Live-Attenuated Measles Virus Vaccine Targets Dendritic Cells and Macrophages in Muscle of Nonhuman Primates

2014 ◽  
Vol 89 (4) ◽  
pp. 2192-2200 ◽  
Author(s):  
Linda J. Rennick ◽  
Rory D. de Vries ◽  
Thomas J. Carsillo ◽  
Ken Lemon ◽  
Geert van Amerongen ◽  
...  
Keyword(s):  
Measles Virus ◽  
Nonhuman Primates ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Vaccine Virus ◽  
Target Cells ◽  
Recombinant Viruses ◽  
Green Fluorescent

ABSTRACTAlthough live-attenuated measles virus (MV) vaccines have been used successfully for over 50 years, the target cells that sustain virus replicationin vivoare still unknown. We generated a reverse genetics system for the live-attenuated MV vaccine strain Edmonston-Zagreb (EZ), allowing recovery of recombinant (r)MVEZ. Three recombinant viruses were generated that contained the open reading frame encoding enhanced green fluorescent protein (EGFP) within an additional transcriptional unit (ATU) at various positions within the genome. rMVEZEGFP(1), rMVEZEGFP(3), and rMVEZEGFP(6) contained the ATU upstream of the N gene, following the P gene, and following the H gene, respectively. The viruses were comparedin vitroby growth curves, which indicated that rMVEZEGFP(1) was overattenuated. Intratracheal infection of cynomolgus macaques with these recombinant viruses revealed differences in immunogenicity. rMVEZEGFP(1) and rMVEZEGFP(6) did not induce satisfactory serum antibody responses, whereas bothin vitroandin vivorMVEZEGFP(3) was functionally equivalent to the commercial MVEZ-containing vaccine. Intramuscular vaccination of macaques with rMVEZEGFP(3) resulted in the identification of EGFP+cells in the muscle at days 3, 5, and 7 postvaccination. Phenotypic characterization of these cells demonstrated that muscle cells were not infected and that dendritic cells and macrophages were the predominant target cells of live-attenuated MV.IMPORTANCEEven though MV strain Edmonston-Zagreb has long been used as a live-attenuated vaccine (LAV) to protect against measles, nothing is known about the primary cells in which the virus replicatesin vivo. This is vital information given the push to move toward needle-free routes of vaccination, since vaccine virus replication is essential for vaccination efficacy. We have generated a number of recombinant MV strains expressing enhanced green fluorescent protein. The virus that best mimicked the nonrecombinant vaccine virus was formulated according to protocols for production of commercial vaccine virus batches, and was subsequently used to assess viral tropism in nonhuman primates. The virus primarily replicated in professional antigen-presenting cells, which may explain why this LAV is so immunogenic and efficacious.

Reply to: Ratios and the cycling status of tissue target cells (by J. H. Hendry and C. S. Potten)

1988 ◽  
Vol 12 (1) ◽  
pp. 85
Author(s):  
T.E. Schultheiss ◽  
G.K. Zagars ◽  
L.J. Peters
Keyword(s):  
Target Cells ◽  
Tissue Target

scholarly journals Structure of Adeno-Associated Virus Type 4

2005 ◽  
Vol 79 (8) ◽  
pp. 5047-5058 ◽  
Author(s):  
Eric Padron ◽  
Valorie Bowman ◽  
Nikola Kaludov ◽  
Lakshmanan Govindasamy ◽  
Hazel Levy ◽  
...  
Keyword(s):  
Major Capsid Protein ◽  
Parvovirus B19 ◽  
Structural Similarity ◽  
Surface Topology ◽  
Target Cells ◽  
Adeno Associated Virus ◽  
Cryo Electron Microscopy ◽  
Specific Tissue ◽  
Surface Loops ◽  
Limited Sequence

ABSTRACT Adeno-associated virus (AAV) is a member of the Parvoviridae, belonging to the Dependovirus genus. Currently, several distinct isolates of AAV are in development for use in human gene therapy applications due to their ability to transduce different target cells. The need to manipulate AAV capsids for specific tissue delivery has generated interest in understanding their capsid structures. The structure of AAV type 4 (AAV4), one of the most antigenically distinct serotypes, was determined to 13-Å resolution by cryo-electron microscopy and image reconstruction. A pseudoatomic model was built for the AAV4 capsid by use of a structure-based sequence alignment of its major capsid protein, VP3, with that of AAV2, to which AAV4 is 58% identical and constrained by its reconstructed density envelope. The model showed variations in the surface loops that may account for the differences in receptor binding and antigenicity between AAV2 and AAV4. The AAV4 capsid surface topology also shows an unpredicted structural similarity to that of Aleutian mink disease virus and human parvovirus B19, autonomous members of the genus, despite limited sequence homology.

