Faculty Opinions recommendation of Efficient in-vitro transfer of a 60-Mb mammalian artificial chromosome into murine and hamster cells using cationic lipids and dendrimers.

OBJECTIVES/SPECIFIC AIMS: Despite aggressive chemotherapy, surgical resection, and radiation therapy, glioblastoma remains almost universally fatal. In a pilot, randomized, and blinded clinical trial, we recently demonstrated that administration of RNA-loaded DC vaccines was associated with significantly improved progression-free and overall survival in patients with glioblastoma (Mitchell et al., Nature, 2015). Furthermore, clinical outcomes correlated with DC migration to vaccine-site draining lymph nodes measured by Indium-111 labeling of RNA-loaded DCs and SPECT/CT imaging. Although these studies demonstrated that tracking DC migration may be an important clinical biomarker for response to DC vaccination, the complexity and regulatory requirements associated with nuclear labelling to track DC migration limits widespread application of this technique. We have therefore developed RNA-loaded magnetic nanoparticles (RNA-NPs) to enhance DC migration to LNs and track that migration with a widely available imaging modality (i.e., MRI). METHODS/STUDY POPULATION: Cationic liposomes were loaded with iron oxide nanoparticles with or without cholesterol. The resulting nanoparticles were complexed with RNA and used to transfect DCs ex vivo. RNA-NP-loaded DsRed+ DCs were then injected intradermally into mice and tracked noninvasively with T2-weighted 11T MRI before excision and quantification with flow cytometry. RESULTS/ANTICIPATED RESULTS: In vitro experiments demonstrate that iron oxide loading does not reduce RNA-NP-mediated transfection of DCs. Additionally, replacement of cationic lipids with cholesterol increased RNA-NP transfection of the DC2.4 cell line and enhanced the T cell stimulatory capacity of treated bone marrow-derived dendritic cells (BMDCs). Compared to electroporation, RNA-NPs enhanced DC migration to lymph nodes and reduced T2 MRI intensity in DC-bearing lymph nodes. DISCUSSION/SIGNIFICANCE OF IMPACT: This data suggests that iron oxide-loaded RNA-NPs enable noninvasive cell tracking with MRI and enhance DC migration to lymph nodes. We have further shown that inclusion of cholesterol in RNA-NPs augments the stimulatory capacity of transfected DCs. Future work will consider effects of RNA-NPs on antitumor immune responses and the utility of MRI-detected DC migration as a biomarker of vaccine efficacy.

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Сationic liposomes as delivery systems for nucleic acids

Тонкие химические технологии ◽

10.32362/2410-6593-2020-15-1-7-27 ◽

2020 ◽

Vol 15 (1) ◽

pp. 7-27

Author(s):

A. A. Mikheev ◽

E. V. Shmendel ◽

E. S. Zhestovskaya ◽

G. V. Nazarov ◽

M. A. Maslov

Keyword(s):

Gene Therapy ◽

Nucleic Acids ◽

Serum Proteins ◽

Transfection Efficiency ◽

Biological Fluids ◽

Genetic Material ◽

Cationic Liposomes ◽

Delivery Systems ◽

Cationic Lipids

Objectives. Gene therapy is based on the introduction of genetic material into cells, tissues, or organs for the treatment of hereditary or acquired diseases. A key factor in the success of gene therapy is the development of delivery systems that can efficiently transfer genetic material to the place of their therapeutic action without causing any associated side effects. Over the past 10 years, significant effort has been directed toward creating more efficient and biocompatible vectors capable of transferring nucleic acids (NAs) into cells without inducing an immune response. Cationic liposomes are among the most versatile tools for delivering NAs into cells; however, the use of liposomes for gene therapy is limited by their low specificity. This is due to the presence of various biological barriers to the complex of liposomes with NA, including instability in biological fluids, interaction with serum proteins, plasma and nuclear membranes, and endosomal degradation. This review summarizes the results of research in recent years on the development of cationic liposomes that are effective in vitro and in vivo. Particular attention is paid to the individual structural elements of cationic liposomes that determine the transfection efficiency and cytotoxicity. The purpose of this review was to provide a theoretical justification of the most promising choice of cationic liposomes for the delivery of NAs into eukaryotic cells and study the effect of the composition of cationic lipids (CLs) on the transfection efficiency in vitro.Results. As a result of the analysis of the related literature, it can be argued that one of the most promising delivery systems of NAs is CL based on cholesterol and spermine with the addition of a helper lipid DOPE. In addition, it was found that varying the composition of cationic liposomes, the ratio of CL to NA, or the size and zeta potential of liposomes has a significant effect on the transfection efficiency.Conclusions. Further studies in this direction should include optimization of the conditions for obtaining cationic liposomes, taking into account the physicochemical properties and established laws. It is necessary to identify mechanisms that increase the efficiency of NA delivery in vitro by searching for optimal structures of cationic liposomes, determining the ratio of lipoplex components, and studying the delivery efficiency and properties of multicomponent liposomes.

