Methods of Transfection with Messenger RNA Gene Vectors

Non-viral gene delivery vectors with messenger RNA (mRNA) as a carrier of genetic information are among the staple gene transfer vectors for research in gene therapy, gene vaccination and cell fate reprogramming. As no passage of genetic cargo in and out of the nucleus is required, mRNA-based vectors typically offer the following five advantages: 1) fast start of transgene expression; 2) ability to express genes in non-dividing cells with an intact nuclear envelope; 3) insensitivity to the major gene silencing mechanisms, which operate in the nucleus; 4) absence of potentially mutagenic genomic insertions; 5) high cell survival rate after transfection procedures, which do not need to disturb nuclear envelope. In addition, mRNA-based vectors offer a simple combination of various transgenes through mixing of several mRNAs in a single multi-gene cocktail or expression of a number of proteins from a single mRNA molecule using internal ribosome entry sites (IRESes), ribosome skipping sequences and proteolytic signals. However, on the downside, uncontrolled extracellular and intracellular decay of mRNA can be a substantial hurdle for mRNA-mediated gene transfer. Procedures for mRNA delivery are analogous to DNA transfer methods, which are well-established. In general, there are three actors in the gene delivery play, namely, the vector, the cell and the transfer environment. The desired outcome, that is, the efficient delivery of a gene to a target cell population, depends on the efficient interaction of all three parties. Thus, the vector should be customised for the target cell population and presented in a form that is resistant to the aggressive factors in the delivery milieu. At the same time, the delivery environment should be adjusted to be more vector-friendly and more cell-friendly. The recipient cells should be subjected to a specific regimen or artificially modified to become receptive to gene transfer with a particular vector and resistant to the environment. As a rule, barriers outside tissues (e.g. mucus) and an aggressive intercellular environment complicate gene delivery in vivo, which, therefore, requires more complex gene transfer procedures than transfection of tissue culture cells. This review is focused on transfection methods for mRNA vectors, which rely either on the forceful propulsion of mRNA inside the target cells (e.g. by electroporation or gene gun) or on the complexing of mRNA with other substances (e.g. polycationic transfection reagents) for delivery via endocytic pathways

Chronically shortened rod outer segments accompany photoreceptor cell death in Choroideremia

X-linked choroideremia (CHM) is a disease characterized by gradual retinal degeneration caused by loss of the Rab Escort Protein, REP1. Despite partial compensation by REP2 the disease is characterized by prenylation defects in multiple members of the Rab protein family that are master regulators of membrane traffic. Remarkably, the eye is the only organ affected in CHM patients, possibly because of the huge membrane traffic burden of the post mitotic photoreceptors, which synthesise outer segments, and the adjacent retinal pigment epithelium that degrades the spent portions each day. In this study, we aimed to identify defects in membrane traffic that might lead to photoreceptor cell death in CHM. In a heterozygous null female mouse model of CHM (Chmnull/WT), degeneration of the photoreceptor layer was clearly evident from increased numbers of TUNEL positive cells compared to age matched controls, small numbers of cells exhibiting signs of mitochondrial stress and greatly increased microglial infiltration. However, most rod photoreceptors exhibited remarkably normal morphology with well-formed outer segments and no discernible accumulation of transport vesicles in the inner segment. The major evidence of membrane trafficking defects was a shortening of rod outer segments that was evident at 2 months of age but remained constant over the period during which the cells die. A decrease in rhodopsin density found in the outer segment may underlie the outer segment shortening but does not lead to rhodopsin accumulation in the inner segment. Our data argue against defects in rhodopsin transport or outer segment renewal as triggers of cell death in CHM.publishersversionpublishe

Rab27a Contributes to the Processing of Inflammatory Pain in Mice

Tissue injury and inflammation may result in chronic pain, a severe debilitating disease that is associated with great impairment of quality of life. An increasing body of evidence indicates that members of the Rab family of small GTPases contribute to pain processing; however, their specific functions remain poorly understood. Here, we found using immunofluorescence staining and in situ hybridization that the small GTPase Rab27a is highly expressed in sensory neurons and in the superficial dorsal horn of the spinal cord of mice. Rab27a mutant mice, which carry a single-nucleotide missense mutation of Rab27a leading to the expression of a nonfunctional protein, show reduced mechanical hyperalgesia and spontaneous pain behavior in inflammatory pain models, while their responses to acute noxious mechanical and thermal stimuli is not affected. Our study uncovers a previously unrecognized function of Rab27a in the processing of persistent inflammatory pain in mice.publishersversionpublishe

Chromosomal mapping, gene structure and characterization of the human and murine RAB27B gene

