Slide 1

After these interesting clinical talks, now I will move to a pre-clinical setting because I will try to address what has been done in gene therapy in the treatment of acute renal failure which is a pre-clinical state as you know.

Slide 2

You already have heard the complications and the increased mortality of acute renal failure and in native kidneys but on the other hand even in renal transplantation acute renal failure entails a poor prognosis, those patients displaying a DGF, delayed graft function have poorer graft outcomes.

Slide 3

On the other hand, it has been hypothesized that renal ischemic injury may originate or trigger inflammation, chronic inflammation which may lead to chronic renal damage.

Slide 4

On the other hand, you know that there has been a lot of concerns around gene therapy in general but more recently there is a renovated interest of this therapy especially in the oncology field. On the other hand, some inherited diseases or even acquired diseases are considered that could benefit from the gene therapy approach. On the other hand, I would like to review with you the potential promising uses of gene therapy in acute renal failure.

Slide 5

When we decided to start with this research, what we thought was which target should be addressed and then by which technique.

Slide 6

Considering the targets to be addressed by gene therapy we could target mediators of ischemia/reperfusion injury but on the other hand also those involved in nephrotoxicity.

Slide 7

To maintain needs you know exists in gene therapy the initial one was based on the use of viral vectors that have a clear advantage which is the great efficiency but on the other hand there are some important disadvantages among them perhaps the high immunogenicity of the viruses used. On the other hand and because of that non-viral strategies were started with the potential advantages that these strategies are cheaper and you can transfer large amounts of DNA but the main disadvantages are the low efficiency and the variability of these techniques for gene transfer and because of that you’ll see later on some modifications, some what we could say galenic modifications have been introduced to increase the efficiency of these non-viral transfection techniques.

Slide 8

Among the targets one of them has been hepatocyte growth factor. Hepatocyte growth factor you know is a pleiotrophic growth factor with multiple activities, it’s motogenic, mitogenic, has a very clear anti-apoptotic effect and even has been considered to be morphogenic. However, hepatocyte growth factor protein per se is very unstable in blood circulation because of its very short half life.

Slide 9

Nevertheless, it has been studied in several models of inflammation and ischemic injury. Among them you may see this rat model of inflammatory bowel disease in which they used hepatocyte growth factor of protein reduces the histological score and also the expression of pro-inflammatory molecules.

Slide 10

In a model of myocardial infarction in mice the injection of a hepatocyte growth factor, the protein overexpresses, an anti-apoptotic protein reducing the apoptosis in cardiomyocytes. In the renal filed we have also seen even more than 10 years ago 2 interesting papers. One of them a nephrotoxic model of acute renal failure in which the use of hepatocyte growth factor resulted protective in this kind of model and the same was true in an ischemia/reperfusion injury model because this growth factor accelerates the recovery of renal function.

Slide 11

In another step forwards some authors this Japanese team started using gene transfer not the protein, the gene transfer of hepatocyte growth factor directly in the kidney in a model of cyclosporine

Slide 12

A nephrotoxicity displaying that the use of the gene transfer of HGF reduced epithelial to mesenchymal transition estimated by the alpha-smooth muscle actin staining and decreased the inflammation evaluated by the ED-1 staining.

Slide 13

The group of Doctor Liu from Pittsburgh who has led the research around hepatocyte growth factor showed that a single injection of naked plasmid encoding hepatocyte growth factor exerted a protective effect in this model of acute renal failure in mice.

Slide 14

More recently and in this case using a viral vector, adenovirus the transfer of the gene of an anti-apoptotic molecule bcl-2 gene transfer resulted protective in this model of acute renal failure and this beneficial effect was associated with a reduction in the apoptosis, in the renal cell apoptosis and also interestingly in the reduction in the generation of reactive oxygen species reducing the oxidative stress.

Slide 15

Another model of renal failure as you know, is in the context of sepsis, by injecting LPS uremic renal asepsis and these authors interestingly evaluated the non-viral delivery efficiency of the gene of β2- adrenoreceptor and they estimated that by this technique they can induce an overexpression of this receptor in different parenchyma.

Slide 16

Another approach has been addressed, the MCP-1 and in this experiment they reported also a beneficial effect on renal function.

Slide 17

Another way, another non-viral strategy in gene therapy is the use of electroporation. Electroporation has been shown to be effective introducing DNA into the organs in animal models such as liver, kidney, skeletal muscle. It is free from oncogenicity and improves the efficiency of the transfer by 2-3 orders of magnitude.

Slide 18

In this regard this Japanese group has also studied the electroporation of electroporation-mediated hepatocyte growth factor gene transfer in this model of cyclosporine nephrotoxicity. You can see here in the picture the clear reduction in the inflammation in the interstial inflammation which was persistent 2-3 weeks after electroporation.

Slide 19

Our group was interested also in how to increase gene transfer of hepatocyte growth factor by using electroporation and we published this galenic experiment using a study in two different voltages and you can see here that by using the injection of the gene in the muscle and the electroporated muscle we increased the exposure to hepatocyte growth factor protein and also we were able to detect significant concentrations of the protein in the different organs. Because of that we used this approach to treat diabetic nephropathy in a type II 1 model of diabetes in rats.