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α‐Tocopherol‐Conjugated, Open Chain Sugar‐Mimicking Cationic Lipids: Design, Synthesis and In–Vitro Gene Transfection Properties

ChemistrySelect ◽

10.1002/slct.202102648 ◽

2021 ◽

Vol 6 (46) ◽

pp. 13025-13033

Author(s):

Venkatesh Ravula ◽

Venkanna Muripiti ◽

Shireesha Manthurthi ◽

Srilakshmi V. Patri

Keyword(s):

Gene Transfection ◽

Cationic Lipids ◽

Open Chain ◽

Design Synthesis

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Development of Caco-2 cells expressing four CYPs via a mammalian artificial chromosome

BMC Biotechnology ◽

10.1186/s12896-020-00637-8 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Yumi Ohta ◽

Kanako Kazuki ◽

Satoshi Abe ◽

Mitsuo Oshimura ◽

Kaoru Kobayashi ◽

...

Keyword(s):

Artificial Chromosome ◽

Mammalian Artificial Chromosome

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Dramatic influence of the hydroxy functionality of azasugar moiety in the head group region of tocopherol-based cationic lipids on in vitro gene transfection efficacies

New Journal of Chemistry ◽

10.1039/d0nj03717f ◽

2021 ◽

Author(s):

Venkanna Muripiti ◽

Brijesh Lohchania ◽

Venkatesh Ravula ◽

Shireesha Manturthi ◽

Srujan Marepally ◽

...

Keyword(s):

Gene Transfection ◽

Cationic Lipids ◽

Head Group ◽

Nonviral Vectors

Cationic lipids have been effectively used as nonviral vectors for the delivery of polynucleic acids into the cytosol.

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MyD88-5 links mitochondria, microtubules, and JNK3 in neurons and regulates neuronal survival

Journal of Experimental Medicine ◽

10.1084/jem.20070868 ◽

2007 ◽

Vol 204 (9) ◽

pp. 2063-2074 ◽

Cited By ~ 124

Author(s):

Younghwa Kim ◽

Ping Zhou ◽

Liping Qian ◽

Jen-Zen Chuang ◽

Jessica Lee ◽

...

Keyword(s):

Hippocampal Neurons ◽

Artificial Chromosome ◽

Adaptor Proteins ◽

Innate Immune ◽

Molecular Structures ◽

Endogenous Expression ◽

Microbial Products ◽

The Brain ◽

Deficient Mice

The innate immune system relies on evolutionally conserved Toll-like receptors (TLRs) to recognize diverse microbial molecular structures. Most TLRs depend on a family of adaptor proteins termed MyD88s to transduce their signals. Critical roles of MyD88-1–4 in host defense were demonstrated by defective immune responses in knockout mice. In contrast, the sites of expression and functions of vertebrate MyD88-5 have remained elusive. We show that MyD88-5 is distinct from other MyD88s in that MyD88-5 is preferentially expressed in neurons, colocalizes in part with mitochondria and JNK3, and regulates neuronal death. We prepared MyD88-5/GFP transgenic mice via a bacterial artificial chromosome to preserve its endogenous expression pattern. MyD88-5/GFP was detected chiefly in the brain, where it associated with punctate structures within neurons and copurified in part with mitochondria. In vitro, MyD88-5 coimmunoprecipitated with JNK3 and recruited JNK3 from cytosol to mitochondria. Hippocampal neurons from MyD88-5–deficient mice were protected from death after deprivation of oxygen and glucose. In contrast, MyD88-5–null macrophages behaved like wild-type cells in their response to microbial products. Thus, MyD88-5 appears unique among MyD88s in functioning to mediate stress-induced neuronal toxicity.

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Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones

Journal of Biomedicine and Biotechnology ◽

10.1155/2012/357147 ◽

2012 ◽

Vol 2012 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Kalpana Dulal ◽

Benjamin Silver ◽

Hua Zhu

Keyword(s):

Bacterial Artificial Chromosome ◽

Homologous Recombination ◽

Human Cytomegalovirus ◽

Artificial Chromosome ◽

Bac Clone ◽

Dna Viruses ◽

E Coli ◽

Recombination System ◽

Capture Method

Bacterial artificial chromosome (BAC) technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV). The HCMV BAC clone propagated and maintained insideE. coliallows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method inE. colifor molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects forin vitrohomologous recombination for genetic cloning.

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