Development of Non-Viral Carrier for Gene and Cell Therapy - Research Paper Example

DEVELOPMENT OF NON VIRAL CARRIER FOR GENE AND CELL THERAPY DISCUSSION Introduction This research was started with the aim of investigating how different methods used in non-viral carriers for gene and cell therapy support the efficacy of gene and cell therapies. The rationale for this aim was based on the weaknesses that had been associated with the use of viral carriers in the same process of gene and cell therapies. Because the study was focusing on the outcome of different methods with the non-viral carriers, the method used in the study involved the testing of the effect of different forms of non-viral carrier components on gene and cell therapies. This was largely done by transfecting the c2c12 and h2kb cells with generation 3 dendrimer/dna, generation 3 dendrimer with arginine attached to it/dna, generation 3 dendrimer with aspartic acid attached to it/dna, then pei/dna. This means that 9 networks of methods were investigated from 9 different tubes for their respective outcomes. In the previous chapter which was the results chapter, the outcome of the methods were produced in the form of images after the researcher had used fluorescence microscope for the experiment. In this chapter which is the discussion chapter, the researcher seeks to discuss the results by comparing the outcomes that were produced for each method with what has already been found in other studies. This is done to find the place of the current study within the larger literature on the use of non-viral carriers for cell and gene therapy. Very importantly, the discussion chapter is also approached in a way that attempts to answer the research questions. This is done to ensure that the whole study flows within a common parameter of goal. The Role of Transfection in Cell and Gene Therapy Over the course of time, researchers have been very concerned about the issue of transfection when it comes to cell and gene therapy. This has actually been the major bow of contention when it comes to making preferences between viral based processes and non-viral carriers (Nabel, 1995). In cell and gene therapy, transfection has been explained to be the process of launching nucleic acids into cells for practical medicinal purposes (Mangi et al., 2003). As transfection involves the processes of transformation and infecting, it is very important that transfection will be carried out in a way that can be considered as most efficient so that there will not be unexpected and terminal consequences in the whole cell and gene therapy process (Yau et al., 2005). It was for this reason that the method and results of the study focused on the outcome with various tubes in terms of their efficacy with transfection. Indeed the fact that the study was aiming to validate the development of non-viral carriers for cell and gene therapy did not mean that methods of the process that did not guarantee efficient outcomes will not be identified and exposed. The role of transfection in cell and gene therapy was therefore clearly identified in the results of the study as a means for which the methods used would not only lead to either the transformation of cells or infection of cells and genes with nucleic acids. In the following critical analysis of results therefore, specifications are made on the non-viral vectors as to which of them produced limited efficiency in transfection and which of them guaranteed efficiency. C2C12 PEI + DNA 48 HRS The first method involved various transfection testing with c2c12 PEI and DNA within the maximum period of 48 hours. Within the time, a number of changes were made to the background images that were set for both the PEI and DNA. For each background image that was used, different results were gathered. Generally, two major outcomes were produced. The first was black images, which showed that transfection was not successful. Such failed transfection were seen in the usage of 2ul PEI + 6.9ul DNA green fluorescent protein (gfp), 4 ul PEI + 6.9ul DNA gfp, 2ul PEI + 13.7ul DNA gfp, and 2ul PEI + 27.5ul DNA gfp. However, the following had green images, which indicated that transfection was successful. Those with successful transfection were 8ul PEI + 6.9ul DNA gfp, 4ul PEI + 13.7ul DNA gfp, 8ul PEI + 13.7ul DNA, 4ul PEI + 27.5ul DNA gfp, and 8ul PEI + 27.5ul DNA gfp. The trend of successful and unsuccessful transfection produced above confirms with a number of studies performed in this area. As polymers that have repeating units made up of the amine and carbon aliphatic spacer, Kircheis, Wightman and Wagner (2001) noted that PEI are needed in higher quantity with DNA to be able to condense the DNA as positively charged participles. This means that PEI is not effective in acting as a transfection