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Gene Therapies and DNA Repair


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#91 John Schloendorn

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Posted 20 January 2005 - 02:42 AM

Sorry, Prometheus, if I sound like I have no respect for your ideas. That's very wrong, I am indeed fond of most! Certainly I can see us hijack DNA repair systems from Deinococcus for future gene therapy, which is just plain cool.
In these biotech posts I argued mainly from the perspective of what I personally should and should not do next, which is much too narrow for a forum like this and/or should be made explicit. Thanks for pointing this out and for the ref, which I'll be looking into soon.

#92 olaf.larsson

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Posted 20 January 2005 - 08:54 AM

Don´t you think its very strange, considering what we know and assume, that all mutationprone phenotypes are not associated with accelerated aging?

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#93

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Posted 20 January 2005 - 09:08 AM

You're welcome, John.

I don't have to tell you, Wolfram, that Werner's syndrome, a disease characterized clinically by premature aging, is on a genomic level characterized by an increased incidence of mutations due to impaired DNA repair. So one could make interesting assumptions on what the aging phenotype would be in an organism with extremely enhanced DNA repair. Decreased incidence of cancer would not be an outrageous conclusion.

Could you tell me one cell population type we would definitely not want an increased rate of DNA repair?

#94 olaf.larsson

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Posted 20 January 2005 - 11:06 AM

"I don't have to tell you, Wolfram, that Werner's syndrome, a disease characterized clinically by premature aging"


I mean: Why are is not every mutated DNA-repair associated whith increased aging????

We dont have a clear explanaition for the fact that WRN is associated with aging but other DNA-repair mutations are only associated to increased cancer rate. The fact that WRN is has strong connections to the telomeres and nucleolus makes the paradigm; "general mutation-->aging" very suspect. It looks like aging is caused directly by some special kind of mutations but not others.
I would take a closer look at the rDNA loss in the nucleolus. There is a rDNA loss which is similar to telomere shortening. The rDNA loss i directly proportional to the lifespan. Everyone knows about telomere shortening, but who knows about the rDNA loss? I have never ever heard about it before untill I read it recently.

Could you tell me one cell population type we would definitely not want an increased rate of DNA repair?


What make you think that Im opposed to the improvement of DNA-repair?

#95

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Posted 21 January 2005 - 02:18 AM

My question on DNA repair and cell populations was a didactical one. It relates to thinking about a cell type in the body where it is imperative that genomic rearrangements occur rapidly for the survival of the organism. A massive hint - antibody diversity.

I'm not following your line of reasoning on WRN and aging. Perhaps if you could make it clearer for me.

Now on to rDNA. It is an important observation that this area has not received enough attention from the research community. As you know, the enormous amount of rDNA produced is by merit of an enormous amount of gene duplication of the rDNA genes. The gradual genomic erosion that occurs across the genome would also affect this stretch of repeating DNA. The reason I am mentioning DNA repair even in this example is that it occurs upstream effect wise. In other words, it is my opinion that increasing the rate of DNA repair would also decrease the rate of rDNA loss because rDNA loss is one of the many effects of unrepaired DNA damage.

#96 kevin

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Posted 21 January 2005 - 05:49 AM

It is interesting to note perhaps that the SOS response causes a down regulation of the transcription of the 5S rRNA, tRNAs and other splicing related RNA genes by the dissociation of Casein Kinase 2 alpha subunits from the TFIIIB complex.

http://www.cell.com/...092867401004731

Here we report that RNA polymerase (pol) III transcription is repressed in response to DNA damage by downregulation of TFIIIB, the core component of the pol III transcriptional machinery. Protein kinase CK2 transduces this stress signal to TFIIIB. CK2 associates with and normally activates the TATA binding protein (TBP) subunit of TFIIIB. The â regulatory subunit of CK2 binds to TBP and is required for high TBP-associated CK2 activity and pol III transcription in unstressed cells. Transcriptional repression induced by DNA damage requires CK2 and coincides with downregulation of TBP-associated CK2 and dissociation of catalytic subunits from TBP-CK2 complexes. Therefore, CK2 is the terminal effector in a signaling pathway that represses pol III transcription when genome integrity is compromised.


It wouldn't surprise me that phenotypes associated with aging might be a result of transcriptional repression via this pathway.

