Development of Novel Non-viral Carrier for Gene and Cell Therapy - Essay Example

Development of novel non-viral carrier for gene and cell therapy Introduction One of the main objectives of gene and cell therapy is the treatment of gene mutations. These mutations can lead to diseases or loss of functions, and gene therapies help manage these mutations through the introduction of therapeutic genes into the cells, allowing for the expression of the deficient gene products within the physiological setting (Bolhasani and Rafati, 2011). However, to ensure sustainability, the therapeutic gene must be introduced in the nucleus, replicated and then secured with subsequent cell divisions. Issues in securing sufficient gene delivery processes have implied that, gene therapy has manifested insufficient medical advantages (Bolhasani and Rafati, 2011). Viruses are considered the preferred method for gene delivery due to their better in vivo transfection when compared to non-viral vectors. Better transgene expression in most clinical environments have only been secured with the application of retroviral vectors including the murine leukaemia virus, which incorporates their DNA into the genome, sufficiently managed and grouped into daughter cells (Roesler, et.al., 2002). This study is significant because it seeks to provide a framework for the development of a new non-viral carrier for gene and cell therapy. At present, the developments in gene therapy relate to issues in viral carriers. As such, this study is significant as it considers the inclusion of non-viral carriers for gene and cell therapy. The various issues in the use of viruses as carriers which include immunogenicity and cytotoxicity have affected the clinical efficacy of this transfer process (Hollon, 2000). One of the first therapy-related deaths for instance was due to an inflammatory reaction to an adenovirus carrier. Another issue in the use of viral vectors is insertional mutagenesis where ectopic chromosomal integration for viral DNA may interrupt the tumour-suppressor gene or it may trigger the oncogene, then leading to malignant changes in the cells (Glover, et.al., 2005). In general, it has been perceived that retroviruses have been used randomly and did not have much of a chance in being integrated into a gene in the human genome. Still, it has also become more apparent that viruses like the MLV and the lentivirus seek integration with active regions of the genome (Glover, et.al., 2005). MLV prefers to integrate within gene promoters and the lentivirus seeks to integrate with transcriptional factors including the BCRA1 breast cancer tumour-suppressor gene (Glover, et.al., 2005). Non-viral vectors have significant safety advantages in relation to the viral methods, including the fact that they have less pathogenicity and capacity for insertional mutagenesis, including low cost as well as easy production (Glover, et.al., 2005). The use of non-viral vectors for humans has been however suppressed by inefficiencies in the delivery to the cells including the temporary expression of transgenes. This study shall partly consider the recent developments in cell and non-viral gene therapy. In order to support non-viral gene therapy, several issues must be managed including issues in securing sustained gene expression as well as preventing gene silencing. In order to secure such goal, the primary focus of this study is to consider the favourable developments on therapeutic gene expression in humans in relation to non-viral approaches in gene delivery. Gene therapy, including both the study of viral and non-viral carriers, also presents with new methods in the management of diseases, including, the different types of cancer (Stone, 2010). The most significant challenge for gene therapy is the establishment of appropriate vectors or carriers. The different viral and non-viral methods in gene transfer have already been established and in general, the viral vectors are more effective in supporting gene delivery as well as gene expression when compared to the non-viral methods. However, viral vectors present more risks when compared to non-viral methods. Also, the viral methods cannot be applied to the delivery of drugs (Kaneda and Tabata, 2006). In effect, the non-viral vectors can deliver the anticancer reagents, including the synthetic oligonucleotides, antibodies, as well as RNA (Kaneda and Tabata, 2006). As such, the non-viral vectors are effective methods by which cancer therapy can be secured. Still, different barriers are present, including the processes which prevent exogenous molecules from attacking the body. The non-viral methods must manage such issues in order to ensure effective cancer and disease therapy (Kaneda and Tabata, 2006). Laboratories have developed non-viral processes in order to introduce genes into cells in human gene therapy. To ensure such efforts, the simplicity and better safety of non-viral has been proposed (Cotten and Wagner, 1993). Recombinant viruses may apply in the laboratory setting and may indeed be safe for clients, but the safety assessment of these vectors may take a lot of time and may be laborious. Non-viral methods secure more flexibility in DNA sequencing (Cotten and Wagner, 1993). The time for simple and effective non-viral delivery processes call for a better