reagent when its combination with DNA is not significant as measured in ul in this study. This is because all values of 2ul PEI that were used failed to successfully transfect. Also, once 4ul PEI was used, there was no transfection until the DNA was increased from 6.9ul to 13.7ul or better. Furthermore, all other cases of 4ul PEI used with the DNA and 8ul PEI succeeded with the transfection. This means that for PEI to bind to the anionic cell surface of the DNA, it reinforcement to cause a cytotoxic reaction (Andre and Mir, 2004). C2C12 G3K 48 HRS Still focusing on the C2C12 cell, transfection was attempted with G3K within a maximum time frame of 48 hours. As usual, two major lines of results were expected, which were combination quantum with DNA that would successfuly lead to transfection and those that will not lead to successful transfection. This is because Ohki and Duax (1986) explained that even though 3 generation dendrimers or G3K have been noted to have the potential of trafficking genes into cells without causing damage to the cell does not mean that this process will always be successful under every condition. For example, in almost all when G3K was combined with DNA, transfection was not successful, no matter the amount of DNA used. Even when transfection took place, only very minimal green image was produced, indicating instability with transfection (Goncalves, Pichon, Guerin and Midoux, 2002). Generally, this shows the efficacy of PEI over G3K. The fact that 8ul G3K were not combined with higher DNA such as 27.5ul could also have influenced dehydration process during the process (Tanaka et al., 1983). Some cases where minimal green lights were seen were 8ul G3K + 16.6ul DNA, 2ul G3K + 16.6ul DNA, and 1ul G3K + 16.6ul DNA. Indeed, in line with the research topic, it is expected that as a type of non-viral carrier, the use of C2C12 with G3K for cell and gene therapy would be successful without damaging cells and genes or deactivating the DNA. The results have however showed that there are specific conditions needed to achieve this. This is in direct confirmation to what Weyts and Goethals (1988) noted when it was found that combination processes that leads to dehydration processes that are not well handled results in hampering the fast degrading functional polymer in 3GK that makes it suitable for localised transfection. To this point, a typical instance when wrong handling of dehydration can be said to have taken place is when the forms of outcomes that failed to lead to successful transfection as noted above results. When combining G3K with DNA for better dehydration outcome, it is expected that both solubility and deposition at fast degrading rates will be achieved but lower quantum is not able to ensure this. C2C12 G3K ARG 48hrs Whiles focusing on C2C12 cells again, transfection with G3K and G3K with arginine with (Arg) was attempted. The procedure also had conjugated dye introduced. The maximum processing time used in this situation was 48 hour as with most of the other methods. Here, transfection was possible for most of the attempted procedures. Indeed, three major procedures were used, involving 4ul G3K 8.3ul DNA GFP, 4ul G3K 16.6ul DNA GFP and 8ul G3K 16.6ul DNA GFP. Out of the three, only the combination with 4ul G3K DNA GFP had a negative outcome. In Wågberg (2000), a similar outcome was produced and explained in terms of the presence of the arginine rather than the raw combination processes that took place. Madkour (1999) supported this position and posited that to act as an effective transfection reagent, G3K need the stimulation of external molecular genes such as the arginine, which comes as a natural form of amino acid. Boussif et al. (1995) also explained the relationship between protein synthesis and gene therapy and noted that when combined with DNA, G3K would need the nucleotide bases which code for arginine in the course of protein synthesis. In effect, the introduction of Arg ensured that the function of protein synthesis was replicated while engaging the DNA for transfection. It was for this reason that the differences of quantum as showed in the three different procedures did not register significant impact or outcome. A typical example of the importance of Arg in ensuring a complete gene or cell function is the role it plays in preterm babies who are not able to on their own synthesise arginine (Rudolph et al., 2000). C2C12 G3K ASP Another pair of combination that was considered was the G3K with aspartic acid (Asp). Independently, Asp is also an α-amino acid as Arg is. in the study though, no form of combination with DNA under GFP was successful for transfection. Indeed, there were high quantum combined in some cases such as 8ul G3K and 8.3ul DNA, and 8ul G3K and 16.6ul DNA but none of these