Dr. Arote Rohitas

Seoul National University

Dr. Professor Rohidas completed his Ph.D. in Biotechnology in 2009 as Brain Korea 21 Fellow (2005–2009) from Seoul National University, Republic of Korea. After that, he worked as a postdoctoral research associate in the Institute of Agriculture and Life Sciences of the College of Agriculture and Life Sciences at Seoul National University, South Korea. From December 2010, he moved to the School of Dentistry, Department of Molecular Genetics, Seoul National University. Professor Arote explored biomaterials, nanobiotechnology, nanomedicine for gene therapy, drug delivery applications for cancer, neurodegenerative diseases, and inflammatory bowel diseases. Currently, Prof. Arote is working as an Associate Professor in the Department of Molecular Genetics. Dental Research Institute, School of Dentistry, Seoul National University, and his research group are pursuing various nanomedicine research projects to develop advanced nanomaterials for gene therapy applications and nanoparticles-based drug delivery systems (DDS) for cancer treatment, neurodegenerative disorders and also using nanoparticles for imaging and theranostics.

Aug 4, 2021 Presentation at the Global Conference for Lipid Nanoparticles & Other Non-viral Nanocarriers

Development of Multifunctional Nanomaterials for Gene Therapy Applications

Talk Transcript:


T&T Scientific Dr. Arote: Development of Multifunctional Nanomaterials for Gene Therapy Applications

[00:12]

Dr. Professor Rohidas

Thank you. So, thank you for inviting me on this particular T&T Scientific forum of this particular conference. Today I will be presenting all of our research work what we have done in a decade or so. So, it is typically about the development of biomaterials, specifically for gene therapy applications. So, I will share the screen, one minute.

[00:41]

Nima Tamaddoni, Ph.D.

Yes. You should be able.

[00:50]

Dr. Professor Rohidas

Yeah, this is. Well, it says, I hope everyone can see the slide. So, we are working on specifically these all different types of biomaterials, the nanomaterial for the gene therapy application. So, it is a DNA delivery, RNA, cell RNA, and even all the protein as well. So, going to detail as we all know a history of gene delivery, like in case of, obviously a generation one, like we have all these conventional drug delivery systems without any kind of particular applications but then coming to the specific applications and controlling the release of the drugs from the drug matrix, we have a generation second which is a targeted or controlled drug delivery and here we can control the relays of drugs which are highly, which have a low PK value as well as the others. Actually, we can get easily clear from the body and to avoid the repeated administration and the generation three obviously is gene delivery systems where we can use all this nucleic acid, therapeutics like DNA, RNA, SHRNA, SIRNA, and even micro RNA, and even now they are coming, the latest technology which is a crisper genetic, crisper Cas9. So, as we all know this gene therapy, it is nothing but the collection of different types of approaches for the treatment of human disease. So, within our genes, our instructions for the synthesis and control of the protein productions, which function to maintain the homeostasis of each cell? So, the sequence of genes which will be changed caused by the different elements which results in human being evolution

On the other hand, some will change will cause the production of dysfunctional proteins and the results that sometimes results in a cell sick or abnormal conditions or sometimes in a dead. So, once a human offspring begins an independent exercise, the genetic blueprint received from its parents determines many aspects of the way, the course of its life will run, and that what kind of illness will affect it. So, these are all gene therapeutic applications. So we have to correct what the wrong gene has been present in the body which is causing the particular disease. So, as we know that there are different types of applications of this gene therapy. So, more recently the genetic components of many other common diseases have been established such as cancer or heart disease and diabetes. So, many of these abnormalities require environmental stimulations to initiate them but without the genetic predisposition, the disease might never develop. So, these are the kind of inherited as well as the equations are the main target of the gene therapy.

