 
|
|
| SLIDES
& TRANSCRIPTS
Saturday,
December 6, 2003
Highlights of GU SPORES
John C. Morris, M.D. |
| Slide
1: |
 Thank you, Dr. Gomez.
TOP
|
| Slide
2: |
|
 As the other speakers have preceded me, I will very briefly review the projects in the Mayo prostate SPORE and then highlight the projects I am most familiar with, which is the one at the bottom of the page that we call project number six.
The Mayo prostate SPORE consists of this list of six major projects. There are also two developmental projects and career development awards that are not listed on this.
Dr. Steve Thibideaux
is a cancer geneticist who leads project number one, entitled genetic
susceptibility in prostate cancer. The second project is a biomarker
study led by Charles Young and George Klee. The third project is another
cytogenetics and cancer genetics study led by Dr. Bob Jenkins. Project
four is an immunotherapy project led by Dr. Estaban Selles, and the
last two, including the one I will highlight are both gene therapy
protocols. Project number five uses fusigenic membrane glycoproteins
as a therapeutic gene. That is led by Richard Vyle,
TOP |
| Slide
3: |
|
 and my project, which I will spend the rest of my few minutes highlighting, uses the sodium iodide symporter as a therapeutic gene for prostate cancer.
TOP |
| Slide
4: |
|  This is an I-131 total body scan of a patient with metastatic thyroid cancer. You can see the head here, the legs would be down here. This is excretion of the tracer in the bladder. The shoulders are here. You would expect to see a large black spot here at the base of the neck, but you don't see that because this patient has already had a thyroidectomy to remove their thyroid. What you do see that is abnormal here is diffuse uptake over both lung fields of the radio iodine.
This not only allows the demonstration of the presence of these metastases in this patient, but also allows effective therapy, using therapeutic doses of I-131. However, this therapy although quite effective for thyroid cancer, is only effective for thyroid cancer because other cell types do not express the sodium iodide symporter sufficiently to allow uptake of radio iodine to this degree.
This is the basis of our project, which is to induce uptake of radio iodine by gene transfer, the sodium iodide symporter to prostate cancer.
TOP |
| Slide
5: |
|
 This is a cartoon of the sodium iodide symporter. It is a membrane bound protein. The amino terminus lives outside the cell. The carboxy terminus is inside the cell. It expands the membrane 13 times.
TOP |
| Slide
6: |
|  It normally resides at the basal lateral surface of the thyroid follicular cell, where its expression is driven by the interaction of TSH with the TSH receptor through cyclic EMP dependent mechanisms.
The symporter mediates the movement of iodide from the extracellular space into the cytoplasm. In cell thyroid cells there are other channels that mediate the movement of iodine then outside the cell into the follicular lumen, where it is used for the protection of thyroid hormones.
The most commonly recognized mechanism of this movement at the apical surface is the pendra gene. The reason I highlight this is that other cells that don't normally metabolize iodine do not have these efficient mechanisms for moving the iodine out of the cell. So if one can induce uptake of iodine by expressing the NIS gene, the iodine resides in the cell for a quite reasonable amount of time.
TOP |
| Slide
7: |
|
The NIS gene is expressed primarily in the thyroid. It is expressed to lower levels in salivary, lachrymal glands, gastric and colonic mucosa and in the breast, particularly during lactation.
TOP |
| Slide
8: |
|  We found early that one could induce a very high level uptake of radioactive iodine in LNCAP cells after stable transfection, using PSA promoter to drive expression. This uptake is considerably higher than one normally sees in thyroid cells, at least in part because of this absence of flux that occurs once one induces uptake in non-thyroid cells.
This uptake, because it was PSA promoter driven, was androgen sensitive, and it is completely inhibitable by perchlorate, which is a competitive inhibitor of the NIS gene. TOP |
| Slide
9: |
|
 The protein is expressed and not expressed in the absence of androgen,
TOP |
| Slide
10: |
|  and it is targeted correctly to the membrane through this expression.
TOP |
| Slide
11: |
|  We move very quickly to in vivo preclinical studies using a nude mouse model, initially using stably transfected LNCAP tumors compared to control tumors.
TOP |
| Slide
12: |
|  We found that one could induce enough uptake of iodine through this expression to image these tumors very well.
This is an I-131 total body scan of a mouse harboring a NIS expression tumor over the right hip and a control tumor over the left hip. You can see uptake in the thyroid and stomach and excretion of the tracer in the bladder.
TOP |
| Slide
13: |
|
 After a single therapeutic dose of three millicuries of I-131, you can see a very dramatic volume reduction in the tumor as compared to continued growth of the control tumors, or in the NIS expressing tumor in the absence of radioactive iodine.
TOP |
| Slide
14: |
| 
We constructed
then an adenovirus to drive this expression, fusing a non-specific
promoter to the CMV promoter. Our initial studies in stably transfected
cells used PSA promoter, but that promoter was too large to incorporate
into the expression cassette of the adenovirus, so the studies I
will show you next used non-specific promoter to the CMV promoter
to drive this expression.
