Selective gene silencing in activated leukocytes by targeting siRNAs to the integrin lymphocyte function-associated antigen-1

Peer, Dan; Zhu, Pengcheng; Carman, Christopher V.; Lieberman, Judy; Shimaoka, Motomu

doi:10.1073/pnas.0608491104

Cited by 258 publications

(204 citation statements)

References 41 publications

(48 reference statements)

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“…52 Antibody-protamine fusion proteins constructed using scFvs that bind to the lymphocyte function-associated antigen-1 integrin, which is expressed exclusively and universally on all leukocytes, can be used for selective targeting of leukocytes both in vitro and in vivo. 53 A fusion protein constructed with an scFv that recognizes all conformations of lymphocyte function-associated antigen-1 delivers siRNAs to both resting and activated leukocytes, whereas an scFv that only binds to the activated conformation selectively targets only activated leukocytes, potentially providing a way to manipulate unwanted immune activation without causing global immunosuppression. Importantly, these fusion protein-siRNA complexes do not activate the cells they transfect or induce innate immunity.…”

Section: Introductionmentioning

confidence: 99%

Special delivery: targeted therapy with small RNAs

2011

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Harnessing RNA interference using small RNA-based drugs has great potential to develop drugs designed to knock down expression of any disease-causing gene, thereby greatly expanding the universe of possible drug targets. However, delivering small RNAs into specific tissues and cells is still a hurdle. Here, we review recent progress in overcoming systemic, local and cellular barriers to RNA drug delivery, focusing on strategies for targeted uptake.

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Section: Introductionmentioning

confidence: 99%

Special delivery: targeted therapy with small RNAs

2011

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“…Although lymphocytes can be transfected by electroporation in vitro, this method so far has been rather inefficient, limited to activated cells and was complicated by a severe impairment of cell function and cell viability [9,10]. Recently, a promising approach for the in vivo targeting of lymphocytes has been reported using antibody-protamine fusion proteins to deliver siRNA [11]. However, recent data from small-hairpin RNA transgenic mice indicate that in T lymphocytes the RNAi machinery itself works inefficiently as compared with other cell types [12].…”

Section: Introductionmentioning

confidence: 99%

“…in vivo targeting of lymphocytes has been reported using antibody-protamine fusion proteins to deliver siRNA [11]. However, recent data from small-hairpin RNA transgenic mice indicate that in T lymphocytes the RNAi machinery itself works inefficiently as compared with other cell types [12].…”

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confidence: 99%

siRNA stabilization prolongs gene knockdown in primary T lymphocytes

et al. 2008

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RNA interference (RNAi)-mediated knockdown of target gene expression represents a powerful approach for functional genomics and therapeutic applications. However, for T lymphocytes, central regulators of immunity and immunopathologies, the application of RNAi has been limited due to the lack of efficient small interfering RNA (siRNA) delivery protocols, and an inherent inefficiency of the RNAi machinery itself. Here, we use nucleofection, an optimized electroporation approach, to deliver siRNA into primary T lymphocytes with high efficiency and negligible impairment of cell function. We identify siRNA stability within the cells as the critical parameter for efficient RNAi in primary T cells. While generally short-lived and immediately lost upon T-cell activation when conventional siRNA is used, target gene knockdown becomes insensitive to cell activation and can persist for up to 2 wk in non-dividing cells with siRNA stabilized by chemical modifications. Targeting CD4 and the transcription factor GATA-3, we show that the use of stabilized siRNA is imperative for functional gene analysis during T lymphocyte activation and differentiation in vitro as well as in vivo.Key words: Gene expression . Immune regulation . T cells Introduction RNA interference (RNAi) is an evolutionarily conserved process by which double-stranded small interfering RNA (siRNA) induces sequence-specific, post-transcriptional gene silencing [1][2][3]. Given its ease of application, its high efficiency and remarkable specificity, RNAi holds great promise for broad in vitro and in vivo application in all biomedical areas. Most importantly, RNAi is directly applicable to essentially all somatic cell types including human primary cells, which makes it an invaluable tool to study human gene function and may enable new therapeutic approaches [4,5].Within the immune system T lymphocytes are one major target for siRNA-based gene silencing, since they are key regulators of immune responses and involved in many immune related disorders, like autoimmunity, chronic inflammation or lymphoma.Despite this high potential, the lack of protocols for efficient and sustained siRNA delivery into primary mammalian cells is currently the major obstacle to the use of RNAi. In particular, primary lymphocytes are highly resistant to non-viral transfection using cationic lipids and polymer reagents [6][7][8]. Although lymphocytes can be transfected by electroporation in vitro, this method so far has been rather inefficient, limited to activated cells and was complicated by a severe impairment of cell function and cell viability [9,10]. Recently, a promising approach for the 2616in vivo targeting of lymphocytes has been reported using antibody-protamine fusion proteins to deliver siRNA [11]. However, recent data from small-hairpin RNA transgenic mice indicate that in T lymphocytes the RNAi machinery itself works inefficiently as compared with other cell types [12]. In fact, due to the aforementioned problems with siRNA delivery, the parameters determining RNAi efficien...

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“…Integrins, adhesion molecules that bind to components of the extracellular matrix and act as sensor/signalling molecules, can also be used as targeting moieties [68][69][70] . In particular, the integrin lymphocyte function-associated antigen-1 (LFA-1) expressed on all leukocytes, which mediates heterotypic adhesion to intercellular adhesion molecules on other immune cells, undergoes remarkable conformational changes in response to stimulation [68] .…”

Section: Serum Stabilitymentioning

confidence: 99%

“…Peer et al [68] utilised this approach to target siRNAs to primary leukocytes, more specifically those expressing the high affinity adhesive form of LFA-1 as opposed to the low affinity non-adhesive LFA-1 found on naive cells and thus interrupting unwanted pathogenic immune stimulation. Intravenously injected siRNA complexes have been shown to selectively target K562 cells expressing the high affinity LFA-1 engrafted in the lungs of mice [68] .…”

Section: Serum Stabilitymentioning

confidence: 99%

Therapeutic targeting in the silent era: advances in non-viral siRNA delivery

et al. 2010

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Access to the full text of the published version may require a subscription. Rights AbstractGene silencing using RNA-interference, first described in mammalian systems almost a decade ago, is revolutionizing therapeutic target validation efforts both in vitro and in vivo. Moreover, the potential for using short interfering RNA (siRNA) as a therapy in its own right is also progressing at a significant pace. However, the widespread use of such approaches is contingent on having appropriate delivery systems to achieve clinically appropriate, safe, and efficient delivery of siRNA. There are many physicochemical and biological barriers to such delivery, and a growing emphasis on the design and characterisation of non-viral technologies that will overcome these barriers and expedite targeted delivery. This review discusses the considerations and challenges associated with use of siRNA-based therapeutics, including stability and off-target effects. Speculation is made on the properties of an ideal delivery system and the non-viral delivery approaches used to date, both in vitro and in vivo, are classified and discussed. Moreover, the ability of cyclodextrin-based delivery vectors to fulfil many of the criteria of an ideal delivery construct is also elaborated.3 Introduction:

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Selective gene silencing in activated leukocytes by targeting siRNAs to the integrin lymphocyte function-associated antigen-1

Cited by 258 publications

References 41 publications

Special delivery: targeted therapy with small RNAs

Special delivery: targeted therapy with small RNAs

siRNA stabilization prolongs gene knockdown in primary T lymphocytes

Therapeutic targeting in the silent era: advances in non-viral siRNA delivery

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