understanding of the DNA-delivery processes. The knowledge of proper delivery of DNA in order to efficiently target cells will help provide more data on viral entry, gene maintenance, and endocytosis (Cotten and Wagner, 1993). The effective delivery of long and slender DNA molecules into three lipid bilayers calls for the management of different obstacles (Cotten and Wagner, 1993). The DNA must first manage physical shearing and degradation. Second, it must also manage the transient stay within the extracellular setting, ex vivo in the cell medium or in vivo in the blood. Under these conditions, there may be exposure to the defence cells from the immune system including the elements of the complement system. Third, the DNA complexes must seek to bind the target cell and must have an internalizable size (Cotten and Wagner, 1993). Fourth, the elements of the complex must support the release of the DNA from the vesicles and ensure that the DNA can escape the endosomes to the cytosol. Fifth, as soon as the DNA is in the cell, it must seek the nucleus. Lastly, where the DNA has reached the nucleus, prolonged gene expression must be supported (Cotten and Wagner, 1993). With advancements in medicine, treatments have developed well into new fields and areas. The focus has now been on the establishment of methods which are able to eliminate the causes, rather than the symptoms of diseases. In effect, studies have mostly considered knowledge from genetics. Gene mutation and deletion can sometimes cause genetically-caused diseases. Issues relating to gene disorders within metabolic pathways or cell skeleton and extracellular proteins have all been associated with serious diseases (Yaron, et.al., 1997). The genetically-based and acquired diseases may be managed through gene therapy. Genetic diseases are often due to the deletion or mutation of single cells. In general, for acquired diseases, single genes cannot be considered the only cause of diseases. Gene therapy has become more prominent and has represented significant possibilities in management through acquired as well as genetic diseases (Conwell and Huang, 2005). Gene delivery systems may be viral, non-viral or a combination of these systems. The viral methods include viruses which are modified and transformed to be replication-deficient, but also able to secure DNA for expression. Adenoviruses, retroviruses, as well as lentiviruses are applied in viral-gene delivery processes (Escors and Breckpot, 2010). The non-viral delivery systems have been secured as another possibility in the viral-based processes. An advantage for these systems is better transfection. The non-viral systems are grouped based on preparation – either as physical or chemical preparations. The most observed methods include microinjection, electroporation, ultrasound, gene gun and hydrodynamic assimilations (Cevher, et.al., 2012). In general, the physical processes relate to the delivery of the gene thru physical force in order to ensure permeability for the cell membrane. On the other hand, the chemical applications use natural or the synthetic carriers to promote the efficient delivery of genes into the cells. Under this process, the polymers, liposomes, and lipids are applied for gene delivery (Miyazaki, et.al., 2006). Some human diseases have been attributed to mutations or deletions in genes which have presented as disorders in the metabolic processes, issues in ligand/receptor functions, cell cycle management, and extra-cellular protein structures as well as functions (Sullivan, 2003). Through gene therapy, the diseases may be managed using exogenous nucleic acid sequences targeting the affected tissues in the body (Yaron, et.al., 1997). Diseases which can be managed using gene therapy are considered as mutation or deletions in single cells. Also, single cell genes are not considered as the only cause for acquired diseases. While gene therapy was first applied to manage genetic disorders alone, it is now being applied to manage various diseases including cancer, arthritis, and AIDS (Mhashilkar, et.al., 2001). Expressions in single cells directed to the cells via the gene delivery system are able to treat diseases. Before gene therapies, there were no other treatments available for genetic disorders. At present, it is possible to manage genetic mutation via gene therapies (Sullivan, 2003). Gene therapy was first used by the International Congress of Genetics (Yaron, et.al., 1997). The processes applied by Gregor Mendel in the 1800s became a major development for genetics. With the additional work of Ronald Fischer and Wilhelm Johannsen, the essential elements of genetics were established (Yaron, et.al., 1997). Gene therapy gained momentum in the 1970s and gene therapy trials were carried out. It was established that naturally occurring DNA and RNA tumour were able to secure new gene data into the genomes of mammal cells (Escors and Breckpot, 2010). With more genetic developments, a better understanding of genetic diseases was secured. Moreover, other diseases like cancer, diabetes, and retinoblastoma were also studied based on their genetic connections (Cevher, et.al., 2012). Gene transfers to human cells soon became routine applications with some retrovirus-based therapies ensuring major advantages, bringing about a better integration