TOP |
| Slide
15: |
|  To make a longer story short, in the same animal model, but now with direct injection into LNCAP tumors of the adeno CMV construct as compared to control construct injected into tumors on the opposite hip, and a single therapeutic dose of I-131, three millicuries, one can see very dramatic volume reduction in the tumor over a period of one to five weeks, with continued growth of the control tumors and the tumors injected with NIS, but not exposed to radioactive iodine.
TOP |
| Slide
16: |
|
Just an example of a volume reduction, about 85 percent reduction in volume, as compared to continued growth of the controls.
TOP |
|
Slide 17: |
|  These are the reasons that we believe NIS has significant potential as a therapeutic gene for gene therapy for prostate cancer, but also of course for other tumor types. First of all, it is a normal human protein. It is not toxic in itself and is normally expressed in the thyroid. As I have shown you, it induces highly effective uptake of radioactive iodine in prostate cancer, but we have also shown to date this is true for breast cancer, colon cancer, myeloma, and a couple of other tumor types.
It is possible through targeting, although targeting is still problematic, to have very high specificity. More recently we have used a probasin promoter to induce prostate specific expression of the NIS gene.
As compared to other trans genes currently used in gene therapy, it is possible as I have shown you to directly and non-invasively monitor NIS expression using diagnostic doses of radioactive iodine. So one can monitor where the gene is after it is injected, and equally importantly, monitor where it is not, by using non-invasive means.
Because radioactive iodine is a beta particle emitter, I-131, and this beta particle traverses the tissues up to two millimeters, one achieves a significant bystander effect, meaning that not every cell needs to be transfected to be killed by the cell.
Finally, the active moiety, the therapeutic moiety of this, is not the gene itself or the NIS gene, but it is radioactive iodine, which of course is currently FDA approved for treatment of human disease, thyroid disease in particular, and we have more than 50 years' experience using it to treat thyroid cancer, and we understand therefore very well the therapeutic window and safety issues regarding radioactive iodine.
TOP |
| Slide
18: |
|  We have therefore designed a phase one clinical trial in which this construct, the adeno CMV NIS construct, will be directly injected into recurrent prostate cancer in the prostate bed in men who have failed external beam radio therapy, using a setup identical to that practiced routinely for prostate brachytherapy.
TOP |
| Slide
19: |
|  These will be men that have locally recurrent prostate cancer after external beam radio therapy, using multiple doses of the adeno CMV NIS, ranging from ten to the eight to ten to the 12th particles. Delivery method, directly into the tumor using a setup similar to prostate brachytherapy. The protocol calls for measuring uptake and monitoring expression of the gene non-invasively, using diagnostic doses of I-123, performing dosimetry, calculating a predicted dose effect, and then administering therapeutic I-131 up to a maximum of 200 millicuries, which is a standard dose given to patients with metastatic prostate cancer.
The protocol also calls for protecting the thyroid by giving the patients t3 cytomel prior to the administration of the virus, because as I showed you earlier, NIS expression in the thyroid is regulated by TSH, and if one suppresses TSH by thyroid hormone, one can reduce expression and therefore uptake of radio iodine in the thyroid. TOP |
| Slide
20: |
 This is the status of the protocol, the current status of the review process and regulatory issues that are quite rigorous, as you would expect for gene therapy protocols. We have completed a series of toxicity and biodistribution studies in normal rats that were designed in collaboration with the FDA. We have developed protocol and consent forms. We made a RAC presentation last summer and have completed that process. We have had local review by the Mayo Cancer Center , and we have a pre-IND meeting that is currently scheduled for next month. We will be depending upon the outcome of the pre-IND meeting.
We are prepared to submit our IND application. We are in the process of producing clinical grade virus in the Mayo core production facility, led by Dr. Mark Federsville, and the IRB review will come after that. We are hopeful of recruiting our first patients in the summer of next year.
TOP
|
| Slide
21: |
|  At the last few seconds here, I'll show you some more recent data using a prostate specific adenovirus that I alluded to earlier. This construct uses a probasin promoter, the construct that we obtained through collaboration with Dr. Bob Matusik of Vanderbilt.
TOP |
| Slide
22: |
|  You can see that in vitro, this construct induces very high level uptake of radioactive iodine, even higher in LNCAP cells than that induced by the CMV construct, but it is much more specific, in other words, not active in other tumor types that are not prostate in origin, as compared to the CMV construct, as one might expect.
We are currently completing additional preclinical studies with this, and have shown to date also therapeutic effects in the animal model.
TOP |
| Slide
23: |
|  This is the last slide, which lists at least a partial list of the investigators involved in this particular project. I think the comments from Dr. Gomez earlier regarding multidisciplinary thyroid supported by SPORES, this represents a very good example of that.
Thank you.
TOP |
|
