Rajendra K. Pandey

Bio: Rajendra K. Pandey is an academic researcher from Alnylam Pharmaceuticals. The author has contributed to research in topics: Small interfering RNA & Gene silencing. The author has an hindex of 13, co-authored 21 publications receiving 4344 citations. Previous affiliations of Rajendra K. Pandey include Binghamton University.

Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs

Abstract: RNA interference (RNAi) holds considerable promise as a therapeutic approach to silence disease-causing genes, particularly those that encode so-called 'non-druggable' targets that are not amenable to conventional therapeutics such as small molecules, proteins, or monoclonal antibodies. The main obstacle to achieving in vivo gene silencing by RNAi technologies is delivery. Here we show that chemically modified short interfering RNAs (siRNAs) can silence an endogenous gene encoding apolipoprotein B (apoB) after intravenous injection in mice. Administration of chemically modified siRNAs resulted in silencing of the apoB messenger RNA in liver and jejunum, decreased plasma levels of apoB protein, and reduced total cholesterol. We also show that these siRNAs can silence human apoB in a transgenic mouse model. In our in vivo study, the mechanism of action for the siRNAs was proven to occur through RNAi-mediated mRNA degradation, and we determined that cleavage of the apoB mRNA occurred specifically at the predicted site. These findings demonstrate the therapeutic potential of siRNAs for the treatment of disease.

Mechanisms and optimization of in vivo delivery of lipophilic siRNAs

Abstract: Cholesterol-conjugated siRNAs can silence gene expression in vivo. Here we synthesize a variety of lipophilic siRNAs and use them to elucidate the requirements for siRNA delivery in vivo. We show that conjugation to bile acids and long-chain fatty acids, in addition to cholesterol, mediates siRNA uptake into cells and gene silencing in vivo. Efficient and selective uptake of these siRNA conjugates depends on interactions with lipoprotein particles, lipoprotein receptors and transmembrane proteins. High-density lipoprotein (HDL) directs siRNA delivery into liver, gut, kidney and steroidogenic organs, whereas low-density lipoprotein (LDL) targets siRNA primarily to the liver. LDL-receptor expression is essential for siRNA delivery by LDL particles, and SR-BI receptor expression is required for uptake of HDL-bound siRNAs. Cellular uptake also requires the mammalian homolog of the Caenorhabditis elegans transmembrane protein Sid1. Our results demonstrate that conjugation to lipophilic molecules enables effective siRNA uptake through a common mechanism that can be exploited to optimize therapeutic siRNA delivery.

Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits

Abstract: Huntington's disease (HD) is an autosomal dominant disease caused by a CAG repeat expansion in the Htt gene (1). Mutant Htt causes neuronal death, dementia, and movement dysfunction; there is no effective treatment. In an inducible transgenic mouse model of HD, turning off transgene expression reversed neuropathology and motor deficits (2). Lowering mutant Htt gene expression in brain may treat HD. In mice, viral vector delivery of short hairpin RNAs (shRNAs) against mutant Htt gene exon 1 or genes that cause other neurodegenerative disorders reduced neuropathology and motor deficits (3–10). Brain delivery of adeno-associated virus (AAV)-shRNA against mutant Htt improved signs of disease in HD transgenic models (7, 11). In the inaugural study on RNAi targeting Htt in vivo, shRNA against Htt in AAV2, delivered to the N171–82Q transgenic model of HD, improved ambulation at 4 months and rotarod performance at 10 and 18 weeks after injection (7). Five and one-half months after shRNA administration, quantitative RT-PCR revealed a 50% reduction in striatal Htt mRNA. Statistical changes in quantification of Htt protein reduction and inclusions were not reported. AAV5 delivery of shRNA against Htt in the R6/1 murine model of HD showed a 25% decrease in Htt protein and an 80% reduction in Htt mRNA 10 weeks after shRNA injection (10). The shRNA delayed onset of clasping by 2 weeks (20–22 weeks), and treated mice had fewer clasps. No difference in rotarod performance was detected. Inclusion size and number decreased in the striatum, but not in the cortex, compared with the corresponding contralateral brain regions. The authors provided an important caveat that one of the shRNAs had off-target effects; the cause of the off-target effects was not established. shRNA in AAV2 or AAV5 was used to target EGFP to knock down EGFP-Htt in another transgenic model of HD (11). shRNA reversed pathology after the onset of pathologic changes; however, behaviors were not studied. Administration of large amounts of siRNA against Htt in a Lipofectamine 2000 suspension into the lateral ventricle of newborn R6/2 transgenic mice (exon 1 of Htt) reduced whole-brain levels of mutant Htt in two mice and Htt mRNA up to 7 days posttreatment, delayed the onset of clasping, rotarod, and open-field phenotypes, and improved survival (12). Statistical quantification of neuropathology was not reported. Thus, prior studies examining RNAi against Htt provided the groundwork for therapeutic gene silencing in HD. Most of the studies used viral delivery of shRNA, and the study using siRNA required liposome delivery to newborns, with the potential liposome neuronal toxicity. Caveats attend the use of shRNAs, which can be toxic when integrated into the host genome (13, 14), in part because shRNA production is unregulated. Long siRNAs (>29 nt) and shRNAs are prone to activate off-target gene expression (15). For patient safety, shRNA will need to be able to be switched off, currently a hurdle in viral delivery systems. An alternative strategy for HD therapy is the use of small-interfering RNAs (siRNAs), ≈21-nt RNA duplexes. siRNA has been administered into cerebroventricles, vasculature, intrathecal space, and parenchyma (16–20). siRNAs were found effective and safe when introduced into mice and non-human primates (19, 21, 22). Several limitations impede progress in using siRNAs as a treatment for HD: entry and effectiveness in adult neurons without the use of potentially toxic transfection reagents; a clear demonstration that gene silencing reduces protein expression; and an improvement in behavioral deficits and neuropathology, especially neuron survival. Because bioactive molecules conjugated to cholesterol have improved cellular uptake in vitro (23), LDL receptors have been detected in brain (24), and cholesterol conjugation enhances siRNA uptake in cells outside of the central nervous system (16), we speculated that cholesterol-conjugated (cc) siRNA might enter neurons in vivo. An in vivo, rapid-onset model of HD would be optimal to test gene silencing in brain. The current rapid mouse model of HD shows mutant Htt-induced pathology after 2 months. Transgenic mice expressing exon 1 of mutant Htt [R6/2 (25)] develop nuclear inclusions throughout the brain at 2 months of age and exhibit a rapidly progressing, severe phenotype. Other transgenic or knock-in mice expressing mutant Htt exhibit late-onset, mild phenotypes, often after 6 months of age (26, 27), and lack prominent neuronal loss. Neither model is ideal to test transient effects of a single injection of siRNA against Htt introduced directly to the striatum. We therefore developed an acute, in vivo, HD mouse model tailored to addressing the efficacy of siRNA. Here, we used AAV to deliver a 1,395-nt cDNA fragment of human mutant Htt into mouse striatum to evaluate the effectiveness of an siRNA targeting human Htt.

Unexpected origins of the enhanced pairing affinity of 2′-fluoro-modified RNA

Abstract: Various chemical modifications are currently being evaluated for improving the efficacy of short interfering RNA (siRNA) duplexes as antisense agents for gene silencing in vivo. Among the 2'-ribose modifications assessed to date, 2'deoxy-2'-fluoro-RNA (2'-F-RNA) has unique properties for RNA interference (RNAi) applications. Thus, 2'-F-modified nucleotides are well tolerated in the guide (antisense) and passenger (sense) siRNA strands and the corresponding duplexes lack immunostimulatory effects, enhance nuclease resistance and display improved efficacy in vitro and in vivo compared with unmodified siRNAs. To identify potential origins of the distinct behaviors of RNA and 2'-F-RNA we carried out thermodynamic and X-ray crystallographic analyses of fully and partially 2'-F-modified RNAs. Surprisingly, we found that the increased pairing affinity of 2'-F-RNA relative to RNA is not, as commonly assumed, the result of a favorable entropic contribution ('conformational preorganization'), but instead primarily based on enthalpy. Crystal structures at high resolution and osmotic stress demonstrate that the 2'-F-RNA duplex is less hydrated than the RNA duplex. The enthalpy-driven, higher stability of the former hints at the possibility that the 2'-substituent, in addition to its important function in sculpting RNA conformation, plays an underappreciated role in modulating Watson-Crick base pairing strength and potentially π-π stacking interactions.