of genomes to support the host-cell chromosomes (Cevher, et.al., 2012). DNA was also understood based on its genetic components and later such DNA was modifications secured in genetic codes. Such developments allowed cloning (Escors and Breckpot, 2010). By 2006, melanoma soon became a manageable disease. The treatment included a retroviral management of the melanoma antigen-specific T-cell receptor. More developments in gene therapy were made, including somatic cloning, as well as the human genome project (Escors and Breckpot, 2010). Non-viral therapies were also ensured. Naked plasmid DNA, with gold elements was also indicated for cells via the non-viral gene gun application. Such application was initially applied in plants and has now become available for humans. A successful gene therapy technique is the hydrodynamic injection method (Willemejane and Mir, 2009). The gene delivery system includes different processes which ensures uptake of genes which have been chosen to target specific cells (Conwell and Huang, 2005). The efficient designs for the delivery system cover an efficient understanding of the relationship between the target cells and the delivery method. Understanding cell movement and targeting processes is crucial in ensuring efficient gene delivery applications (Cevher, et.al., 2012). Cell targeting covers the delivery of treatment agents to specific cell organelles. It is often used in endocytosis gene therapy, including cell uptake in non-viral gene delivery processes (Prokop and Davidson, 2007). Following cellular uptake in the delivery system by endocytosis, cellular release can unfold in order to allow for DNA translation and to secure the pertinent proteins (Cevher, et.al., 2012). Effective gene delivery helps reduce inhibitory inflammatory reactions while also managing specific issues in gene delivery, thereby promoting gene activity (Conwell and Huang, 2005). Viral gene delivery methods include viruses which are transformed to be replication-deficient and became unable to replicate through their redesign, ensuring the delivery of genes to cells to ensure expression. Adenoviruses, retroviruses, and lentiviruses were applied for viral gene therapy (Cevher, et.al., 2012). Viral methods were beneficial with their persistent expression of therapeutic genes (Sullivan, 2003). Still, some issues have been seen, especially those which affect the use of these processes, large scale production, and limited optimization (Witlox, et.al., 2007). Non-viral gene delivery processes were secured with alternative to viral-based processes. An important advantage of such processes was seen in the fact that they presented with transfection. Non-viral gene delivery systems are grouped into physical and chemical groups. Microinjection, ultrasound-supported processes, and hydrodynamic methods are the most common physical tools (Cevher, et.al., 2012). Physical tools include physical forces to ensure permeability in the cell membrane, also promoting gene entry into the cell. The main advantage in the physical methods includes their applicability and reliability. Still, they are also beneficial as they promote tissue damage in some cases. Chemical applications include carriers made from synthetic elements in gene delivery into cells, alongside synthetic polymers, natural polymers, and cationic lipids (Cevher, et.al., 2012). One of the advantages of these processes includes the fact that they are considered non-immunogenic. The primary understanding for gene therapy is that it presents gene expression for specific cells in order to promote the management of human diseases, allowing for transfers in genetic materials and the inhibition of target protein (He, et.al., 2010). In general, viral carriers can reach rapid transfection in foreign materials incorporated into viral genome with high transfection. Still, various researches using viral vectors have indicated unfavourable results, mostly because of immunologic and oncogenic adverse reactions to these vectors (Cevher, et.al., 2012). On the other hand, the non-viral vectors are advantageous with their easy fabrication and low immune reaction. A major disadvantage for non-viral vectors in the clinical setting is its limited efficiency in transfection (He, et.al., 2010). In effect, the most crucial elements which must be considered in gene therapy include the introduction of the gene into the cell and promoting transfection efficiency. With transfection, cells are allowed to produce chemicals for hormone replacement or protein production (Godbey and Mikos, 2001). For the naked DNA molecules, they are not able to enter cells because of their hydrophilic form due to their negatively charged phosphate groups (Cevher, et.al., 2012). Moreover, they are easily affected by nuclease enzymes. Therefore a major issue for gene therapy is the presence of physical processes which help promote gene transfer for target cells in gene delivery vectors and delivered gene (Al-Dosari and Gao, 2009). Natural and synthetic polymers are applied in order to secure non-viral gene delivery processes. In relation to viral delivery applications, the non-viral carriers are not as toxic and immunogenic. Non-viral vectors also promote production (Cevher, et.al., 2012). Most studies established that non-viral methods were not as effective as