Freethought & Rationalism Archive

ps418 · 04-01-2003, 07:36 AM

I didn't think about it before I made my OP, but wouldn't RNAi be the ideal antiviral tool, for instance to slow HIV infection? Couldn't you design dsRNAs that target gag, pol, and env RNAs and thereby prevent the construction of new viruses, assuming you could get the dsRNAs into the cells somehow? I found this Nature Medicine paper on the web for free:

Pomerantz, RJ 2002. RNA interference meets HIV-1: will silence be golden? Nature Medicine 8:659-60. *PDF File*

Patrick

Wounded King · 04-01-2003, 12:38 PM

What I think it really interesting is that this might reflect a common endogenous mechanism of gene regulation.

See Voinnet. O. RNA silencing: small RNAs as ubiquitous regulators of gene expression. Current Opinion in Plant Biology 2002, 5:444�451 for a review.

ps418 · 04-03-2003, 06:59 AM

One thing is certain -- the range of interesting things that RNAs can do and the number of processes they are known or thought to be involved in hs changed dramatically in the last decade!

Quote:

Originally posted by ps418
I didn't think about it before I made my OP, but wouldn't RNAi be the ideal antiviral tool, for instance to slow HIV infection? Couldn't you design dsRNAs that target gag, pol, and env RNAs and thereby prevent the construction of new viruses, assuming you could get the dsRNAs into the cells somehow?

After reading a bit more, it appears that the dsRNA interference mechanisms do in fact have an antiviral function (amongst others). Chicas and Macino (2001) write:

Quote:

Although in the cases described above PTGS is triggered by artificial stimuli, the biological role of PTGS seems to be to prevent viral infection in plants and the mobilization of transposons in C. elegans. This conclusion has come from the observation that in C. elegans transposons are more active in RNAi defective mutants (Ketting et al., 1999; Tabara et al., 1999). Likewise, mutants defective in PTGS in plants are more susceptible to viral infection (Mourrain et al., 2000). In addition, viruses encode a number of factors that suppress PTGS (Voinnet et al., 2000), further supporting the notion that PTGS is a defense mechanism against viruses.

Characteristics of post-transcriptional gene silencing. EMBO Reports 2, 11, 992�996 (2001).

In my OP I speculated that RNAi may have potential as a biological weapon paradigm, assuming that the right dsRNAs could be introduced reliably into human cells. Subsequently I have read that in C. elegans, you can simply feed them bacteria expressing the dsRNAs, and the PTGS effect will spread to all the cells in their body. I wonder if this is possible in Drosophila or mice?

Patrick

Wounded King · 04-03-2003, 01:24 PM

I think feeding was tried in drosophila using GM yeast, it wasn't successful, I don't recall my source for this though. I think the fact that C. elegans is such a small organism makes a difference, even immersing the C. elgans in a solution with the dsRNA is enough to produce the inhibitory effect, and the effect can be heritable to at least the f1 progeny.

ps418 · 04-07-2003, 07:51 AM

An article in the new The Scientist about the boom of interest in RNAi:

Shhh: Silencing Genes with RNA Interference: Banking on RNAi's promise, academic and private researchers flock to the field requires free registration.

The article gives information on the "delivery issues" I brought up in my last two posts. Apparently it will be much more difficult to introduce dsRNAs to vertebrates and permanently silence genes, unless you do it at the zygote stage, though it is apparently possible even now to nearly silence some genes in vertebrates for a short period of time.

Quote:

Yet another method makes use of viral vectors to infect cells with the dsRNA-expression construct. Some researchers exploit retroviral vectors,6 but recently, Luk Van Parijs' team at MIT,8 and Inder Verma's group at the Salk Institute9 independently described lentiviral-based systems. This approach enabled these investigators to deliver shRNAs to primary cells, stem cells, and noncycling cells, all of which are traditionally hard to transfect. By infecting zygotes, both groups made transgenic mice in which RNAi-directed gene downregulation occurs stably throughout the animal.

Delivering siRNAs directly to whole vertebrate animals is more problematic than it is for invertebrates or cell lines. "Animals can't absorb [the siRNA] through the skin," Mirus' Lewis explains, "and simply injecting it into the bloodstream has been ineffective." Last year, Hannon in collaboration with Mark Kay at Stanford University,10 and Lewis and colleagues,11 independently employed a "hydrodynamic transfection method" to deliver naked siRNAs to mice via tail-vein injection. These authors observed downregulation of a reporter gene by 80%-90% in the liver, kidney, spleen, lung, and pancreas, but the effect is relatively short-lived, lasting only a few days.

