Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting

Paula, Daniel de; Bentley, Maria Vitória Lopes Badra; Mahato, Ram I.

doi:10.1261/rna.459807

Cited by 204 publications

(173 citation statements)

References 154 publications

(244 reference statements)

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“…A number of chemical modifications (reviewed in [19]) have been reported to stabilize siRNA. Modifications are frequently tested for serum stability with regard to subsequent in vivo applications [20].…”

Section: Discussionmentioning

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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Section: Discussionmentioning

confidence: 99%

siRNA stabilization prolongs gene knockdown in primary T lymphocytes

et al. 2008

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“…Thermal stability is increased by introducing a 2'-fluoro, a 2'-O-methyl or a 2′-O-methoxyethyl (2'-MOE) on the position 2 of the ribose or by using Locked Nucleic Acid (LNA). Stability regarding nuclease digestion is enhanced by placing these chemically modified bases at the 5' or 3' end of the RNA sequence [118,119]. siRNA can be complexed with nanoparticles, peptidic or lipidic complexes to promote pharmacokinetics and In pulmonary diseases involving MMP-12, targeting endothelial cells can be easily obtained.…”

Section: A C C E P T E D Article In Pressmentioning

confidence: 99%

Matrix metalloproteinase 12 silencing: A therapeutic approach to treat pathological lung tissue remodeling?

Garbacki¹,

Valentin²,

Piette³

et al. 2009

Pulmonary Pharmacology & Therapeutics

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“…A variety of strategies for siRNA modification have been pursued including alterations in the backbone chemistry [6], 2'-sugar modification [7], nucleobase modifications [8] and other means [9], as recently reviewed. Another way to address this problem is to assemble carbohydrate-modified ONs, exemplified by hexitol nucleic acids [10][11][12][13][14], 2'-O-(2-methoxy)ethyl ONs [15,16] and bicyclic ONs [17], with the LNA monomers of the Wengel group [18] showing the strongest affinity for RNA, and having many alternative structures [19].…”

Section: Introductionmentioning

confidence: 99%

Hybridization potential of oligonucleotides comprising 3′-O-methylated altritol nucleosides

2012

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Abstract:A series of 3'-O-methylated D-altrohexitol nucleoside analogs (MANA) was synthesized comprising all four base moieties, adenine, cytosine, uracil and guanine. These monomers were incorporated into While the specificity of these new synthons is slightly lower compared to mismatches within RNA double strands, it is similar to the discrimination potential of other hexitol modifications (HNA and ANA) which already proved their biologic interest, highlighting the potential of MANA constructs in antisense and in siRNA applications.3

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Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting

Cited by 204 publications

References 154 publications

siRNA stabilization prolongs gene knockdown in primary T lymphocytes

siRNA stabilization prolongs gene knockdown in primary T lymphocytes

Matrix metalloproteinase 12 silencing: A therapeutic approach to treat pathological lung tissue remodeling?

Hybridization potential of oligonucleotides comprising 3′-O-methylated altritol nucleosides

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