So, as we all know, actually I am not going to repeat more in detail but then just like a basic, like this mechanism of gene therapy as DNAs and nucleic acids are ionic in nature, so we need the chronic polymers to ionically bound them in a complex and then this particular polyplex which we call a DNA polymer complex or DNA carrier complex, goes on the cell surface and depending on these times as well as the rate limiting state we call it, get absorbed on the cell surface and crosses the plasma membrane and undergoes the endocytosis. So, this endosome, early endosome late and then probably within that time if it the cargo or the DNA polymer cargo comes out of the endosome quickly, then it is good enough to enter into the nucleus and if it does not come out of the endosome even it is late endosome then it ultimately leads to the lysosome, and then degradation. So, our entire gene therapy applications and the designing of different types of biomaterials and Nano carriers depends on all these particular steps, how these nanoparticles are undergoing in this particular figure? So, there are four major elements of gene delivery, like a targeted disease, we have a therapeutic gene, we have a gene carrier and a gene expression finally. So, among these four, we are majorly focusing on gene carrier development, that is what we have a different types of. So, here we know there are viral vectors and non-viral vectors. So, till now many scientists have reported these retroviruses, lengthy viruses, Adeno viruses and all different type of viral vectors which are having a very successful result, specifically in terms of the delivering genes to the target site. Apart from that they have a various types of disadvantages because of their immunogenicity, toxicity, or carcinogenicity, or self-replications or sometimes the insertional mutagenesis and so on. So, substitutions have focus, I mean shifted their view towards the non-viral vectors, probabilities, a lot of lipids, nanoparticles or sometimes naked DNA as well, sometimes in some cases it works well and the combination of lipids and polymers that is the lipopolyplexes. So, by till today, this entire era, there are different types of viruses been used in clinical trials? And now in recent, I would say probably a five years or even seventy ten years around this time, there are a lot of non-viral vectors are also in the clinical trial, and they are very successful and this particular conference is about the lipid nanoparticles, so since this pandemic probably in a couple of years, we can see this tremendous use of lipid nanoparticles for the nucleic acid therapeutics.

So, in case of gene to be delivered successfully, we have extracellular barrier, intracellular barriers. So, once we overcome these extracellular barriers like this at the nucleus attack which is a system of circulations or tightly packed, endothelial cells prevent sometimes diffusion, and also sometimes these reticular endothelial systems which clear the materials easily. So, once we overcome these barriers and these particular steps by designing a versatile carrier or certainly not a carrier. We can overcome and we can achieve a success in delivering genes at the target side and then once this genes along with the polymers as the polyplex goes in the cell, then endocytosis at two, three different stages. Particularly in the acidic PH and even the late endosomes, little bit kind of a neutral towards the neutral. So, these kinds of technicalities, if you can achieve in a specific way, we could be able to achieve a good transfection and better transfection delivery, and have a better gene expression. So, as we can see the timeline of the gene therapy since 1990 in September, the first report of the successful treatment of brain channels and patients that with this severe combined immunodeficiency syndrome which has a particular adrenocindiamine deficiency.

So, since then actually there are a lot of success stories of this gene therapy except these two black boxes we can see in June 1999 and the October 2002, where there were death of the patients during the clinical trials. So, there were strict limitations been put on the gender because ensuring all the safety? So, in terms of any gene therapeutic material where we would like to develop, so we have to specifically think about these three major factors, like the specificity, efficacy and the safety. As we know this specifically is very important, like not only the desired cells nature needed to be targeted and not the entire cell to enter body. The efficacy is also very unique parameter where we can have a small dose and the high effect and the safety finally is like all the materials, all the vectors as well as the systems what we are using should be biocompatible non-toxic, non-immunogenic and should not generate any kind of a tumor formation. So, keeping in mind all these factors we have decided to use a carriers as a copolymer of polytechnic because we know that is polythene mean is something like very golden unique and golden standard polymer in case of the non-viral gene therapy and it has been widely used in vitro, in vivo as well and in by different types of roots of administrations. But the disadvantage of this polyethylene is that it is a toxic and it is a non-biodegradable and that is the reason it is not able to clear out the body easily. So, once it gets deposited, it creates a lot of toxicity, and it also because of the high charge density it has, I mean. So, it interacts with the different kind of blood components and the non-target cells and it ultimately leads to toxicity.

So, we have decided to use the polymers. I mean synthesize the polymers, hyper branch polystyrene polymers with the low molecular weight polyethylene. I mean since high molecular polymerization had a high toxicity, so we decided to use a low molecular weight polyphenol means and the copolymers of different monomers like polycaprolactone or glycerol. So, we have a long library of the glycerol or sugar based polymers. All these polystyrenes means will be, these are all things, and they have ester linkage, so they are very good soluble in water, they have a better DNA condensation and also because of the presence of the PI, we have a high buffering capacity and, you know, ultimately its endosomal escape. So, our first project was something to develop the polycaprolactone based polymers with the help of low molecular weight polyurethane. So, here as we know this polycaprolactone is a non-toxic hydrophobic and bio regular branches, so USFD Afroed polymer. So, the copolymer of this polycaprolactone and the polyethylene amine will lead to the synthesis of this polyester amine which will be bio gradable, will have high transfection efficiency, and the reduced high toxicity. So, we decided to use this polycaprolacton diacrylate with the molecule of 530-nanometer… Sorry. 530 Dalton, by the simple micro addition reaction, we synthesize this polyester amine. We confirm its synthesis by NMR and all of the spectroscopic techniques.

