Animal Research International (2019) 16(2): 3367 – 3392 3367

GENE THERAPY, PHYSIOLOGICAL APPLICATIONS, PROBLEMS AND PROSPECTS - A REVIEW

1UGWU, Godwin Chigozie, 1EGBUJI, Jude Victor Ifeanyi, 1OKANYA, Laureta Chinagorom, 2OMEJE, Joy Nwamaka and 1EYO, Joseph Effiong 1Department of Zoology and Environmental Biology, University of Nigeria, Nsukka, Enugu State, Nigeria. 2Department of Pharmacognosy and Environmental Medicine, University of Nigeria, Nsukka, Enugu State, Nigeria.

Corresponding Author: Ugwu, G. C. Department of Zoology and Environmental Biology, University of Nigeria, Nsukka, Enugu State, Nigeria. Email: [email protected] Phone: +234 8064005944

Received: June 16, 2017 Revised: May 23, 2018 Accepted: July 19, 2019

ABSTRACT

Gene therapy can be defined as the use of DNA as a pharmaceutical agent to treat disease. It is also an experimental medical treatment that manipulates a gene or genes within cells in order to produce proteins that change the function of those cells. The physiological applications, problems and prospects of gene therapy are reviewed in this study. The different types of gene therapy such as germline gene therapy, somatic gene therapy and chimeraplasty gene therapy are discussed. Polymerase chain reaction (PCR), nanoparticles, sonoporation, electroporation and gene gun are the techniques used in gene therapy. Polymerase chain reaction (PCR) is used in medical and biological research. Nanoparticles have been widely used in the field of drug and gene delivery to target cells. Sonoporation allows uptake of large molecules of DNA into the cell, in a process called cell transformation. Electroporation is highly efficient for the introduction of foreign genes in tissue culture cells, in tumor treatment and cell-based therapy. A gene gun is a device for injecting cells with genetic information to plant cells. Gene therapy is applied in medicine, agriculture, loss and gain of function, tracking and expression studies. Some problems bedeviling gene therapy include insertional mutagenesis, mutagenic disorders, problem of viral vectors, immune response etc. Gene therapy has the potential to eliminate and prevent hereditary diseases such as cystic fibrosis and is a possible cure for Alziehmer’s disease and cancer, enhance agricultural productivity of farm animals, and in the production of genetically modified animals (GMOs) which will further help in medical and biomedical research.

Keywords: Gene therapy, Chimeraplasty, Nanoparticles, Sonoporation, Electroporation, Gene gun, Polymerase chain reaction

INTRODUCTION derives its name from the idea that DNA can be used to supplement or alter genes within an In gene therapy, DNA is use as a individual cells. The most common form of gene pharmaceutical agent to treat disease. It is also therapy involves using DNA strands that encode an experimental medical treatment that functional therapeutic gene in order to replace a manipulates a gene or genes within cells in mutated gene. In gene therapy, DNA that order to produce proteins that change the encodes a therapeutic protein is packaged function of those cells (Alberts et al., 2002). It within a “vector” which is used to get the DNA