#97

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Posted 23 January 2005 - 10:46 AM

Kevin, I think this type of repression may be transient and designed to temporarily hold off transcription until the DNA lesion has had an opportunity to be repaired. This is likely considering that the role of CK2 is to facilitate the assembly of DNA repair complexes by coupling DNA damage sensors to DNA repair systems which in turn repress transcription mechanisms.

From the article you cited:


Why Repress Pol III Transcription in Response to DNA Damage?

CK2-mediated pol III repression could serve three functions in cells with DNA damage. Toczyski et al. (1997) suggested that CK2 is important in the adaptation response to genotoxic stress because it plays a critical role in enhancing survival during checkpoint arrest. By extension, one function of CK2-mediated pol III (and pol I) repression in cells subjected to genotoxic stress could be to divert cellular resources from the energetically costly process of transcription to DNA repair and other processes required to maintain viability while the damage is repaired.

Two possible functions are more proximally related to the DNA damage events. In yeast, DNA repair is inhibited by pol III transcription, perhaps because elongating polymerase obstructs the repair machinery (Aboussekhra and Thoma, 1998). It follows that CK2-signaled repression of pol III transcription might be important in the DNA damage response because this repression ensures adequate access of the repair machinery to damaged DNA. Finally, we do not rule out a role for tRNA/rRNA downregulation in establishment of DNA damage checkpoints. Translational capacity will decrease in vivo when these RNAs are depleted in response to genotoxic stress, perhaps to levels at which key cell cycle regulators are not synthesized in sufficient amounts to promote cell cycle progression (Neufeld and Edgar 1998 and Polymenis and Schmidt 1999). In this respect, it is noteworthy that CK2 has been implicated in the regulation of translation and G1/S and G2/M progression (Hanna et al., 1995). Besides its downregulation functioning to repress pol III transcription in response to DNA damage, we presume that reactivation of CK2 after damage repair is necessary for restoring the cell's full capacity for translation


I would suggest the aging phenotype is the result of more permanent transcriptional repressors where DNA lesions have resulted in deliberate mechanisms of damage control such as altered methylation or incidental mechanisms such as loss of regulatory sites (such as promoters).

#98 kevin

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Posted 23 January 2005 - 04:06 PM

I think this type of repression may be transient and designed to temporarily hold off transcription until the DNA lesion has had an opportunity to be repaired. This is likely considering that the role of CK2 is to facilitate the assembly of DNA repair complexes by coupling DNA damage sensors to DNA repair systems which in turn repress transcription mechanisms.


I think you are right that this method of transcriptional control is meant to be transient however, it occurs to me that the systemic inflammation which increases with age might also indiciate that there could be a constitutive activation of this signal with concommitant systemic repression of these genes.

DNA damage is likely the initiator and at the top of many aging phenotype processes but this would be one mechanism by way of which DNA damage could affect a vast array of genes.

#99

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Posted 23 January 2005 - 10:24 PM

I agree, and can also see where regulatory aberrations of CK2 as well as many other related factors could make a transient system behave as if stuck on a repetitive loop resulting in a more permanent effect. Such transcriptional delays resulting from normal or aberrant CK2 et al regulation could propagate throughout the genome resulting in deficits of essential factors requiring expression and consequently decelerate or even bring to a halt the entire mitotic machine (senescence).

#100 kevin

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Posted 24 January 2005 - 03:10 AM

It is an interesting question... the only reason I know about it is because one of authors of the paper, Michael Schultz, mentioned it in his section of a eukaryotic gene regulation course I took last term. It might be worth pursuing with him if he's interested..

#101 Cyto

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Posted 27 January 2005 - 01:17 AM

Im moving something I was poking in about on the IBG thread.

-------------------
Hopefully we can take care of the flipase action occurring to the hydrophobic drugs soon.

Delivery of MDR1 Small Interfering RNA by Self-Complementary Recombinant Adeno-Associated Virus Vector

While they don't use any engineered knobs, fibers, fabs or moded capsids for target specificity the use of a self-complementary Adeno-assoc. Vector (scAAV) allows for it to no longer be dependent on second strand DNA synth for expression. Course next it would be nice to have a more targeted approach in a whole system, targeting the transcriptional profile sounds feasible.
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#102 Cyto

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Posted 24 August 2005 - 07:11 PM

More Insight to Photolesions in DNA

http://www.bio.com/n...ml?cid=13500002



In the current issue of the journal Nature, Bern Kohler and his colleagues report that DNA dissipates the energy from ultraviolet (UV) radiation in a kind of energy wave that travels up the edge of the DNA molecule, as if the energy were climbing one side of the helical DNA "ladder."