We have found that this particular polymer has a really a good half-life that is almost 4.5 to 5 days. So, it is the ultimate applications as well for the control release delivery. It has a particle size less than 200 nanometer indicating that it is a wonderful candidate for intracellular delivery and also the zeta potential and its surface charge leads, I mean shows that it has a moderate or kind of a within the limit surface charge in order to get absorbed on the hydrophobic cell surface. So, we have found that this synthesized polymer have a low toxicity and all this 293 FGM and a cell lines in all different concentration, I mean in gross dependent manner. But ultimately if you compare with the polyethylene mine with 25 kilohertz positive, you can see that it is highly toxic but compared with that carrier shows a low toxicity and much better cell liberty. Almost around 80 percent and that is a good sign to go for the animal studies as well. So, we checked the transfection efficiency by the luciferase by using the PGL3 as they put a gene and in all these three different cell lines it was found that this particular polymer which is in different, three different polymers we have using different feed ratios, showing the high transaction efficiency sign compared with lipopectamine and also PI 25, kilo Dalton P as a positive control. With another report region like a GFP, we calculated, we found that by using the flow cytometer also, like it shows a similar train and it indicates that the superiority of the carrier over the positive controls. So, by the errors of administration with using GFPN2, we found that a particular gene expression of fluorescence after the aerosol administration, it is even better than the PI 25 kilo Delta has a positive control. So, this all indicates that because of the presence of hydro this ester linkage, the polymer is not shrinking any kind of a toxicity and similarly, it has a high buffering capacity as well because of the presence of PI backbone.

So, it shows a severity in transfection as well. So, later we, further we decided to use the PG as a linker and conjugate the folic acid because until this stage we had no specific targeting. So, here we decided to go for the specific way to target the folate receptors. So, we decided to conjugate the folic acid to this particular polycaprolactone PI polymer. So, PG was used as a linker, as we know that PG is a non-toxic hydrophilic and ad fruit molecule and it increases solubility and as well stability as well, so that is the reason actually we decided to use this particular piece as a linker. So, we initially use a little high molecular PG which is a bifunctional PG, and it has a molecule of 5000 Dalton. So, this is the kind of a scheme of conjugation of this folic acid to the polycaprolactone to the PI copolymer. So, initially we have activated the folic acid using this GCCNHS chemistry and this activated folic acid in the basic PH, using pyridine we conjugated to this bifunctional PG which is 5000 Dalton molecular weight. So, this folate PG which will have this amide linkage was then added to this polyester amine, our carrier which is EDCNHS catalyst and this is a representative structure where we can see the folic acid moiety at the particular end. So, we confirmed the synthesis by NMR and it was found that almost 11.2 mole percent of the folic acid was conjugated to this polymer which is sufficient enough to target the folate receptors in the cell.

So, here we confirm the transfection efficiency with and without folic acid polymers and we found that obviously the folate conjugate polymers have a high a superior edge over the non-folate polymers indicating that it has a specific receptor made to endocytosis application. Later, we chose cells which are abundant in folic acid and which are less in folic acids like FJ2 and KB. So, KB is a mouth carcinoma cell line where the folate receptors are over expressed in abundant number. So, it was found that this particular cell line we can see the transfection diffusion is very, very high and this is a kind of application that indicates this particular, our carrier follows this reciprocated interceptors pathway. We also later decided to block those folate receptors by external folic acid and after blocking we again transfected the cells. So, we can see here in the figure C that with and without folic acid, the difference. So, this all figure shows that this particular system followed the receptor material in the site of this, specifically by a fully pathway and it was a good application. Then later we went down further using another carrier that is a glycerol based carriers and where we have used glycerol time, calculate. As we all know the glycerol is a non-toxic, hydrophilic and biodegradable polymer and also it is, moreover it is osmotically active. So, again the similar way, we use the low molecular weight polythene amine and the polyester amine was synthesized by the micro addition reaction. It was been confirmed by the spectroscopic techniques like NMR and we have found this all these biophysical properties such as half-life and zero potential, and the particle size as well. Those are within the limit and however much age over the other systems. So, transfection, we found that, in case of the transfection all these cell lines have G2 hela and also 293.

