scholarly journals In vivo self-assembled small RNAs as a new generation of RNAi therapeutics

Cell Research ◽  
2021 ◽  
Author(s):  
Zheng Fu ◽  
Xiang Zhang ◽  
Xinyan Zhou ◽  
Uzair Ur-Rehman ◽  
Mengchao Yu ◽  
...  
Keyword(s):  
Self Assembly ◽  
Direct Injection ◽  
Sirna Delivery ◽  
Genetic Circuit ◽  
Genetic Circuits ◽  
Rnai Therapeutics ◽  
Two Stages ◽  
New Generation ◽  
Rnai Therapy

AbstractRNAi therapy has undergone two stages of development, direct injection of synthetic siRNAs and delivery with artificial vehicles or conjugated ligands; both have not solved the problem of efficient in vivo siRNA delivery. Here, we present a proof-of-principle strategy that reprogrammes host liver with genetic circuits to direct the synthesis and self-assembly of siRNAs into secretory exosomes and facilitate the in vivo delivery of siRNAs through circulating exosomes. By combination of different genetic circuit modules, in vivo assembled siRNAs are systematically distributed to multiple tissues or targeted to specific tissues (e.g., brain), inducing potent target gene silencing in these tissues. The therapeutic value of our strategy is demonstrated by programmed silencing of critical targets associated with various diseases, including EGFR/KRAS in lung cancer, EGFR/TNC in glioblastoma and PTP1B in obesity. Overall, our strategy represents a next generation RNAi therapeutics, which makes RNAi therapy feasible.

scholarly journals In vivo self-assembled siRNA: a new modality for combination therapy of ulcerative colitis

2021 ◽  
Author(s):  
XinYan Zhou ◽  
Mengchao Yu ◽  
JinYu Fu ◽  
Luzhen Ma ◽  
Jingwei Guo ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Combination Therapy ◽  
Self Assembly ◽  
Intestinal Inflammation ◽  
Genetic Circuit ◽  
Complex Nature ◽  
Genetic Circuits ◽  
Tnf Α ◽  
Integrin Α4

Abstract Given the complex nature of ulcerative colitis (UC), combination therapy targeting multiple pathogenic genes and pathways of UC may be required. Unfortunately, current therapeutic strategies based on independent chemical compounds or monoclonal antibodies are not applicable for combination therapy of UC. Here, we developed a synthetic biology strategy that integrates the naturally existing exosome-circulating system with artificial genetic circuits for self-assembly and delivery of multiple siRNAs for the combination therapy of UC. Intravenous injection of genetic circuit (in the form of DNA plasmid) designed for inhibition of TNF-α, B7-1 and integrin α4 successfully reprogrammed the host liver to direct the self-assembly of TNF-α, B7-1 and integrin α4 siRNA into secretory exosomes. The multitargeted genetic circuit could rapidly relieved intestinal inflammation and exert a synergistic therapeutic effect against UC through suppressing the proinflammatory cascade in colonic macrophages, inhibiting the costimulatory signal to T cells and blocking T cell homing to sites of inflammation. More importantly, we designed an AAV-driven genetic circuit to induce substantial and lasting inhibition of TNF-α, B7-1 and integrin α4. Overall, this study established a feasible combination therapeutic strategy for UC, which is superior to the conventional biological therapies requiring.

An Autonomous In Vivo Dual Selection Protocol for Boolean Genetic Circuits

2015 ◽  
Vol 21 (2) ◽  
pp. 247-260 ◽  
Author(s):  
David Beneš ◽  
Petr Sosík ◽  
Alfonso Rodríguez-Patón
Keyword(s):  
Rational Design ◽  
Selection Process ◽  
Logic Gates ◽  
Genetic Circuit ◽  
Evolutionary Engineering ◽  
Genetic Circuits ◽  
E Coli ◽  
Search And Selection ◽  
Selection Of

Success in synthetic biology depends on the efficient construction of robust genetic circuitry. However, even the direct engineering of the simplest genetic elements (switches, logic gates) is a challenge and involves intense lab work. As the complexity of biological circuits grows, it becomes more complicated and less fruitful to rely on the rational design paradigm, because it demands many time-consuming trial-and-error cycles. One of the reasons is the context-dependent behavior of small assembly parts (like BioBricks), which in a complex environment often interact in an unpredictable way. Therefore, the idea of evolutionary engineering (artificial directed in vivo evolution) based on screening and selection of randomized combinatorial genetic circuit libraries became popular. In this article we build on the so-called dual selection technique. We propose a plasmid-based framework using toxin-antitoxin pairs together with the relaxase conjugative protein, enabling an efficient autonomous in vivo evolutionary selection of simple Boolean circuits in bacteria (E. coli was chosen for demonstration). Unlike previously reported protocols, both on and off selection steps can run simultaneously in various cells in the same environment without human intervention; and good circuits not only survive the selection process but are also horizontally transferred by conjugation to the neighbor cells to accelerate the convergence rate of the selection process. Our directed evolution strategy combines a new dual selection method with fluorescence-based screening to increase the robustness of the technique against mutations. As there are more orthogonal toxin-antitoxin pairs in E. coli, the approach is likely to be scalable to more complex functions. In silico experiments based on empirical data confirm the high search and selection capability of the protocol.

scholarly journals A Fluorescent Split Aptamer for Visualizing RNA-RNA Assembly In Vivo

2017 ◽  
Author(s):  
Khalid K. Alam ◽  
Kwaku D. Tawiah ◽  
Matthew F. Lichte ◽  
David Porciani ◽  
Donald H. Burke
Keyword(s):  
Self Assembly ◽  
Living Cells ◽  
Rna Aptamers ◽  
Genetic Circuits ◽  
Fluorescent Reporter ◽  
Indirect Measures ◽  
Reporter Proteins ◽  
Circuit Function