Retroviral transduction efficiency of G-CSF+SCF–mobilized peripheral blood CD34+ cells is superior to G-CSF or G-CSF+Flt3-L–mobilized cells in nonhuman primates

Blood ◽  
2003 ◽  
Vol 101 (6) ◽  
pp. 2199-2205 ◽  
Author(s):  
Peiman Hematti ◽  
Stephanie E. Sellers ◽  
Brian A. Agricola ◽  
Mark E. Metzger ◽  
Robert E. Donahue ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Peripheral Blood ◽  
Nonhuman Primates ◽  
Transduction Efficiency ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Retroviral Transduction ◽  
Peripheral Blood Cd34 ◽  
Cd34 Cells ◽  
Clinical Gene Therapy

Gene transfer experiments in nonhuman primates have been shown to be predictive of success in human clinical gene therapy trials. In most nonhuman primate studies, hematopoietic stem cells (HSCs) collected from the peripheral blood or bone marrow after administration of granulocyte colony-stimulating factor (G-CSF) + stem cell factor (SCF) have been used as targets, but this cytokine combination is not generally available for clinical use, and the optimum target cell population has not been systematically studied. In our current study we tested the retroviral transduction efficiency of rhesus macaque peripheral blood CD34+ cells collected after administration of different cytokine mobilization regimens, directly comparing G-CSF+SCF versus G-CSF alone or G-CSF+Flt3-L in competitive repopulation assays. Vector supernatant was added daily for 96 hours in the presence of stimulatory cytokines. The transduction efficiency of HSCs as assessed by in vitro colony-forming assays was equivalent in all 5 animals tested, but the in vivo levels of mononuclear cell and granulocyte marking was higher at all time points derived from target CD34+ cells collected after G-CSF+SCF mobilization compared with target cells collected after G-CSF (n = 3) or G-CSF+Flt3-L (n = 2) mobilization. In 3 of the animals long-term marking levels of 5% to 25% were achieved, but originating only from the G-CSF+SCF–mobilized target cells. Transduction efficiency of HSCs collected by different mobilization regimens can vary significantly and is superior with G-CSF+SCF administration. The difference in transduction efficiency of HSCs collected from different sources should be considered whenever planning clinical gene therapy trials and should preferably be tested directly in comparative studies.

Tumor Therapeutics via Highly Efficient miRNA Delivery to Human Nasopharyngeal Cancer Cells by Multifunctional Magnetic Nanoparticles

2020 ◽  
Vol 12 (1) ◽  
pp. 69-78
Author(s):  
Weimin Wu ◽  
Zhongwen Chen ◽  
Xia Li ◽  
Jianjun Wang ◽  
Lihong Fan ◽  
...  
Keyword(s):  
Nasopharyngeal Cancer ◽  
Cell Counting ◽  
Nuclear Antigen ◽  
Target Cells ◽  
Antitumor Effects ◽  
Efficient Delivery ◽  
Fcm Analysis ◽  
The Stability ◽  
Pei Nanoparticles ◽  
Proliferating Cell

Efficient delivery of miRNA to target cells remains a significant challenge in clinical applications. In this research, we constructed a multifunctional miRNA (miR) delivery system composed of miRexpressing plasmids mediated by Fe3O4-polyethyleneimine (PEI) nanoparticles (NPs). Fe3O4-PEImiR-expressing plasmid NPs targeting HIF-1 or Survivin gene were respectively constructed and transfected into human nasopharyngeal carcinoma (NPC) CNE-II cells. The stability of Fe3O4 PEI-miR NPs was experimentally confirmed by serum protection assay. The antitumor effects of Fe3O4-PEI-miR NPs on CNE-II cells proliferation, apoptosis, and radiosensitivity were studied by employing the Cell Counting Kit (CCK-8) experiment, flow cytometry (FCM) analysis and radiosensitivity test. The antitumor efficiency of Fe3O4-PEI-miR NPs was investigated by qRT-PCR and Western blot. The Fe3O4-PEI-miR-expressing plasmid NPs were shown to be successfully established with favorable stability, enhanced biocompatibility and lower cytotoxicity, compared with Lipofectamine 2000. In addition, down-regulation of HIF-1 or Survivin was validated to improve antitumor effects and radiosensitivity of NPC cells through proliferating cell nuclear antigen (PCNA). Transfection of miR-HIF-1 /Survivin by Fe3O4-PEI NPs to CNE-II cells effectively inhibited NPC cell proliferation, induced cell apoptosis, and increased radiosensitivity. Fe3O4-PEI NPs were shown to be ideal gene carriers capable of novel gene therapy for treating human NPC.

scholarly journals Vehicles for Small Interfering RNA transfection: Exosomes versus Synthetic Nanocarriers

2013 ◽  
Vol 1 (1) ◽  
Author(s):  
Markus Duechler
Keyword(s):  
Small Rnas ◽  
Sirna Delivery ◽  
Target Cells ◽  
Messenger Rnas ◽  
Small Interfering Rnas ◽  
Rna Transfection ◽  
Efficient Delivery ◽  
Micro Rnas ◽  
Delivery Vehicles ◽  
Interfering Rna

AbstractTherapies based on RNA interference (RNAi) hold a great potential for targeted interference of the expression of specific genes. Small-interfering RNAs (siRNA) and micro-RNAs interrupt protein synthesis by inducing the degradation of messenger RNAs or by blocking their translation. RNAibased therapies can modulate the expression of otherwise undruggable target proteins. Full exploitation of RNAi for medical purposes depends on efficient and safe methods for delivery of small RNAs to the target cells. Tremendous effort has gone into the development of synthetic carriers to meet all requirements for efficient delivery of nucleic acids into particular tissues. Recently, exosomes unveiled their function as a natural communication system which can be utilized for the transport of small RNAs into target cells. In this review, the capabilities of exosomes as delivery vehicles for small RNAs are compared to synthetic carrier systems. The step by step requirements for efficient transfection are considered: production of the vehicle, RNA loading, protection against degradation, lack of immunogenicity, targeting possibilities, cellular uptake, cytotoxicity, RNA release into the cytoplasm and gene silencing efficiency. An exosomebased siRNA delivery system shows many advantages over conventional transfection agents, however, some crucial issues need further optimization before broad clinical application can be realized.

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