ISSN: 1597 – 3115 ARI 2019 16(2): 3367 – 3392 www.zoo-unn.org Ugwu et al. 3368 inside the cells within the body. The DNA now lead to the production of a protein or enzyme inside becomes expressed by the cell machinery that functions improperly, disrupting cellular resulting in the production of a therapeutic biological activities such as codons for proteins protein which in turn treats the patient’s or enzymes production (Isenbarger et al., diseases (Brivanlou and Darnell, 2002). 2008). Genes correspond to regions within DNA Gene therapy was first conceptualized which is composed of a chain of four different as a result of efforts to treat and cure some types of nucleotides: adenine, guanine, thymine known genetic disorders, most of which lack an and cytosine. The sequence of these nucleotides effective therapy. The original goal of gene is the genetic information organisms inherit DNA therapy was to substitute a healthy gene for a naturally occurs in a double stranded from, with defective one, or to repair a faulty gene, the nucleotide base pairs on each strand thereby dominating symptoms of the disease or complementary to each other. Each strand can defect (Cole-Strauss et al., 1996). However, act as a template for creating a new partner researchers have moved beyond inherited strand. This is the physical method for making genetic disorders to treat other kinds of copies of genes that can be inherited. Viruses diseases, with nearly 75 % of all clinical trials on the other hand use a similar molecule aimed at treatments for cancer, acquired ribonucleic acid, RNA, instead of DNA as their immunodeficiency syndrome (AIDS), Alzheimer’s genetic material (Hershey and Chase, 1952). disease, diabetes mellitus and arthritis, all of The nucleotides sequence in a gene is which involve genetic susceptibility to illness. translated by cells to produce a chain of amino Although gene therapy offers seemingly acids; the order of amino acids in a protein limitless possibilities, researchers have been corresponds to the order of nucleotides in the thwarted by many technical problems. Most gene. The relationship between nucleotide clinical trials of gene therapy have not resulted sequence and amino acids is known as the in enough improvement in the patients genetic code. The types of amino acids in a underlying condition to consider it an protein determine how it folds into a three- unqualified success and to justify treating large dimensional shape. This structure is in turn numbers of people (Coghlan, 1999). responsible for the protein’s function. A change The extraordinary potential of gene to the DNA in a gene can change a protein’s therapy had also raised alarms among critics amino acid changing its shape and function; this who warn that the technology could go too far. can have a dramatic effect on the whole They note that gene therapy could offer wealthy organism (Gura, 1999). families opportunities for genetic enhancement unavailable to the poor. More troubling still for Gene Expression: Genes generally express some critics is gene therapy’s potential to their functional effect through the production of narrow the human gene pool, producing proteins which are complex molecules unknown and possibly harmful consequences responsible for most functions in the cells. (Cheng et al., 1994). Proteins are chains of amino acids, and the DNA sequence of a gene (through an RNA Overview of Gene Therapy: A gene is a long intermediate) is used to produce a specific segment of the molecule deoxyribonucleic acid protein sequence (Mayeux, 2005). The process (DNA). This segment, composed of minute called transcription begins with the production subunits called nucleotide bases, serves as the of an RNA molecule with a sequence that blueprint for manufacturing a single protein or matches the gene’s DNA sequence. The enzyme needed for the structure or function of messenger RNA (mRNA) molecule is then used cells (Griffiths et al., 2000). In humans, genes to produce a corresponding amino acid are compressed and bundled into a set of 23 sequence through a process called translations. pairs of chromosomes which stabilize and Each group of three nucleotides in the sequence protect the DNA. Any tiny error in the called a codon corresponds either to one of the arrangement of a genes nucleotide bases can twenty possible amino acids in a protein or an