was successful with transfection. While reviewing literature, some answers were found as to why this situation was recorded and not what was experienced with the Arg. Firstly, Astruc, Boisselier and Ornelas (2010) noted the high pervasive nature of Asp in biosynthesis. What this means is that whenever Asp is introduced, it takes a dominant posture, overshadowing the presence of almost all other elements present. Meanwhile in cell and gene therapy, Roland et al. (2002) stressed on the need for there to be the uptake of DNA into the hepatocytes when no mechanical stretching of the endothelial barrier is present. Meanwhile, because the presence of acid protons with Asp is determined by the local chemical environment, which has already been described as pervasive, there is the creation of this mechanical stretching which inhibits the uptake of DNA. In effect, Tomalia et al. (1985) indicated that even though the role of Asp in biosynthesis would have been an impressive characteristic with the combination of G3K, the overly reaction of Asp inhibits other natural processes that should have taken place. Elaborating on the failue of Asp in transfection, Hawker and Fréchet (1990) also mentioned that Asp breaks down hydrostatic pressure within the system, refusing the DNA from entering into the cells before the blood. H2KB PEI+DNA 48HRS A new set of cells were focused on, which were H2KB. Schatzlein (2001) noted that the H2KB acts in antigen presentation to T-cells which express themselves in different formats including CD3/TCR and CD8. It is therefore expected that as the study is focusing on cell and gene therapy, the use of H2KB will be very necessary with its role played with antigens, which are generally toxic to the human body. For transfection to be recorded in this area was therefore very important. The first area with this cell was the combination of PEI with DNA for a total of48 hours. The outcome of this method showed very high numbers of successful transfection based on five different parameters. As showed in the results section, the only parameters where black images were produced throughout were with 4ul PEI + 1.66ul DNA and 4ul PEI + 6.62ul DNA. Generally, this confirms the strength and potency of PEI as a transfection reagent (Sullivan, 2003). Giving the premise to why PEI exhibit such positive behaviour or reaction when paired with DNA, Willemejane and Mir (2009) indicated that there is an excellent condensation process between PEI and DNA which makes it possible for the PEI to condense DNA into positively charged particles. As a result, the transfection process is eased right at the onset when the condensation has taken place. This is because once the DNA is condensed int positively charged particles within the H2KB, there is easy binding to the anionic cell surfaces of the nuclear. It is for this reason that Witlox et al. (2007) referred to the PEI as a transfection facilitator. H2KB PEI + DNA 72HRS After the first line of results obtained with the H2KB cells with PEI and DNA in 48 hours in the tube, the researcher became concerned with further investigating the impact of the same variables when the time of the procedure was increased. As a result, the time was increased to 72 hours, which was 24 additional hours. As depicted in the results in the earlier chapter, there were six different parameters under which the procedures were undertaken. For these six parameters, there was only one instance that transfection was not recorded at all. This was with 4ul PEI + 1.66ul DNA under GFP. This line of result gives the first indication of the use of PEI as a very active transfection reagent. This line of result consolidates the literature by Yaron, Kramer, Johnson and Evans (1997) which noted that PEI is extremely cytotoxic, giving it a unique characteristic that instigates the combined processes of transformation and infection as seen in transfection. Because of this extreme cytotoxic nature of PEI, they make transfection possible and easier causing disruption of cell membranes during the cell and gene therapy process (Escors and Brecpot, 2010). What is more, it is possible for the cytotoxic nature of the PEI to lead to disruption of the mitochondrial membrane even when apoptosis have taken place as part of the transfection process (Gao, Kim and Liu, 2007). With all these noted, the extent of efficiency with the additional time did not increase very significantly. What this indicates is that time is not a major determining factor in the outcome of the transfection process. H2KB G3K 48HRS Another generation 3 dendrimer was introduced and paired with DNA in the H2KB cell. Out of as many as 15 different pairing parameters, there were only three of these that had green images indicating the success with transfection. Between the positive