The polymer is showing a very good trend in terms of the transfection in a dose-dependent manner starting from this 5, 10, 20, 30 NP ratios, and also by using the GFP by flow cytometer. So, after the aerosol administration in the bulk semis, after the 48 hours of the gene expression time, the mice were dissected and found that this GDMPI polymer which is having high transfection and also I mean the green fluorescence of the GFP compared with the positive control which is a PI25k. So, this is again a non-specific way, then in similar category we have a synthesized another carrier which is a based on the glycerol acrylate. So, since this sugar alcohol, like glycerol di methacrylate, glycerol diacrylate, glycerol tri methacrylate as well as the further like lactitol, manitol, xylitol, all this sugar alcohol library, actually we have successfully synthesized further on and then got a good results in terms of gene delivery. Then there is another new carrier that is the lactol. In similar way we synthesize it in very better and successful way and it was no sign of any kind of toxicity even at high concentration compared with the positive controls and we found that because of the presence of this I mean the sugar alcohol, this particular carrier have a specificity for these special glycoprotein receptors in the liver.

So, since it is a sugar alcohol and in the liver the parenchyma tic cells of the liver under, they over express this issue like glycoprotein receptor. So, this is particularly a mechanism actually we kept in mind and then decided to target the liver cancer. So, initially we synthesized this particular carrier by using the lactitol and then making the lactorel diacrylate and then further this lactorel diacrylate was then added to this branch, PI using the Michael addition reaction. So, this particular lactitol or like a free hydroxyl group, particular part is responsible for this caviar uptake stimulating part and the basic one which is the central one is the extra linkage, it is responsible for the hydrolysis and it is a degradable part. So even there is a long polymer we can get degradation at this particular points and the amine functionality part is responsible for the DNA binding. So, in this way this career was designed and then we have confirmed the toxicity and the different cell lines and it was found that even at a high concentration like a 30 NP ratio, we can see even 80 percent of the area or 90 percent of the cell viability, indicating that we can use this carrier for the cell transfection in the FG2 cells. So, in vitro we found that almost there were 13 fold higher reciprocal, increase higher luciferase transfection was observed. So, later we thought like whether really polymer this, this polymer is really following, this recipromator endosaresis pathway. So, we block this parenchyma tic cells, I mean the liver have G2 cells by the external galactose and even at 10, 20 and 40 millimole concentrations using. You can see here the decreasing trend of the transfection. So, this indicates that like once we block those extra glycopotent receptors, by the external galactose. I mean external material, so there is no increase in the transfection. So, this pathway indicates, this competition assay indicates that this sugar alcohol based polymer that is a polyelectrol-based polymer showed the receptor-made endocytosis for the liver cancer treatment.

Well, because of the presence of this PI backbone, we have a proton sponge effect and as we all know that once we block this protons sponge by using this bactylomycin, so we cannot get high transfer efficiency. So, as for the JP Bear who has, we showed this particular effect in 1997. So, this is again, still it is a very active and we can see that because of the presence of PI, the buffering capacity is very low and with this PI’s buffering capacity we can easily, they count the endosome and the content gets released in the cytosol. So, with this carrier when we got this in vitro success then we decided to conjugate a molecular probe that is SI 5.5 and then delivered to these mice by the interference distribution just like a normal bio distribution and this feature we just wanted to check which organs have been targeted most. So, we can found that actually liver is one of the best one of the organ which is targeted most by this Nano carrier. So, it indicates the specificity for this liver, so we decided to go for further using… his irony, the project is ongoing right now. Then next is basically from this orbital. So, next carrier is from sorbitol, so it is already been published by Dr. Arif-ul-Islam who was in Harvard and I think today he is joining this session I guess.