Animal Research International (2019) 16(2): 3367 – 3392 Gene therapy, physiological applications, problems and prospects 3369 instruction to end the amino acid sequence and et al., 2002). Within the genome of Escherichia this corresponds to the genetic code. The flow coli bacteria, for example, there exists a series of information is unidirectional; a situation in of genes necessary for the synthesis of the which information is transferred from nucleotide amino acid tryptophan. Once tryptophan is sequences into the amino acid sequence of already available in the cell, the genes for proteins, but it never transfers from protein tryptophan synthesis are no longer needed. The back into the sequence of DNA (Lodish et al., presence of tryptophan directly affects the 2000). A single nucleotide difference within DNA activity of the genes. Tryptophan molecules can cause a change in the amino acid sequence bind to the tryptophan repressor (a transcription of a protein. Because protein structures are the factor), changing the repressor's structure such result of their amino acid sequences, some that the repressor binds to the genes. The changes can dramatically change the properties tryptophan repressor blocks the transcription of a protein by destabilizing the structure or and expression of the genes, thereby creating changing the surface of the protein in a way negative feedback regulation of the tryptophan that changes its interaction with other proteins synthesis process (Yang, 2007). and molecules (Saiki et al., 1985). For example, Differences in gene expression are sickle cell anaemia is a human genetic disease especially clear within multicellular organisms that results from a single base difference within where cells all contain the same genome but the coding region for the β-globin section of have very different structures and behaviors due hemoglobin, causing a single amino acid change to the expression of different sets of genes. All that changes hemoglobin's physical properties. the cells in a multicellular organism derive from Sickle-cell versions of hemoglobin stick to a single cell, differentiating into variant cell themselves, stacking to form fibers that distort types in response to external and intercellular the shape of red blood cells carrying the signals and gradually establishing different protein. These sickle-shaped cells no longer flow patterns of gene expression to create different smoothly through blood vessels, having a behaviors. As no single gene is responsible for tendency to clog or degrade, causing the the development of structures within medical problems associated with this disease multicellular organisms, these patterns arise (Nabel et al., 1992; Cole-Strauss et al., 1996). from the complex interactions between many Some genes are transcribed into RNA but are cells (Yoon et al., 1996). not translated into protein products, such RNA Within eukaryotes there exist structural molecules are called non-coding RNA. In some features of chromatin that influence the cases, these products fold into structures which transcription of genes, often in the form of are involved in critical cell functions (e.g. modifications to DNA and chromatin that are ribosomal RNA and transfer RNA). RNA can also stably inherited by daughter cells. These have regulatory effect through hybridization features are called "epigenetic" because they interactions with other RNA molecules (e.g. exist "on top" of the DNA sequence and retain micro RNA) (Sails, 2004). inheritance from one cell generation to the next. These epigenetic features will cause different Gene Regulation: The genome of a given cell types grown within the same medium to organism contains thousands of genes, but not retain very different properties. Although all these genes need to be active at any given epigenetic features are generally dynamic over moment. A gene is expressed when it is being the course of development, some, like the transcribed into mRNA (and translated into phenomenon of paramutation, have protein), and there exist many cellular methods multigenerational inheritance and exist as rare of controlling the expression of genes such that exceptions to the general rule of DNA as the proteins are produced only when needed by the basis for inheritance (Pathak et al., 2009). cell. Transcription factors are regulatory proteins that bind to the start of genes, either promoting Mutations: During the process of DNA or inhibiting the transcription of the gene (Wolf replication, errors occasionally occur in the

Animal Research International (2019) 16(2): 3367 – 3392 Ugwu et al. 33683370 polymerization of the second strand. These resulting in the phenomenon of evolution. errors, called mutations, can have an impact on Selection for beneficial mutations can cause a the phenotype of an organism, especially if they species to evolve into forms better able to occur within the protein coding sequence of a survive in their environment, a process called gene (Alberts et al., 2002). Error rates are adaptation (Griffiths et al., 2000). New species usually very low. One error in every 10 – are formed through the process of speciation, 100 million bases due to the "proofreading" often caused by geographical separations that ability of DNA polymerases (without prevent populations from exchanging genes proofreading error rates are a thousand fold with each other. The application of genetic higher; because many viruses rely on DNA and principles to the study of population biology and RNA polymerases that lack proofreading ability, evolution is referred to as the modern synthesis they experience higher mutation rates). (Imes et al., 2006). Processes that increase the rate of changes in By comparing the homology between DNA are called mutagenic: mutagenic chemicals different species' genomes it is possible to promote errors in DNA replication, often by calculate the evolutionary distance between interfering with the structure of base-pairing, them and when they may have diverged (called while UV radiation induces mutations by causing a molecular clock). Genetic comparisons are damage to the DNA structure. Chemical damage generally considered a more accurate method of to DNA occurs naturally as well, and cells use characterizing the relatedness between species DNA repair mechanisms to repair mismatches than the comparison of phenotypic and breaks in DNA. Nevertheless, the repair characteristics (Jaenisch and Bird, 2003). The sometimes fails to return the DNA to its original evolutionary distances between species can be sequence (Berg et al., 2002). In organisms that used to form evolutionary trees; these trees use chromosomal crossover to exchange DNA represent the common descent and divergence and recombine genes, errors in alignment of species over time, although they do not show during meiosis can also cause mutations. Errors the transfer of genetic material between in crossover are especially likely when similar unrelated species known as horizontal gene sequences cause partner chromosomes to adopt transfer and most common in bacteria (Gura, a mistaken alignment; this makes some regions 1999). in genomes more prone to mutating in this way (Cheng et al., 1994). These errors create large MATERIALS AND METHODS structural changes in DNA sequence such as duplications, inversions, or deletions of entire A comprehensive internet search of literature on regions, or the accidental exchanging of whole gene therapy was undertaken using Google parts between different chromosomes (called search. Literatures recovered were analyzed in translocation) (Newton et al., 1989). pros and relevant cited tables and figures Mutations alter an organism’s genotype adopted. and occasionally this causes different phenotypes to appear. Most mutations have RESULT AND DISCUSSION little effect on an organism's phenotype, health, or reproductive fitness. Mutations that do have Types of Gene Therapy an effect are usually deleterious, but occasionally some can be beneficial. Studies in 1. Germline Gene Therapy: Germline gene the fly Drosophila melanogaster suggest that if therapy involves the modification of germ cells a mutation changes a protein produced by a (gametes) that will pass the change on to the gene, about 70 percent of these mutations will next generation. With germline therapy, genes be harmful with the remainder being either sequence can be corrected in the egg or the in neutral or weakly beneficial (Felgner and sperm that is being used to conceive. The child Ringold, 1989). Over many generations, the that results would be spared certain genetic genomes of organisms can change significantly, problems that might otherwise have occurred