the viral methods. However, in relation to gene therapy with physical applications, gene transfection and therapy improved and the extent of gene expression was also improved (Al-Dosari and Gao, 2009). Various issues have to be managed in order to improve efficacy in non-viral vectors for humans. These issues are grouped into production, the formulation, and the storage issues, also the extracellular issues, and the intracellular issues (Davis, 2002). The anatomic issues include the extracellular coating for the cells, which do not allow for the direct transport of molecules into target cells with epithelium and endothelial cell processes. Phagocytes in the liver and residential macrophage allow for clearance of DNA colloidal particles (Cevher, et.al., 2012). Also, nucleases in the blood allow for free nucleic acids to be inactivated after their use. The most critical issue impacting on DNA transfection is the transition of plasma membrane. In general, the naked nucleic acids do not pass into the cell membrane with cellular uptake processes including endocytosis and phagocytosis without the use of physical applications or loading into carriers (Al-Dosari and Gao, 2009). There is much evidence on the development of effective chemical as well as biological processes and the delivery of transgenes into cells to secure effective expression (Gao, et.al., 2007). Physical processes, alongside gene gun, ultrasound, and hydrodynamic management are founded on the use of force to ensure permeability of cell membrane as well as the promotion of intracellular gene transfer. With chemical applications, synthetic or natural carriers are applied for the transport of genes into the cells. Ideal gene delivery systems must support three requisites (Cevher, et.al., 2012). First, the carriers must protect the gene from the enzymes within the cells; second, they must transport transgenes from the plasma membrane into the target cell nucleus; and lastly, the carriers must not produce any toxicities (Gao, et.al., 2007). Advantages of synthetic carriers include the fact that they do not have any immunogenicity; also large scale production can be possible. Moreover, the size of the gene does not decrease the effective use of these processes. Each human chromosome has been sufficiently transfected with these kinds of carriers (Schatzlein, 2001). The non-viral vectors can cause inflammatory reactions as they do not indicate specific recognition and are not as dangerous as compared to the viral vectors in relation to the antigen specific immune responses (Cevher, et.al., 2012). Still, while the non-viral vectors seem to be more applicable, there are important elements which must be understood in the designs of vectors. The non-viral carriers must be managed based on specific cell targets. Cellular uptake as well as release must also be effectively used and possible immune reactions must be reduced (Conwell and Huang, 2005). The best gene therapy vector in the management of genetic diseases must not only secure intact DNA to the target cell nucleus, but as soon as they are delivered, the transgene must be kept in the nucleus without affecting host gene expression. Securing these vectors have become however more difficult (Glover, et.al., 2005). The conventional non-viral vectors cover different liposomal elements, as well as proteins and polymers like polytethylenimine, reacting with the DNA to ensure cell entry by covering DNA through charged interactions (Glover, et.al., 2005). While these vectors work well in vitro, they do not have sufficient in vivo transfection and only transmit transient gene expression. Such inadequate transfection is mostly attributed to the power of the non-viral vector-DNA compounds to affect the blood plasma proteins, including the extracellular matrix and the unfavourable cells (Glover, et.al., 2005). As soon as they reach the target cells, more elements for transfection cover the importance of DNA leaving the liposome, as well as non-reaction to cytoplasmic degradative enzymes, including nucleases and the exploration of the double-membrane structure of the nucleus (Glover, et.al., 2005). The plasmid DNA introduced into the nucleus is also more or less replicated and may be lost with the destruction of the nuclear envelope during mitosis. To manage these issues, novel non-viral vectors with more in vivo stability have been introduced and these do not have as much attraction to the intracellular proteins as well as cell surfaces (Glover, et.al., 2005). As a result they are able to get through to the target cells within their active structure. Also, ligands for receptor-mediated endocytosis, peptides which help with DNA compaction, endosomal disruption sequence, and nuclear import signs have helped improve the reach of non-viral vectors into the cell, including the nucleus (Glover, et.al., 2005). In general, these different proteins are modular non-viral vectors which echo the power of the virus to manage cellular blocks to the delivery of DNA using resources which match the viral vectors. Since protein domains for these vectors can be used interchangeably, the modular vectors can be fashioned to match various applications including tissues and cell types (Glover, et.al., 2005). Despite such improvements, the non-viral vectors have mostly been focused on