Unique Gene‐Silencing and Structural Properties of 2′‐Fluoro‐Modified siRNAs

Abstract: With little or no negative impact on the activity of small interfering RNAs (siRNAs), regardless of the number of modifications or the positions within the strand, the 2'-deoxy-2'-fluoro (2'-F) modification is unique. Furthermore, the 2'-F-modified siRNA (see crystal structure) was thermodynamically more stable and more nuclease-resistant than the parent siRNA, and produced no immunostimulatory response.

Silencing of microRNAs in vivo with ‘antagomirs’

Abstract: MicroRNAs (miRNAs) are an abundant class of non-coding RNAs that are believed to be important in many biological processes through regulation of gene expression. The precise molecular function of miRNAs in mammals is largely unknown and a better understanding will require loss-of-function studies in vivo. Here we show that a novel class of chemically engineered oligonucleotides, termed 'antagomirs', are efficient and specific silencers of endogenous miRNAs in mice. Intravenous administration of antagomirs against miR-16, miR-122, miR-192 and miR-194 resulted in a marked reduction of corresponding miRNA levels in liver, lung, kidney, heart, intestine, fat, skin, bone marrow, muscle, ovaries and adrenals. The silencing of endogenous miRNAs by this novel method is specific, efficient and long-lasting. The biological significance of silencing miRNAs with the use of antagomirs was studied for miR-122, an abundant liver-specific miRNA. Gene expression and bioinformatic analysis of messenger RNA from antagomir-treated animals revealed that the 3' untranslated regions of upregulated genes are strongly enriched in miR-122 recognition motifs, whereas downregulated genes are depleted in these motifs. Analysis of the functional annotation of downregulated genes specifically predicted that cholesterol biosynthesis genes would be affected by miR-122, and plasma cholesterol measurements showed reduced levels in antagomir-122-treated mice. Our findings show that antagomirs are powerful tools to silence specific miRNAs in vivo and may represent a therapeutic strategy for silencing miRNAs in disease.

Knocking down barriers: advances in siRNA delivery

Abstract: In the 10 years that have passed since the Nobel prize-winning discovery of RNA interference (RNAi), billions of dollars have been invested in the therapeutic application of gene silencing in humans. Today, there are promising data from ongoing clinical trials for the treatment of age-related macular degeneration and respiratory syncytial virus. Despite these early successes, however, the widespread use of RNAi therapeutics for disease prevention and treatment requires the development of clinically suitable, safe and effective drug delivery vehicles. Here, we provide an update on the progress of RNAi therapeutics and highlight novel synthetic materials for the encapsulation and intracellular delivery of nucleic acids.

Non-viral vectors for gene-based therapy

Abstract: Gene-based therapy is the intentional modulation of gene expression in specific cells to treat pathological conditions This modulation is accomplished by introducing exogenous nucleic acids such as DNA, mRNA, small interfering RNA (siRNA), microRNA (miRNA) or antisense oligonucleotides Given the large size and the negative charge of these macromolecules, their delivery is typically mediated by carriers or vectors In this Review, we introduce the biological barriers to gene delivery in vivo and discuss recent advances in material sciences, nanotechnology and nucleic acid chemistry that have yielded promising non-viral delivery systems, some of which are currently undergoing testing in clinical trials The diversity of these systems highlights the recent progress of gene-based therapy using non-viral approaches

MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins

Abstract: Circulating microRNAs (miRNA) are relatively stable in plasma and are a new class of disease biomarkers. Here we present evidence that high-density lipoprotein (HDL) transports endogenous miRNAs and delivers them to recipient cells with functional targeting capabilities. Cellular export of miRNAs to HDL was demonstrated to be regulated by neutral sphingomyelinase. Reconstituted HDL injected into mice retrieved distinct miRNA profiles from normal and atherogenic models. HDL delivery of both exogenous and endogenous miRNAs resulted in the direct targeting of messenger RNA reporters. Furthermore, HDL-mediated delivery of miRNAs to recipient cells was demonstrated to be dependent on scavenger receptor class B type I. The human HDL-miRNA profile of normal subjects is significantly different from that of familial hypercholesterolemia subjects. Notably, HDL-miRNA from atherosclerotic subjects induced differential gene expression, with significant loss of conserved mRNA targets in cultured hepatocytes. Collectively, these observations indicate that HDL participates in a mechanism of intercellular communication involving the transport and delivery of miRNAs.