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(Cole-Strauss et al., 1999). In living organisms, The section of cut DNA has to be intact then a every cell descends from the fertilized egg, thus technique called electrophoresis is used to every cell in the offspring will possess separate the selected pieces of DNA and remove transplanted gene. This would be a far more the genes that contained the DNA sequence effective way of transferring genes than the that coded for the polypeptides needed ones presently used in somatic cell therapies, (Jaroslav et al., 2002). where genes into the cells of children or adults Similar restriction enzymes are used to usually enter only a small portion of the remove a section of enzyme from a plasmid or person’s cells and eventually stop functioning virus and using ligase (a special enzyme used to (Cheng et al., 1994). "glue" a foreign piece of DNA into a donor cell) The technology of gene therapy is the therapeutic DNA sequence is placed in the based on the effective delivery of the corrective DNA of the vector. In the case of a virus, genes and to do this, scientists have developed instead of causing illness the virus carries the gene delivery vehicles called vectors. These normal genes into the target cells where they vectors encapsulate therapeutic genes for begin functioning (Lawyer et al., 1993). delivery into the targeted cells. Many of the Germ line therapy which is the focus of vectors currently in use are based on attenuated this presentation is technically more difficult and or modified versions of viruses. Plasmids, which raises many ethical challenges. The two main are circular pieces of DNA extracted from methods of performing germline therapy would bacteria (Figure 1), are also used as vectors be: i) To treat a pre-embryo that carries a (Gura, 1999). serious genetic defect before implantation into the mother (this requires the use of in vitro fertilization techniques); or ii) to treat the germ cells (sperm or egg cells) of the afflicted adults so that their genetic defects would not be passed on to their offspring (Figure 2). This approach requires the technical expertise to remove the defective genes and insert a properly functioning replacement (Khan et al., 2008).

Figure 1: Mechanism of gene delivery. Source: Gura (1999)

The therapeutic gene to be transferred is extracted from the cell of a healthy individual. The gene is extracted by cutting the DNA using a restriction enzyme (restriction enzymes Figure 2: Illustration showing germline "digest" DNA at designated nucleotide locations gene therapy involving pronuclear along the DNA chain). There are different types transfer in human embryos to prevent of restriction enzymes, each being specific to diseases. Source: Australian Science and the location of the DNA chain that it will cut. Media Centre (2010)