clinical applications which call for specific transient gene expression, including the expression of toxic genes against cancer cells. More improvements in non-viral vectors translate to viral resources which can be repeated and which include nuclear maintenance as well as replication with site-specific integration into the safe regions. Various approaches may be deemed compatible with non-viral delivery and the different methods rely on host cell resources to secure the long-term nuclear gene expression. Most of the approaches mimic specific viral applications. More importantly however, the methods seek to avoid interrupting the host gene expression as well as signalling pathways and are better preferred when compared to the viral-setting vectors which are at present being applied for humans. The general success for the nonviral gene therapy within the clinical setting calls for efficient management of specific target tissue, including the HACs in the management of cystic fibrosis which has to be delivered to the pulmonary epithelium using aerosol management (Glover, et.al., 2005). In the past, the efficacy of transgene delivery into the in vivo objectives have not been easy to identify; however with developments in imaging technology, more efficacy in viral and non-viral transfer tools can be considered in vivo. They must also assist in identifying the unidentified barriers to efficient in vivo therapy, prior to use in humans within the clinical setting. Also another concern in relation to gene transfers is the fact that requisites in non-viral vectors can sustain expression of therapeutic agents in human gene therapy which are not the same as those needed for biotechnical applications, including ectopic gene expression for animals (Glover, et.al., 2005). Moreover, gene therapy is not specifically applied to secure missing gene products for patients having inborn issues in metabolism. For instance, anti-cancer gene therapy trials are being undertaken where the goal is to secure high transgene expression in various tumour cells, as compared to prolonged gene expression. This helps support efficient and specific delivery and gene expression (Glover, et.al., 2005). It is clear that various applications cover different needs but with the developments indicated in this study, fashioning non-viral gene delivery vectors to the specific needs of therapeutic application is gradually becoming a reality. The application of non-viral tools indicated herein to ensure safe, long-term transgene expression may help secure better results relating to human gene therapy. The objectives of this study mostly relates to the development of novel non-viral carrier for gene and cell therapy. Specifically, the following objectives will be considered: 1. What are the current novel developments in non-viral carriers for gene and cell therapy? 2. How well do these therapies contribute to disease management? 3. What are the issues and barriers in the use of gene and cell therapy? 4. What are the possible recommendations in gene and cell therapy for effective disease management? This study likely involves some barriers and limitations, especially in relation to the actual clinical experimentations which can be carried out. Since this study will only serve to gather other data from current and previous studies, the actual clinical setting application of these therapies may not be adequately reviewed. More limitations in this work would likely emerge towards its completion. Conclusion Based on the details indicated above, it is important to note that there are different developments in non-viral carriers for gene and cell therapy. Different methods have been and are still being developed in order to support the efficient delivery of gene and cell therapies. The goals and hopes related to gene therapy support studies in the field of molecular biology. While clinical trials have already been initiated, there are still limitations which must be resolved before any clinical use can be secured. The objective of gene and cell therapy is for the development of efficient, as well as non-toxic gene carriers which can envelope and deliver foreign genetic elements into cells including cancer cells. The viral and non-viral carriers have been developed and assessed in the delivery of therapeutic genes into the cancer cells. Some of the viruses have been modified to cancel out toxicities and secure efficient gene transfer capacities. With limitations on viral carriers, non-viral carriers have been discussed as alternatives to the delivery systems. Their primary non-viral vectors cover cationic polymers, cationic peptides, as well as cationic liposomes. At present, most modifications on delivery systems have been secured in order to ensure transfer efficiency. Moreover, immunization routes can impact on the outcome of the immune reactions by changing interactions between the vaccines and sites of injection. Routes of immunization and formulation of DNA also impacts on therapeutic reactions as these change the immune pathway. It is critical to further assess results from ongoing trials, including aspects where success or failure of specific methods of delivery has been seen in gene and cell therapies. References Al-Dosari, M. and Gao, X. (2009). 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