Patrick

Wounded King · 04-07-2003, 08:19 AM

I dont see any reason why introducing transient siRNA effects should be any harder than standard gene therapy. Of course standard gene therapy is notoriously difficult in terms of delivery as well. I suppose it depends if you want to just deliver the siRNA itself or some sort of vector to allow you to express the SiRNA in a specific population.

A vector seems like a more sensible idea given the natural instability of RNA.

Intavenous injection has not, as your reference suggests, been wholey ineffective.

See the recent Nature Medicine paper.

Song E, Lee SK, Wang J, Ince N, Ouyang N, Min J, Chen J, Shankar P, Lieberman J.
RNA interference targeting Fas protects mice from fulminant hepatitis.
Nat Med. 2003 Mar;9(3):347-51.

Which used intravenous injection as the mode of delivery.

LostGirl · 04-11-2003, 09:31 PM

I'm wondering if lipoplexes might not be a bad way to deliver the RNAi to cells. I believe some targeting has been done with lipoplexes; but I've only heard of them being used to deliver condensed DNA packets for gene therapy.

ps418 · 05-07-2003, 07:39 AM

Quote:

Originally posted by ps418
Think also of the possibilities for treating "genetic" diseases. If a disease is caused by the presence of a particular protein, and the individual is heterozygous for the gene for that protein, then potentially you could actually shut down production of that faulty protein using RNAi, without affecting production of the good protein. And cancer research should benefit greatly too, since overexpression of some genes is common in cancerous cells.

I'm resurrecting this thread because there is an article in the new issue of Clinical Cancer Research that demonstrates the potential of RNAi as a cancer therapy. In this case, dsRNAs were used to interfere with the production of beta catenin, which is often mutated and over-expressed in colon cancer cells, and is associated with colon cancer cell proliferation. The experimental result was positive -a "marked" decrease in beta catenin and inhibition of cell proliferation.

Quote:

The �-catenin and APC genes are key components of the Wnt signaling pathway. Mutation of these genes results in increased levels of the �-catenin protein, which is associated with enhanced cellular proliferation and the development of both colon polyps and colon cancer. Recently, a technique known as RNA interference has been successfully adapted to mammalian cells so that it is now possible to specifically decrease the expression of cellular genes after transfection of annealed small interfering 21-mer RNAs. In the current study, we used small interfering RNA (siRNA) directed against �-catenin to determine the effects of decreasing the high constitutive levels of this protein in colon cancer cell lines with mutations in either �-catenin or APC. Our studies demonstrate that siRNA directed against �-catenin markedly decreased �-catenin-dependent gene expression and inhibited cellular proliferation as reflected in the reduced growth of these colon cancer cells both in soft agar and in nude mice. These results indicate that siRNA can target specific factors whose expression is altered in malignancy and may have the potential as a therapeutic modality to treat human cancer.

Udit N. Verma, Rama M. Surabhi, Aurelia Schmaltieg, Carlos Becerra, and Richard B. Gaynor, Small Interfering RNAs Directed against �-Catenin Inhibit the in Vitro and in Vivo Growth of Colon Cancer Cells. Clin Cancer Res 2003 9: 1291-1300.

Patrick

ps418 · 05-07-2003, 07:52 AM

See also the the following article from the march issue of Clinical Cancer Research, which shows more generally how the combination of DNA microarrays (another awesome technology!) and RNAi provides a whole new paradigm for cancer treatment and research. Basically, you can use the microarrays (gene chips) to get a detailed picture of how gene expression is changed in cancer cells (i.e. which genes are upregulated and downregulated), and then use RNAi to target and downregulate various genes that are being overexpressed (don't know how you fix downregulated gene expression though).

Quote:

Purpose: The purpose of this study was to profile gene expression changes in colorectal tumors to identify new targets and strategies for the management of this disease.

Experimental Design: cDNA microarray analysis was used to detect differences in gene expression between normal tissue and colon tumors and polyps isolated from 20 patients. To identify genes that are important in regulating the growth properties of colorectal cancer, RNA interference (RNAi) was used to disrupt expression of several of the overexpressed genes in a colon tumor cell line, HCT116, which showed similar patterns of gene expression as many of the patient tumors.

Results: Expression changes of 2-fold in approximately one-third of the patients were consistently observed for 2632 of a total of 9592 genes (574 up-regulated genes and 2058 down-regulated genes). Subsequent analysis of 13 genes by quantitative real-time PCR confirmed the reliability of this analysis. RNAi-mediated disruption of the expression of one of these genes, survivin, a potent inhibitor of apoptosis, severely reduced tumor growth both in vitro and in an in vivo xenograft model.

Conclusions: The combined use of microarray analysis and RNAi provides an excellent system to define the role of specific genes that are up-regulated in cancer lead to the increased in vitro and in vivo growth of colon tumors.

