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Posts Tagged ‘specific sequence-site insertion’


Larry H. Bernstein, MD, FCAP, Reviewer and Curator

http://pharmaceuticalinnovation.com/2012-12-09/larryhbern/Silencing Cancers with Synthetic siRNAs

The challenge of cancer drug development has been marker by less than a century of development of major insights into the know of biochemical pathways and the changes in those pathways in a dramatic shift in enrgy utilization and organ development, and the changes in those pathways with the development of malignant neoplasia.  The first notable change is the Warburg Effect (attributed to the 1860 obsevation by Pasteur that yeast cells use glycolysis under anaerobic conditions).  Warburg also referred to earlier work by Meyerhoff, in a ratio of CO2 release to O2 consumption, a Meyerhoff ratio.  Much more was elucidated after the discovery of the pyridine nucleotides, which gave understanding of glycolysis and lactate production with a key two enzyme separation at the forward LDH reaction and the back reentry to the TCA cycle.  But the TCA cycle could be used for oxidative energy utilization in the mitochondria by oxidative phosphorylation elucidated by Peter Mitchell, or it can alternatively be used for syntheses, like proteins and lipid membrane structures.

A brilliant student in Leloir’s laboratory in Brazil undertook a study of isoenzyme structure in 1971, at a time that I was working under Nathan O. Kaplan on the mechanism of inhibition of mitochondrial malate dehydrogenase. In his descripton, taking into account the effect of substrates upon protein stability (FEBS) could be, in a prebiotic system, the form required in order to select protein and RNA in parallel or in tandem in a way that generates the genetic code (3 bases for one amino acid). Later, other proteins like reverse transcriptase, could transcribe it into the more stable DNA. Leloir had just finished ( a few years before 1971 but, not published by these days yet) a somehow similar reasoning about metabolic regions rich in A or in C or .. G or T.  He later spent time in London to study the early events in the transition of growing cells linked to ion fluxes, which he was attracted to by the idea that life is so strongly associated with the K (potassium) and Na (sodium) asymmetry.   Moreover, he notes that while DNA is the same no matter the cell is dead or alive,  and therefore,  it is a huge mistake to call DNA the molecule of life. In all life forms, you will find K reach inside and Na rich outside its membrane. On his return to Brazil, he accepted a request to collaborate with the Surgery department in energetic metabolism of tissues submitted to ischemia and reperfusion. This led me back to Pasteur and Warburg effects and like in Leloir´s time, he worked with a dimorphic yeast/mold that was considered a morphogenetic presentation of the Pasteur Effect.  His findings were as follows. In absence of glucose, a condition that prevents the yeast like cell morphology, which led to the study of an enzyme “half reaction”. The reaction that on the half, “seen in our experimental conditions did not followed classical thermodynamics” (According to Collowick & Kaplan (of your personal knowledge) vol. I See Utter and Kurahashi in it). This somehow contributed to a way of seeing biochemistry with modesty. The second and more strongly related to the Pasteur Effect was the use an entirely designed and produced in our Medical School Coulometer spirometer that measures oxygen consumption in a condition of constant oxygen supply. At variance with Warburg apparatus and Clark´s electrode, this oxymeters uses decrease in partial oxygen pressure and decrease electrical signal of oxygen polarography to measure it (Leite, J.V.P. Research in Physiol. Kao, Koissumi, Vassali eds Aulo Gaggi Bologna, 673-80-1971). “With this, I was able to measure the same mycelium in low and high “cell density” inside the same culture media. The result shows, high density one stops mitochondrial function while low density continues to consume oxygen (the internal increase or decrease in glycogen levels shows which one does or does not do it). Translation for today: The same genome in the same chemical environment behave differently mostly likely by its interaction differences. This previous experience fits well with what  I have to read by that time of my work with surgeons.  Submitted to total ischemia tissues mitochondrial function is stopped when they already have enough oxyhemoglobin (1) Epstein, Balaban and Ross Am J Physiol.243, F356-63 (1982) 2) Bashford , C. L, Biological membranes a practical approach Oxford Was. P 219-239 (1987).”

Of course, the world of medical and pharmaceutical engagement with this problem, though changed in focus, has benefitted hugely from “The Human Genome Project”, and the events since the millenium, because of technology advances in instrumental analysis, and in bioinformatics and computational biology.  This has lead to recent advances in regenerative biology with stem cell “models”, to advances in resorbable matrices, and so on.  We proceed to an interesting work that applies synthetic work with nucleic acid signaling to pharmacotherapy of cancer.

