The Design and Exogenous Delivery of siRNA for Post-transcriptional Gene Silencing

Gilmore, Ian R.; Fox, Stephen; Hollins, Andrew John; Sohail, Muhammad; Akhtar, Saghir

doi:10.1080/10611860400006257

Cited by 67 publications

(52 citation statements)

References 147 publications

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“…Once targeted to the complementary mRNA, the RNase activity of the complex degrades the message [Hutvagner and Zamore, 2002;Haley and Zamore, 2004;Martinez and Tuschl, 2004]. However, only *10-30% of the siRNAs targeted against a single mRNA are effective in triggering mRNA degradation [Gilmore et al, 2004;Tomari and Zamore, 2005]. Therefore, we chose to target N-RAP for knockdown using a cocktail of siRNAs generated by in vitro transcription and cleavage by RNase.…”

Section: Specificity and Time Course Of N-rap Knockdown By Rnaimentioning

confidence: 99%

Targeted disruption of N-RAP gene function by RNA interference: A role for N-RAP in myofibril organization

Dhume

Horowits

2006

Cell Motil. Cytoskeleton

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N-RAP is a muscle-specific protein concentrated in myofibril precursors during sarcomere assembly and at intercalated disks in adult heart. We used RNA interference to achieve a targeted decrease in N-RAP transcript and protein levels in primary cultures of embryonic mouse cardiomyocytes. N-RAP transcript levels were decreased by approximately 70% within 2 days following transfection with N-RAP specific siRNA. N-RAP protein levels steadily decreased over several days, reaching approximately 50% of control levels within 6 days. N-RAP protein knockdown was associated with decreased myofibril assembly, as assessed by alpha-actinin organization into mature striations. Transcripts encoding N-RAP binding proteins associated with assembling or mature myofibrils, such as alpha-actinin, Krp1, and muscle LIM protein, were expressed at normal levels during N-RAP protein knockdown, and alpha-actinin and Krp-1 protein levels were also unchanged. Transcripts encoding muscle myosin heavy chain and nonmuscle myosin heavy chain IIB were also expressed at relatively normal levels. However, decreased N-RAP protein levels were associated with dramatic changes in the encoded myosin proteins, with muscle myosin heavy chain levels increasing and nonmuscle myosin heavy chain IIB decreasing. N-RAP transcript and protein levels recovered to normal by days 6 and 7, respectively, and the changes in myofibril organization and myosin heavy chain isoform levels were reversed. Our data indicate that we can achieve transient N-RAP protein knockdown using the RNA interference technique and that alpha-actinin organization into myofibrils in cardiomyocytes is closely linked to N-RAP protein levels. Finally, N-RAP protein levels regulate the balance between nonmuscle myosin IIB and muscle myosin by post-trancriptional mechanisms.

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Section: Specificity and Time Course Of N-rap Knockdown By Rnaimentioning

confidence: 99%

Targeted disruption of N-RAP gene function by RNA interference: A role for N-RAP in myofibril organization

Dhume

Horowits

2006

Cell Motil. Cytoskeleton

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“…Cationic polymers readily bind and condense polyanionic nucleic acids and have thus been widely used as transfection reagents for genes, oligonucleotides, and siRNAs (34)(35)(36)(37).…”

Section: Cationic Polymeric Delivery Systemsmentioning

confidence: 99%

Efficient siRNA Delivery with Non-viral Polymeric Vehicles

2008

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Sequence-specific gene silencing using small interfering RNA (siRNA) provides a potent and specific method for gene expression, thus is now being evaluated in clinical trials as a novel therapeutic strategy. As a results, there has been a significant surge of interest in the application of siRNA in therapeutics as a means of silencing the specific gene function. However, for siRNA technology to be valuable and effective, the development of efficient siRNA delivery strategy is essential for improving biological activities such as stability, cellular uptake, sequence-specificity, devoid of nonspecific knockdown and toxic side effects. Accordingly, a number of delivery systems, both viral and nonviral, have been reported and some of them successfully used for the introduction of siRNA into cells both in vitro and in vivo. Here, we discuss the current understanding of synthetic siRNA delivery mechanism and strategies of siRNA delivery by non-viral polymeric vehicles which are currently used in vitro and in vivo.

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“…Indeed several different types of cationic synthetic vectors have been investigated for gene therapy applications including lipids/liposomes, polymers, dendrimers (branched-like polymer structures) and cell-penetrating peptides ( Figure 1a). 2,3 The complexes that synthetic vectors form with DNA are often specifically referred to as lipoplexes, dendriplexes or polyplexes depending on whether the vector used is a cationic lipid, dendrimer or polymer, respectively. A vesicular transport system (endocytosis/macropinocytosis) generally takes these complexes into the cell and they are subsequently transported into the nucleus where the delivered gene is expressed.…”

mentioning

confidence: 99%

Non-viral cancer gene therapy: Beyond delivery

Akhtar

2005

Gene Ther

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The Design and Exogenous Delivery of siRNA for Post-transcriptional Gene Silencing

Cited by 67 publications

References 147 publications

Targeted disruption of N-RAP gene function by RNA interference: A role for N-RAP in myofibril organization

Targeted disruption of N-RAP gene function by RNA interference: A role for N-RAP in myofibril organization

Efficient siRNA Delivery with Non-viral Polymeric Vehicles

Non-viral cancer gene therapy: Beyond delivery

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