Noelle Sevilir Williams, Richard B. Gaynor, Shane Scoggin, Udit Verma, Tunc Gokaslan, Clifford Simmang, Jason Fleming, Denise Tavana, Eugene Frenkel, and Carlos Becerra, Identification and Validation of Genes Involved in the Pathogenesis of Colorectal Cancer Using cDNA Microarrays and RNA Interference. Clin. Cancer Res. 2003 9: 931-946.

ps418 · 06-03-2003, 06:23 AM

There is an paper published online yesterday in the PNAS demonstrating the potentially perfect specificity of RNAi. This is important, as it demonstrates that it is possible to silence a dominant, disease-causing allele without affecting expression of the other, 'normal' allele, even if the other allele differs by only a single SNP.

Quote:

Small interfering RNA (siRNA) holds therapeutic promise for silencing dominantly acting disease genes, particularly if mutant alleles can be targeted selectively. In mammalian cell models we demonstrate that allele-specific silencing of disease genes with siRNA can be achieved by targeting either a linked single-nucleotide polymorphism (SNP) or the disease mutation directly. For a polyglutamine neurodegenerative disorder in which we first determined that selective targeting of the disease-causing CAG repeat is not possible, we took advantage of an associated SNP to generate siRNA that exclusively silenced the mutant Machado-Joseph disease/spinocerebellar ataxia type 3 allele while sparing expression of the WT allele. Allele-specific suppression was accomplished with all three approaches currently used to deliver siRNA: in vitro-synthesized duplexes as well as plasmid and viral expression of short hairpin RNA. We further optimized siRNA to specifically target a missense Tau mutation, V337M, that causes frontotemporal dementia. These studies establish that siRNA can be engineered to silence disease genes differing by a single nucleotide and highlight a key role for SNPs in extending the utility of siRNA in dominantly inherited disorders.

Miller et al, Allele-specific silencing of dominant disease genes. PNAS, Published online before print June 2, 2003.

Patrick

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04-01-2003, 07:36 AM	#11
ps418 Veteran Member Join Date: Mar 2001 Location: Louisville, KY, USA Posts: 1,840	I didn't think about it before I made my OP, but wouldn't RNAi be the ideal antiviral tool, for instance to slow HIV infection? Couldn't you design dsRNAs that target gag, pol, and env RNAs and thereby prevent the construction of new viruses, assuming you could get the dsRNAs into the cells somehow? I found this Nature Medicine paper on the web for free: Pomerantz, RJ 2002. RNA interference meets HIV-1: will silence be golden? Nature Medicine 8:659-60. PDF File Patrick

04-01-2003, 12:38 PM	#12
Wounded King Veteran Member Join Date: Mar 2003 Location: Edinburgh Posts: 1,211	What I think it really interesting is that this might reflect a common endogenous mechanism of gene regulation. See Voinnet. O. RNA silencing: small RNAs as ubiquitous regulators of gene expression. Current Opinion in Plant Biology 2002, 5:444�451 for a review.

04-03-2003, 01:24 PM	#14
Wounded King Veteran Member Join Date: Mar 2003 Location: Edinburgh Posts: 1,211	I think feeding was tried in drosophila using GM yeast, it wasn't successful, I don't recall my source for this though. I think the fact that C. elegans is such a small organism makes a difference, even immersing the C. elgans in a solution with the dsRNA is enough to produce the inhibitory effect, and the effect can be heritable to at least the f1 progeny.

04-07-2003, 08:19 AM	#16
Wounded King Veteran Member Join Date: Mar 2003 Location: Edinburgh Posts: 1,211	I dont see any reason why introducing transient siRNA effects should be any harder than standard gene therapy. Of course standard gene therapy is notoriously difficult in terms of delivery as well. I suppose it depends if you want to just deliver the siRNA itself or some sort of vector to allow you to express the SiRNA in a specific population. A vector seems like a more sensible idea given the natural instability of RNA. Intavenous injection has not, as your reference suggests, been wholey ineffective. See the recent Nature Medicine paper. Song E, Lee SK, Wang J, Ince N, Ouyang N, Min J, Chen J, Shankar P, Lieberman J. RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med. 2003 Mar;9(3):347-51. Which used intravenous injection as the mode of delivery.

04-11-2003, 09:31 PM	#17
LostGirl Junior Member Join Date: Mar 2003 Location: Guelph, Ontario Posts: 45	I'm wondering if lipoplexes might not be a bad way to deliver the RNAi to cells. I believe some targeting has been done with lipoplexes; but I've only heard of them being used to deliver condensed DNA packets for gene therapy.

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