BACKGROUND: Rab GTPases are regulators of intracellular membrane traffic. The Rab27 subfamily consists of Rab27a and Rab27b. Rab27a has been recently implicated in Griscelli Disease, a disease combining partial albinism with severe immunodeficiency. Rab27a plays a key role in the function of lysosomal-like organelles such as melanosomes in melanocytes and lytic granules in cytotoxic T lymphocytes. Little is known about Rab27b. RESULTS: The human RAB27B gene is organised in six exons, spanning about 69 kb in the chromosome 18q21.1 region. Exon 1 is non-coding and is separated from the others by 49 kb of DNA and exon 6 contains a long 3' untranslated sequence (6.4 kb). The mouse Rab27b cDNA shows 95% identity with the human cDNA at the protein level and maps to mouse chromosome 18. The mouse mRNA was detected in stomach, large intestine, spleen and eye by RT-PCR, and in heart, brain, spleen and kidney by Northern blot. Transient over-expression of EGF-Rab27b fusion protein in cultured melanocytes revealed that Rab27b is associated with melanosomes, as observed for EGF-Rab27a. CONCLUSIONS: Our results indicate that the Rab27 subfamily of Ras-like GTPases is highly conserved in mammals. There is high degree of conservation in sequence and gene structure between RAB27A and RAB27B genes. Exogenous expression of Rab27b in melanocytes results in melanosomal association as observed for Rab27a, suggesting the two Rab27 proteins are functional homologues. As with RAB27A in Griscelli Disease, RAB27B may be also associated with human disease mapping to chromosome 18

Functional expression of Rab escort protein 1 following AAV2-mediated gene delivery in the retina of choroideremia mice and human cells ex vivo

Choroideremia (CHM) is an X-linked retinal degeneration of photoreceptors, the retinal pigment epithelium (RPE) and choroid caused by loss of function mutations in the CHM/REP1 gene that encodes Rab escort protein 1. As a slowly progressing monogenic retinal degeneration with a clearly identifiable phenotype and a reliable diagnosis, CHM is an ideal candidate for gene therapy. We developed a serotype 2 adeno-associated viral vector AAV2/2-CBA-REP1, which expresses REP1 under control of CMV-enhanced chicken β-actin promoter (CBA) augmented by a Woodchuck hepatitis virus post-transcriptional regulatory element. We show that the AAV2/2-CBA-REP1 vector provides strong and functional transgene expression in the D17 dog osteosarcoma cell line, CHM patient fibroblasts and CHM mouse RPE cells in vitro and in vivo. The ability to transduce human photoreceptors highly effectively with this expression cassette was confirmed in AAV2/2-CBA-GFP transduced human retinal explants ex vivo. Electroretinogram (ERG) analysis of AAV2/2-CBA-REP1 and AAV2/2-CBA-GFP-injected wild-type mouse eyes did not show toxic effects resulting from REP1 overexpression. Subretinal injections of AAV2/2-CBA-REP1 into CHM mouse retinas led to a significant increase in a- and b-wave of ERG responses in comparison to sham-injected eyes confirming that AAV2/2-CBA-REP1 is a promising vector suitable for choroideremia gene therapy in human clinical trials. © 2013 The Author(s)

Methods of Transfection with Messenger RNA Gene Vectors

Abstract Non-viral gene delivery vectors with messenger RNA (mRNA) as a carrier of genetic information are among the staple gene transfer vectors for research in gene therapy, gene vaccination and cell fate reprogramming. As no passage of genetic cargo in and out of the nucleus is required, mRNA-based vectors typically offer the following five advantages: 1) fast start of transgene expression; 2) ability to express genes in nondividing cells with an intact nuclear envelope; 3) insensitivity to the major gene silencing mechanisms, which operate in the nucleus; 4) absence of potentially mutagenic genomic insertions; 5) high cell survival rate after transfection procedures, which do not need to disturb nuclear envelope. In addition, mRNA-based vectors offer a simple combination of various transgenes through mixing of several mRNAs in a single multi-gene cocktail or expression of a number of proteins from a single mRNA molecule using internal ribosome entry sites (IRESes), ribosome skipping sequences and proteolytic signals. However, on the downside, uncontrolled extracellular and intracellular decay of mRNA can be a substantial hurdle for mRNAmediated gene transfer. Procedures for mRNA delivery are analogous to DNA transfer methods, which are well-established. In general, there are three actors in the gene delivery play, namely, the vector, the cell and the transfer environment. The desired outcome, that is, the efficient delivery of a gene to a target cell population, depends on the efficient interaction of all three parties. Thus, the vector should be customised for the target cell population and presented in a form that is resistant to the aggressive factors in the delivery milieu. At the same time, the delivery environment should be adjusted to be more vector-friendly and more cell-friendly. The recipient cells should be subjected to a specific regimen or artificially modified to become receptive to gene transfer with a particular vector and resistant to the environment. As a rule, barriers outside tissues (e.g. mucus) and an aggressive © 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. intercellular environment complicate gene delivery in vivo, which, therefore, requires more complex gene transfer procedures than transfection of tissue culture cells. This review is focused on transfection methods for mRNA vectors, which rely either on the forceful propulsion of mRNA inside the target cells (e.g. by electroporation or gene gun) or on the complexing of mRNA with other substances (e.g. polycationic transfection reagents) for delivery via endocytic pathways