The finding lends insight into how DNA damage occurs along the ladder's edge.

It also counters what scientists proposed in the 1960s: that UV causes mutations by damaging the bonds between base pairs - the horizontal "rungs" on the ladder. The new study shows that UV energy moves vertically, between successive bases.

In undamaged DNA, there are no chemical bonds between vertically stacked bases. But the bases do interact electronically, which is why Kohler thinks they form an efficient conduit for UV energy to flow through.

"Even though paired bases are connected by weak chemical bonds, it's the interactions that take place without chemical bonds - the interactions between stacked bases - that are much more important for dissipating UV energy," Kohler said.

The Nature paper builds on work from five years ago, when the associate professor of chemistry and his team first discovered that single DNA bases convert harmful UV energy to heat to prevent sun damage in the same way that sunscreen molecules protect sunbathers.

Back then, they studied only single bases floating in water. They hit the bases with a kind of UV strobe light, and saw that the energy was released as heat in less than one trillionth of a second.

Their new experiments show that the behavior of full DNA differs profoundly from that of isolated bases. When the chemists turned their strobe light on whole strands of novel DNA, the UV energy still changed to heat eventually, but the energy dissipated a thousand times more slowly.

That's an eternity in the DNA universe, where scientists need to use special equipment just to see these ultra-fast chemical reactions happen. Yet, Kohler's team saw no evidence that the UV affected the chemical bonds between the base pairs. They surmised that the UV energy was leaving the molecule by traveling along the edges instead.

"This slow relaxation of energy is utterly different from the mechanism in single bases that transforms the energy into heat in less than a trillionth of a second," Kohler said.

"Eventually, the energy does turn into heat, but the important point is that the energy is retained within the molecule for much longer times," he added. "This can cause all kinds of photochemical havoc."

It could be that when base pairs are aligned in their natural state in a DNA strand, the electronic interactions along the stack provide an easier way for DNA to rid itself of UV energy, compared to passing the energy back and forth between the two bases in a base pair as scientists have previously thought.

In fact, it was the brilliance of James Watson and Francis Crick's discovery of the structure of DNA that kept this secret hidden for so many years, Kohler said. Their work revealed that the DNA helix was composed of paired bases, and that discovery led researchers to focus on how UV energy might interact with base pairs.

"In fact, so much attention has been paid to base pairing that this other interaction, base stacking, has been neglected," Kohler said.

Base stacking is frequently overlooked, he admitted, because the ladder terminology that we use to describe DNA structure makes us think that there are open spaces between successive rungs of base pairs.

A better analogy would be a stack of coins, he said. Bases are stacked right on top of each other.

Here's what he and his team suspect is happening during the UV energy wave: as sunlight warms our skin, UV photons are absorbed by the bases, causing their electrons to vibrate. These high-energy vibrations nudge the atoms in the bases around, but only along one edge of the DNA ladder at a time.

If all goes well, the DNA returns to normal after the energy wave passes. But some of the time, the atoms don't return to their original positions, and new chemical bonds are formed.

Scientists know that such accidental bonds create "photolesions" - injuries that prevent DNA from replicating properly. The details of the process aren't fully understood, but studies suggest that photolesions cause genetic mutations that lead to diseases such as cancer.

This new research helps explain why most photolesions are formed between bases on the same side of the DNA strand.

Scientists believe that proteins in the body repair DNA by removing photolesions and filling in new material, using the remaining DNA strand as a template.

If UV damage is confined to one side of the DNA double helix or the other, then the undamaged side makes an easy template for the proteins to follow. But if both sides of a strand were damaged, then the template would effectively be missing.

The Nature paper highlights the shortcomings of previous studies, which applied results from isolated base pairs to the full DNA molecule. "It turns out that you can't extrapolate the results of base pairs to whole strands of DNA," Kohler said.

The discovery has clear implications for biology, since it can help explain the DNA repair process.

"The ability to observe what happens to electronic energy in DNA on such short time scales also extends the hope that methods such as ours can finally determine how DNA is damaged by UV light in the first place," Kohler said.

The Ohio State chemists are now testing other DNA strands. For simplicity, they first wanted to compare the behavior of single Adenine and Thymine bases with strands entirely composed of those two bases. That is the work described in Nature.