control and negative control, transfection was possible and successful in the positive control but failed in the negative control. This trend led to the search for literature to explaining why this situation might have resulted. In this, Godbey and Mikos (2001) made reference to the history of dendrimers and noted that dendrimers have for long showed lack of independence in their mechanisms, leading to the synthesis of this agent. In the first generation for instance, dendrimers were discovered as macromolecules, which were further synthesised through various processes such as nucleophilic substitution of 1-bromopentane by benzene (Glover, Lipps and Jans, 2005). Meanwhile, for transfection to take place, there are various anatomic issues that are very important to be considered. A typical example of such issues is the extracellular coating of the cells, which inhibit the direct transport of molecules into target cells. Here, the notion is that the absence of synthesising creates further blockage in the transport of molecules into target cells especially when this is done with epithelium and endothelial cell processes (He, Tabata and Gao, 2010). The third generation was therefore expected to be the solution to all these limitations, only for the results gathered to show that further synthesis of the process may be needed as with the positive control used in the study. The inefficiency of G3K was therefore confirmed further in this study. H2KB G3K ARG 48HRS With the major fallout that was experienced with the G3K, and by noting that additional stimulation of the agent may lead to some forms of success created the need to add Arg to the G3K and engage in the process for another 48 hours. In four different parameters, involving separate forms of combinations between G3K and Arg and the DNA, not a single indication of success with transfection was recorded. Without missing words, this is a direct indication of how weak these combinations manifest as non-viral carriers for cell and gene therapy. Gao, Kim and Liu (2007) found that there are a number of medical uses that Arg have for very long time being used in. It was therefore expected that as this was introduced alongside the G3K and the DNA, some of the various combinations would have recorded successful transfection. This failure has however been defended by Glover, Lipps and Jans (2005) who stressed that even though the efficacy of Arg in medical procedures may not be in doubt, a major question that must always be emphasised by researchers is the means or procedure used in the medical process. This is because while administering oral Arg alone, He, Tabata and Gao (2010) noted the stimulated release of GH, with further response seen in the instance of exercising alone. To generalise this position for non-viral carriers as a whole, it can be said that even though some of these carriers may have other forms of medical uses, this does not mean that when procedures are specified for transfection alone, there will certainly be success with the procedure. H2KB G3K ASP 48HRS Another reagent that was attempted together with G3K was Asp, which is aspartic acid. In this instance also, there were four different parameters that were tested and tried for in combination with DNA. As was the case with Arg, none of the procedures resulted in a successful transfection. To a large extent, this trend of result confirms what Witlox et al. (2007) found when it was indicated that in mammals, Asp acts as a non-essential as it is produced from oxaloacetate by transamination. The implication that this gives to the transfection process is that there are forms of non-viral carriers that inhibit successful transfection due to the mode through which they are derived. Instead of a successful transfection procedure, Asp was noted to have very high potency when used in other biochemical procedures such as when used as a metabolite in urea cycle. It can be accepted therefore that even though non-viral carriers have confirmed possibility with cell and gene therapy through the intentional modulation of gene expression, the DNA which is introduced as an exogenous nucleic acid require specific components of acid and non-viral carriers to make transfection possible (Sullivan, 2003). The need to continue clinical trials that aim at verifying viability with transfection is very important. This is because DNAs come as macromolecules with very large size and negative charge, which require accommodating forms of mediated carriers or vectors to make transfection successful. References Andre F. and Mir LM. (2004). DNA electrotransfer: its principles and an updated review of its therapeutic applications. Gene Ther.;11(Suppl 1):S33–42. Astruc D., Boisselier E. and Ornelas C. (2010). 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