So, he already reported this particular carrier and it has a high success because of this particular objective we can see here this endosomal escape, a less cell toxicity and also it avoids electron degradation because of this high buffering capacity and the targeting of this particular moiety. So, these all parameters, what actually we need in particular carrier in order to develop a successful gene therapy system. So, this orbital dimethylate was using in this particular synthesis step and then we use a linear polyphenolumin here by the micro addition, we can see here this transporter part that is called as a PSOAT transporter part which has been synthesized from this orbital diameter and the low molecular weight polyethylene. Then later we went on increasing a more specificity by conjugating the folic acid. So, here also again like, you know, we conjugated the folic acid carrier and then decided to go to target the folate receptors. Then we have used the opponent as a SIRNA where it, this open actually paralyzes the mitochondrial dysfunction and ultimately leads to the apoptosis. So, here by the western blot we can see the expression of this O-Parma Protein which has been kind of regulated comparedly with the other polymers, other candidates and the number of tumors which you can see here, decreasing the number of tumors indicates that the cells are… the tumors is undergoing the apoptosis and compared with the control and the even the scramble part. So, scramble its RNA. So, this is kind of a comparative study which indicates that this particular carrier already been reported for the DNA delivery successfully but here we have used it for the SIRNA. So, this particular part actually of the research is under communication right now. Then further one, the last part actually I will just summarize in a quickly. So, there are the two new polymers we are using right now and we have synthesizer and character as well, there is a diuretin dimethylate, another is a triton glycol dimethylate. So, these two carriers have a unique, specificity that they are responsible; they are delivering material to the nervous system, specifically for the microglia.

So, as we all know this microglial polarization, once we activate this microglia because microglials are the immune cells of the nervous system. So, there are, after the activation or polarization of this microglia, there are two stages of the microbial that is M1 and M2. So, M1 is responsible for the pro-inflammatory properties such as damage or cell death and so on and there is the other stage that is M2 which is anti-inflammatory; it has intermediate properties like neurogenesis for the repair or kind of a new synthesis of the cells. So, both the stages of the microglia have their own advantages, and they are like very important to target and polarize this microglia. So, here we have decided to use this two particular carrier that is kind of a diuretin dimethylate based polymer and another is a triphylline glycol dimethylate. So, this polymer in the similar way, we synthesize characterizing spectroscopically well, check the toxicity and there is no sign of toxicity, also check the physical chemical parameters like particle size and zeta potential, and these are very unique and fantastic to use for this intracellular delivery.

So, here we, initially we have used this to check transfection efficiency and then later on we went on… later we went on using those for the particular microglia specific cell lines. I mean the mix, primary cell culture of the mix glia cells. So, here by the intrathecal administration, initially we checked the transfection in case of the mix microglia primary cell lines and then later on we decided to use SRNA to target the microglia using this intrathecal administration. So, initially we use one microgram and five microgram concentrations and comparatively use like which is the best one. So, using the five micro, five microgram and as well as one microgram, there was not much difference found here. So, equally the microglia, the astrocytes and even the other neurons were being targeted. So, here our purpose is to target only a microglia but not the astrocyte. So, we decided to go to use a specific peptide or antibodies which are responsible for… which are high specificity for the microbial. But before that we got an, even when in the kind of initial raw results, we found that it is something very specifically targeting the microglia and not much content or not much percentage of the astrocytes and even neurons are being targeted. So, even at low concentration like one microgram, so one microgram here henceforth we use for this particular cell studies and then again we check the transfection in the primary mixed clear cells here and it was found that I even had and ratio 20 and 30 which is the higher input ratio, you can see this particular polymer that is a urethane dioxide dimethylate. This carrier shows very good signs of transmission almost 20 to 30 fold higher transfection compared with PI25 Dalton. So, this indicates the superiority of carrier compared with the other carriers and also the positive control like a lipofactamine which is a market and commercially available. So, this indicates that, again the neuronal cell because those are again is very difficult to transfect compared with the other cell lines like BB to cell line.

Both the polymer shows high transfection efficiency in all possible concentrations even the 5, 10, 20, and 30 in a dose different manner. Indicating that these are all kind of a library of polyester in base carriers and the sugar alcohol based carriers, like a polycarpalactin and the glycerol di methylate, glycerol tri methylate sorbitol and we have even the xylitol, manitol and also erythritol. So, these are kind of a sugar-based, sugar alcohol based carriers and we have a huge library and all are like successfully been used in vitro and innovative as well. So, I just would like to say specifically that a unique ability of this polyester means to target the microglia in brain. Actually brain is very significant platform for this neurodegenerative disease and now we are working on this neuropathic pain, traumatic brain injury as well as this spinal cord injury. So, thank you very much for your attention. So, these are our collaborators from Korea and overseas. Thank you. Thank you for your attention.

[29:49]

Interviewer

Yes. Thank you very much, it is very informative. We have a few questions. The first is great talk one of the slides said aerosol administration, why the inhalation route and not Ivy subcutaneous or intramuscular?