#103 kevin

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Posted 27 August 2005 - 02:33 PM

Link: http://www.medicalne...=29625&nfid=mnf


Novel lipoplex nanoparticle to be used in 1st human trial treating advanced solid cancer
25 Aug 2005

The first clinical trial of a biologic nanoparticle designed to give back to cancer patients the tumor-busting gene they have lost is expected to start in September at Georgetown University Medical Center.

The phase I clinical study will enroll 20 patients with advanced solid cancers (including most common tumor types), and is the culmination of more than a decade of work by a team of researchers led by Professor Esther H. Chang, Ph.D. at the Lombardi Comprehensive Cancer Center.

Their research has led to development of a tiny structure -- measuring a millionth of an inch across -- that resembles a virus particle that can penetrate deeply into the tumor and move efficiently into cells. The device is a "liposome" -- a microscopic globule made of lipids -- that is spiked on the outside with antibody molecules that will seek out, bind to, and then enter cancer cells including metastases wherever they hide in the body. These molecules bind to the receptor for transferrin that is present in high numbers on cancer cells.

Once inside, the nanoparticle, which the researchers call a "immunolipoplex," will deliver its payload -- the p53 gene whose protein helps to signal cells to self-destruct when they have the kind of genetic damage characterized by cancer and by cancer therapies.

More than half of all cancer patients have cancer cells that have lost normal functioning of the p53 gene, so-called "guardian of the genome," and the Georgetown researchers believe that restoring the gene will improve the tumor-killing ability of traditional treatments.

"We are excited about the promise this nanoparticle has shown in animal tumor models, and are anxious to offer it to patients," said Chang, Professor in the Department of Oncology and Co-director of the Molecular Targets & Developmental Therapeutics Program at Georgetown.

The federal Food and Drug Administration granted approval for the trial to begin in late July. The work is being sponsored by grants from the National Institutes of Health and private foundations. Additional support comes from SynerGene Therapeutics, a biotech research firm with which Chang collaborates.

John Marshall, M.D., Director of Developmental Therapeutics and GI Oncology at Georgetown, will serve as the trial's principal investigator.

The researchers believe that immunolipoplex represents an advance over the viral "vectors" that have been used to deliver gene therapy, because these liposomes do not produce the kinds of immunologic response seen when disabled viruses are used to carry the payload. They also say that the nanoparticle is of a small uniform size and consistency, and has been proven to work in animals bearing tumor.

In preclinical research, Chang and long-term research colleague Kathleen Pirollo, Ph.D. have found that these nanoparticles substantially improve the tumor-fighting power of both chemotherapy and radiation therapy. These agents work synergistically with traditional therapies because the newly restored p53 protein helps push cancer cells that are now damaged to self-destruct.

"We believe this approach will make it difficult for the cancer cells to become resistant to therapy," Chang said. "As a result, cancers treated with these liposomal formulations should be less likely to recur after therapy is complete."

For example, use of these p53-loaded liposomes in combination with radiation therapy eliminated prostate and head and neck tumors in mice, which then survived cancer-free for more than 200 days -- until they all died of old age. Similar promising results were seen when the nanoparticles were combined with chemotherapy to treat animal models of melanoma and aggressive breast cancer.

Among the solid tumors approved for testing in the clinical trial are head and neck, prostate, pancreatic, breast, bladder, colon, cervical, brain, melanoma, liver and lung cancers.

Laura Cavender
lsc6@georgetown.edu
202-687-5100
Georgetown University Medical Center
http://gumc.georgetown.edu

#104 Matt

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Posted 27 August 2005 - 04:38 PM

Although a good step forward and hopefuly early results are encouraging... The fact that you still have to go through traditional toxic chemotherapy is a downside... But if the method saves more lives then great! ! ! I am looking more forward to hearing results back on the more targeted therapies where the treatment doesn't involve killing healthy cells in the process. I hope that the treatment is just as effective in human as when used in mice.

How long can we expect results from this trial?

#105 Cyto

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Posted 11 October 2005 - 10:56 PM

And the reality of Site Specific Recombinases come to fruition.

SSR's in action

Wonderful.

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#106 Karomesis

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Posted 04 November 2007 - 06:20 PM

can any of you pro's shed some light on why this may/may not actually work? http://web.mit.edu/n...ivery-0907.html

it seems like a very exciting development. [glasses]

I really think as soon as just ONE of these therapies works in human trials, SENS will become one of the hottest topics almost instantly.




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