Slide 20

Also we have employed this technique in the prevention of chronic allograft --- model, a very well established model of chronic allograft nephropathy recently reported. In the next few slides I would like to show you our experience in the direct electrotransfer of hepatocyte growth factor into the kidney in comparison with the use of the muscle route.

Slide 21

We did that, we injected the gene into the renal artery and the kidney was electroporated by using 6 pulses in comparison with injection in the muscle also electroporated.

Slide 22

We used a plasmid pCL-neo vector.

Slide 23

As you can see here, after the electroporation, after 45 minutes of warm ischemia you can see that the protein is observed in a vascular distribution and mainly around – in the renal parenchyma. Here on the right side you have the levels at 10 days post electroporation which are quite significant and you may see the evolution of the hepatocyte growth factor kinetics after electroporation with sustained levels until day 10.

Slide 24

Concerning the expression of the rat protein because we transferred human gene, you may see that this technique increases the expression of rat hepatocyte growth factor and when we use both routes muscle electroporation and kidney electroporation without significant changes of the expression of rat TGF-β than when we analysed the ratio between the rat TGF-β and rat hepatocyte growth factor you see that the most beneficial balance between these two growth factors is in the group of kidney electroporation which suggests that in this group we reach the best anti-fibrotic balance.

Slide 25

Concerning the evolution of renal function you may see that the kidney erythropoietic group displayed a faster recovery of renal function in comparison with the 2 other groups.

Slide 26

Concerning the apoptosis, cellular death we also saw that the gene transfer reduced apoptosis by using two techniques.

Slide 27

And this was also true for tubular necrosis but there is a trend for a better or a lower reduction of the apoptosis in the kidney electroporated group.

Slide 28

Here you have an illustration of what I was saying in the previous slide. Concerning cell proliferation and regeneration you see that the faster regeneration was observed in the kidney, the electroporated group, much less in the muscle electroporated in comparison with the ischemic control group. But there was a less severe effect concerning regeneration evaluated by tubular mitosis.

Slide 29

Concerning the inflammation there was also a transfer of lower inflammation in the kidney electroporated group. Now and after this properly considered gene transfer I would like to address the opposite strategy which is the gene silencing by using a small interferon RNA.

Slide 30

The group of Zheng published last year a very interesting approach silencing two main genes involved in ischemia/reperfusion injury, complement 3 and caspases 3 genes.

Slide 31

You may see here that in both instances by silencing C3 or both at the same time these authors observed a functional protection according to the lower levels of blood – and also serum creatinine levels but interestingly it seems that the combined strategy provided or displayed a longer graft survival, a higher survival. They also evaluated the efficiency of this gene silencing in which you may see very low levels of C3 RNA and the C3 caspases in the treatment groups.

Slide 32

Finally this group has been awarded in the last congress of the American Transplant Society in San Francisco last month because of this interesting and elegant work. They decided to target 3 molecules, 3 genes involved in ischemia/reperfusion injury; TNF-α, Fas and C3 and they diluted these small interferences of RNA for these genes in their university with a containing solution in a model of heart transplantation and after an extended cold ischemia time 2 days is not a clinical cold ischemia time for heart transplantation as you know and despite that these animals displayed a cardiac normal function and the main findings of this work was prevention in the apoptosis and necrosis and also a reduction in the inflammation of these organs.

Slide 33

So I would conclude very briefly saying that by using gene therapy in the treatment of acute renal failure there are several potential candidate genes to be targeted. It seems we have to optimise the transfer efficiency and in the case of organ transplantation we would be able to manipulate ex vivo the organs in promoting the transfer in an ex vivo model allowing the transfer by using electroporation and perhaps in the future we’ll rely on the combined strategy, combined gene and cell therapy and perhaps this will lead us to have promising pre-clinical data.

Slide 34

Thank you very much for you attention and I would be delighted to answer your questions.

Slide 35

Question: Hello, may I have a question Doctor Grinyo? Thank you very much. My name is Marc De Broe. Thank you very much for this very important and very interesting overview of this new application. I just want to join you that we have to focus on combined therapy because all the treatments which we have developed in experimental medicine be it growth factors, be it anti-inflammation, be it stimulation of perfusion never have worked in human medicine. It works in animals, it works in mice I think the only solution is that to combine gene therapy of anti-inflammation for example, combining that with regeneration genes or genes generating the circulation I think that’s the future. I thank you very much.

Dr , Grinyo: Thanks for this comment I fully agree with your suggestion in fact, we have already started doing that because we think that gene therapy using this kind of genes can allow or enhance the properties of the cells injected and in that way we could increase the efficiency especially in the regeneration of the organs which could be very, very important in the case of native acute renal failure. Thanks for this comment.

Chairman: I think that if there are no other questions, I would like to thank you all for your active participation and the previous speakers for their clear contributions. Thank you very much.