[30:10]

Dr. Professor Rohidas

Well, Ivy probably again like we need a very small concentration to check in, in case of Ivy and there is a high chances of even death or casualty as well. But since aerosol, why aerosol because we know that lung is having a high surface area, so it is better and it has because of the high surface area, it can be even more possible to go and having more transfection efficiency and also, you know the area to work on for the gene expression. So, now I realize that actually this was work I think long back but now I can see that in case of the COVID pandemic, probably if you can use in particular genes or kind of SRRNS to combat the COVID, this particular, in case of the aerosol administration, this kind of a carrier will probably have a high age. I mean probably will have a success in case of the error administration, kind of just a speculation. 

[31:11]

Nima Tamaddoni, PhD

Yes. Alright, so there is another, there are more than 300 gene delivery, I imagine candidates in clinical use for an adeno-associated virus and few and market. How many gene delivery uses are there for polymer lipid-based carriers in the clinic and in the market? Thank you.

[31:32]

Dr. Professor Rohidas

Well, recently, I mean this particular decade have seen a lot of surge of these non-viral carriers since they have a, there is a lot wide variety of the scope. I mean there is a lot of scope for the modifications in different way, like depending on the disease requirements, so that is one of the best advantage but in case of the viruses we cannot have that. Again the self-replication is a major disadvantage and limitations for all the science scientists to focus on so that is the reason actually the shift has been focused towards the non-viral carriers, there are a lot of, so many since starting from the dogs electric, dogs is one of the first Nano medicine. I mean material been used in long back. But since then again in case of the gene therapy, there are plenty of gene therapy carriers around the clinical trial, and specifically modern and then lipid as well. Yeah 

[32:26]

Nima Tamaddoni, PhD

Yeah. So, another question, delivering the formulations by intra fecal administration is painful. Are you considering alternative delivery vehicles proud inspection? 

[32:40]

Dr. Professor Rohidas

That is a good question because at the initial point actually we thought like let us target the microglia, so rather than using the systemic circulation by crossing the BBB, we thought let us initially have this, whether our carrier is really targeting microglia or not because in this particular case we have to specifically target the microglia, not the astrocyte, so that is our main objective. So, initially we thought to have the intrathecal and absolutely it is a painful. It needs a skill also to deliver those particular Nanos system. So, now we are actually optimizing the systemic application using the system circulation and crossing the BBB. So, here actually we are finding out the particular lipophilicity of the carrier as well because it needs to have, once they are being used for the BBB crossing, crossing the blood band barrier. So, yes it is in pipeline.

[33:37]

Nima Tamaddoni, PhD

Very good! Alright. What do you think on why the polymeric Nano formulations such as PEI which is in research area for several decades now are not up to date or affect and are not available for clinical use as IMPs progressed recently? 

[33:58]

Dr. Professor Rohidas

Well, PI alone is obviously it is very toxic. It is very good in transfection but it is toxic as well because of its non-biodegradability, so there is no way we can use the PI as it is. But then there is a lot of applications as a PI copolymers using different other polymers as well. So, these copolymers having have very good success even the bob langer lab, actually long back, in fact I must remember, I think it was in 2002 itself, they have published a paper in Jax and showing is an almost ten thousand polymers or I mean, sorry two thousand polymers, a library of by hard report screening and those are all like obviously I mean functions and as well as the PI based polymer as well. So, there is a lot of work going on in PI based polymers but yes as it is a PI or alone PI, there is no success. I mean, sorry, there is no chance of kind of a safety because toxicity is very high in case of the PI.

[34:57]

Nima Tamaddoni, PhD

Dr. Rice, thank you so much for your time, it was very informative, and I appreciate that. There is about, we have another Q/A session that goes for another 15 minutes or so on the other side. But we want to thank you for your time and your ability to answer all of our questions, much obliged thank you very much.

[35:17]

Dr. Professor Rohidas

Thank you.

[35:17]

Nima Tamaddoni, PhD

Thank You so much.

[35:18]

Dr. Professor Rohidas

Thank you very much.

[35:19]

Nima Tamaddoni, PhD

Thank you so much. What time is it right there?

[35:20]

Dr. Professor Rohidas

Well, it is 12:20 in India.

[35:27]

Nima Tamaddoni, PhD

Thank you so much. Thank you.

[35:30]

Dr. Professor Rohidas

Thank you. Thank you very much for having me there. Thank you very much, looking forward to interact with you. Thank you. Thank you.

[35:37]

Nima Tamaddoni, PhD

Bye-bye. 

[35:08]

Interviewee

Bye.

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