AbstractRNA-RNA assembly governs key biological processes and is a powerful tool for engineering synthetic genetic circuits. Characterizing RNA assembly in living cells often involves monitoring fluorescent reporter proteins, which are at best indirect measures of underlying RNA-RNA hybridization events and are subject to additional temporal and load constraints associated with translation and activation of reporter proteins. In contrast, RNA aptamers that sequester small molecule dyes and activate their fluorescence are increasingly utilized in genetically-encoded strategies to report on RNA-level events. Split-aptamer systems have been rationally designed to generate signal upon hybridization of two or more discrete RNA transcripts, but none directly function when expressed in vivo. We reasoned that the improved physiological properties of the Broccoli aptamer enable construction of a split-aptamer system that could function in living cells. Here we present the Split-Broccoli system, in which self-assembly is nucleated by a thermostable, three-way junction RNA architecture and fluorescence activation requires both strands. Functional assembly of the system approximately follows second order kinetics in vitro and improves when cotranscribed, rather than when assembled from purified components. Split-Broccoli fluorescence is digital in vivo and retains functional modularity when fused to RNAs that regulate circuit function through RNA-RNA hybridization, as demonstrated with an RNA Toehold switch. Split-Broccoli represents the first functional split-aptamer system to operate in vivo. It offers a genetically-encoded and nondestructive platform to monitor and exploit RNA-RNA hybridization, whether as an all-RNA, stand-alone AND gate or as a tool for monitoring assembly of RNA-RNA hybrids.

scholarly journals A new concept in minimally invasive embryo transfer

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Jarosław Wieczorek ◽  
Ewa Stodolak-Zych ◽  
Krzysztof Okoń ◽  
Jurij Kosejuk ◽  
Magdalena Bryła ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Material Properties ◽  
Histopathological Examination ◽  
Second Stage ◽  
Embryo Cultures ◽  
Low Efficiency ◽  
Two Stages ◽  
New Generation

AbstractConsiderable variation in embryo transfer (ET) catheter types, diverging opinions on their quality and functionality, complications following the insertion of catheters, low efficiency of the application of ET methods in humans, and their widely varying efficiency in animals demonstrate the need to improve ET methods and to look for new types of catheters. Such an opportunity is offered by the introduction of catheters made of new-generation biomaterials. This study was aimed to introduce a new generation of biomaterials into reproductive biotechnology. New-generation materials were compared with materials that have been used for many years, and the functionality of newly produced catheters was compared in vivo. Five types of biomaterials were tested: polycaprolactone (PCL), dibutyryl chitin (DBC), polypropylene (PP), polyethylene (PE) and polylactide (PLA). The study was carried out in two stages. Firstly, the basic utility parameters such as geometric stability, surface structure and catheter resistance were evaluated. Subsequently, the biocompatibility of selected biomaterials in embryo cultures was examined, and the development potential of the obtained blastocysts was evaluated. In the second stage, in in vivo with live animals, the biomaterials were tested for biocompatibility and the obtained catheters were examined for their ET functionality. Efficiency with the use of the newly produced catheters was determined, the quality of the blastocysts obtained after embryo culture in the uterus was assessed, and oviducts were subjected to histopathological examination after embryo transfer. Of the tested biomaterials, only polyethylene (PE) showed adequate biological and material properties and proved suitable for production of ET catheters.

scholarly journals siRNA Delivery: Mastering Dendrimer Self-Assembly for Efficient siRNA Delivery: From Conceptual Design to In Vivo Efficient Gene Silencing (Small 27/2016)

Small ◽  
2016 ◽  
Vol 12 (27) ◽  
pp. 3604-3604 ◽  
Author(s):  
Chao Chen ◽  
Paola Posocco ◽  
Xiaoxuan Liu ◽  
Qiang Cheng ◽  
Erik Laurini ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Conceptual Design ◽  
Self Assembly ◽  
Sirna Delivery ◽  
Efficient Gene

scholarly journals Rekindling RNAi Therapy: Materials Design Requirements for In Vivo siRNA Delivery

2019 ◽  
Vol 31 (49) ◽  
pp. 1903637 ◽  
Author(s):  
Byungji Kim ◽  
Ji‐Ho Park ◽  
Michael J. Sailor
Keyword(s):  
Sirna Delivery ◽  
Materials Design ◽  
Design Requirements ◽  
Rnai Therapy

scholarly journals Mastering Dendrimer Self-Assembly for Efficient siRNA Delivery: From Conceptual Design to In Vivo Efficient Gene Silencing

Small ◽  
2016 ◽  
Vol 12 (27) ◽  
pp. 3667-3676 ◽  
Author(s):  
Chao Chen ◽  
Paola Posocco ◽  
Xiaoxuan Liu ◽  
Qiang Cheng ◽  
Erik Laurini ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Conceptual Design ◽  
Self Assembly ◽  
Sirna Delivery ◽  
Efficient Gene

Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

1978 ◽  
Vol 36 (2) ◽  
pp. 650-651
Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo
Keyword(s):  
Direct Injection ◽  
Skin Pigmentation ◽  
Freeze Fracture ◽  
Pigment Granule ◽  
Time Lapse ◽  
Ultrastructural Level ◽  
Lanthanum Tracer ◽  
A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

2018 ◽  
Author(s):  
Noor H. Dashti ◽  
Rufika S. Abidin ◽  
Frank Sainsbury
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Cell Engineering ◽  
Particle Analysis ◽  
Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both <i>in vitro</i> and <i>in vivo</i> cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for <i>in vivo</i> self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by <i>in vitro</i> self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

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