Synthetic RNAs Designed to Fight Cancer

Fri, 12/06/2013 Biosci Technology
Xiaowei Wang and his colleagues have designed synthetic molecules that combine the advantages of two experimental RNA therapies against cancer. (Source: WUSTL/Robert J. Boston)In search of better cancer treatments, researchers at Washington University School of Medicine in St. Louis have designed synthetic molecules that combine the advantages of two experimental RNA therapies.  The study appears in the December issue of the journal RNA.
 RNAs play an important role in how genes are turned on and off in the body. Both siRNAs and microRNAs are snippets of RNA known to modulate a gene’s signal or shut it down entirely. Separately, siRNA and microRNA treatment strategies are in early clinical trials against cancer, but few groups have attempted to marry the two.   “These are preliminary findings, but we have shown that the concept is worth pursuing,” said Xiaowei Wang, assistant professor of radiation oncology at the School of Medicine and a member of the Siteman Cancer Center. “We are trying to merge two largely separate fields of RNA research and harness the advantages of both.”
 “We designed an artificial RNA that is a combination of siRNA and microRNA, The showed that the artificial RNA combines the functions of the two separate molecules, simultaneously inhibiting both cell migration and proliferation. They designed and assembled small interfering” RNAs, or siRNAs,  made to shut down– or interfere with– a single specific gene that drives cancer.  The siRNA molecules work extremely well at silencing a gene target because the siRNA sequence is made to perfectly complement the target sequence, thereby
  • silencing a gene’s expression.
Though siRNAs are great at turning off the gene target, they also have potentially dangerous side effects:
  • siRNAs inadvertently can shut down other genes that need to be expressed to carry out tasks that keep the body healthy.
 According to Wang and his colleagues, siRNAs interfere with off-target genes that closely complement their “seed region,” a short but important
  • section of the siRNA sequence that governs binding to a gene target.
 “We can never predict all of the toxic side effects that we might see with a particular siRNA,” said Wang. “In the past, we tried to block the seed region in an attempt to reduce the side effects. Until now,
  • we never tried to replace the seed region completely.”
 Wang and his colleagues asked whether
  • they could replace the siRNA’s seed region with the seed region from microRNA.
Unlike siRNA, microRNA is a natural part of the body’s gene expression. And it can also shut down genes. As such, the microRNA seed region (with its natural targets) might reduce
  • the toxic side effects caused by the artificial siRNA seed region. Plus,
  • the microRNA seed region would add a new tool to shut down other genes that also may be driving cancer.
 Wang’s group started with a bioinformatics approach, using a computer algorithm to design
  • siRNA sequences against a common driver of cancer,
  • a gene called AKT1 that encourages uncontrolled cell division.
They used the program to select siRNAs against AKT1 that also had a seed region highly similar to the seed region of a microRNA known to inhibit a cell’s ability to move, thus
  • potentially reducing the cancer’s ability to spread.
In theory, replacing the siRNA seed region with the microRNA seed region also would combine their functions
  • reducing cell division and
  • movement with a single RNA molecule.
 Of more than 1,000 siRNAs that can target AKT1,
  • they found only three that each had a seed region remarkably similar to the seed region of the microRNA that reduces cell movement.
 They then took the microRNA seed region and
  • used it to replace the seed region in the three siRNAs that target AKT1.
The close similarity between the two seed regions is required because
  • changing the original siRNA sequence too much would make it less effective at shutting down AKT1.
 They dubbed the resulting combination RNA molecule “artificial interfering” RNA, or aiRNA. Once they arrived at these three sequences using computer models,
  1. they assembled the aiRNAs and
  2. tested them in cancer cells.
 One of the three artificial RNAs that they built in the lab
  • combined the advantages of the original siRNA and the microRNA seed region that was transplanted into it.
This aiRNA greatly reduced both
  1. cell division (like the siRNA) and
  2. movement (like the microRNA).
And to further show proof-of-concept, they also did the reverse, designing an aiRNA that
  1. both resists chemotherapy and
  2. promotes movement of the cancer cells.
 “Obviously, we would not increase cell survival and movement for cancer therapy, but we wanted to show how flexible this technology can be, potentially expanding it to treat diseases other than cancer,” Wang said.
Source: WUSTL
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