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Germline gene therapy is advancing at a Germ-line gene therapy is a new rapid pace, so fast that new technologies are technology being developed by scientists in the being readied for use without answering forefront of their fields. The final form that this concerns about safety. The safety concern of technology will take depends on new advances this type of engineering arises because of a lack and political influences by decision-makers. of knowledge about the human genome. It is Concerns about gene therapy include safety, quite possible that a valuable gene could be effectiveness, impact on the genome, funding of erased (Saiki et al., 1985). However, only research, the future applications and direction of having one copy of the gene confers added research (Cui et al., 2012). The total elimination resistance to malaria, making the sickle cell of all disease-causing alleles is an unrealistic gene beneficial to people living in regions where goal, and is unobtainable. It is not possible to malaria is prevalent. Since both the function and eradicate genetic disease completely as many interaction of many genes are still not fully people are carriers for genetic disease and are understood then it would be unwise to remove not aware of it, and many occur spontaneously them permanently as they may prove to be as quickly as we would be able to get rid of beneficial later (Saiki et al., 1988). them new mutations would occur. Germline Experiments using germline gene therapy hold great potential for the treatment of therapy in animals have been underway for a many devastating genetic disorders (Kato et al., few years. In the initial demonstration of the 2005). use of the technique the human genes for Given the inherent hazard it poses, growth hormone along with a regulator were germ-line therapy is a technology best inserted into mice embryos with the resultant postponed until adequate knowledge of the expression in the recipient mice being a human genome is available, and implications of doubling of their body size. However the this process for the future of the human race. technology still has a high failure rate in terms Then and only then can mankind safely decide of the large losses of egg cells, and the failure whether germ-line engineering is the true to achieve any expression (Vijayanathan et al., answer to our genetic problems (Kamau et al., 2002). With the way genetic information is 2006). transmitted in humans the genetic disease may not be expressed in the children, as they will 2. Somatic Gene Therapy: Somatic gene only receive one of their pair of genes from the therapy is the transfer of genes into the somatic affected parent and would only be a carrier of cells of the patient, such as cells of the bone the disease (Veiseh et al., 2009). Mice that marrow, and hence the new DNA does not enter were lacking the gene for the synthesis of the eggs or sperm. The genes transferred are gonadotrophic releasing hormone were used in usually normal alleles that could ‘correct’ the these trials, in which DNA containing the correct mutant or disease alleles of the recipient. gene was injected into fertilized eggs. The The technique of somatic gene therapy successfully treated animals became normal for involves inserting a normal gene into the the synthesis of gonadotropic releasing appropriate cells of an individual affected with a hormone. Although germ-line therapy is not yet genetic disease, thereby permanently correcting safe enough to use on humans, we must the disorder (Neuman et al., 1982). The consider technology before it is ready to be targeted cells may be bone marrow cells, which used especially when such high stakes are are easily isolated and re-implanted. Bone involved. Germ line therapy will likely become marrow cells continue to divide for a person's available for human applications in the next 10 whole life to produce blood cells, so this to 20 years according to scientists though it will approach is useful only if the gene you want to likely appear under less threatening names. So deliver has a biological role in the blood. the question is really not if this technology will Delivery of a gene that has a biological role in be used but when and in what ways (Brivanlou the lungs, muscle, or liver would have to occur and Darnell, 2002). within those targeted organs. In many cases,

Animal Research International (2019) 16(2): 3367 – 3392 3373 Gene therapy, physiological applications, problems and prospects 3369 accessing the appropriate tissue or, if the gene by injection into a vein. This type of gene is required in multiple tissues (e.g. muscles therapy is called ex vivo (Figure 3), because the throughout the body) ensuring it can be cells are treated outside the organism (Cheng et delivered where it is needed, is a major problem al., 1994). (Sakaguchi et al., 2008). Somatic cells are non-reproductive. Somatic cell therapy is viewed as a more conservative, safer approach because it affects only the targeted cells in the patient, and is not passed on to future generations. In other words, the therapeutic effect of gene therapy ends with the individual who receives the therapy. However, this type of therapy presents unique problems of its own. Often the effects of somatic cell therapy are short-lived. From the fact that living cells of most tissues ultimately die and are replaced by new cells, repeated treatments over the course of the individual's life span are required to maintain the Figure 3: Illustration showing ex vivo therapeutic effect (Pathak et al., 2009). form of somatic gene therapy. Source: Transporting the gene to the target cells or ONGSIMEI (2016) tissue is also problematic. Regardless of these difficulties, somatic cell gene therapy is b) In vivo: which means interior (where genes appropriate and acceptable for many disorders, are changed in cells still in the body). This form including cystic fibrosis, muscular dystrophy, of gene therapy is called in vivo (Figure 4), cancer, and certain infectious diseases. because the gene is transferred to cells inside Clinicians can even perform this therapy in vitro, the patient’s body (Cole-Strauss et al., 1999). potentially correcting or treating a life- threatening disorder that may significantly impair a baby's health or development if not treated before birth. Somatic gene therapy is restricted to the actual patient and is not passed on to his or her children (Cole-Strauss et al., 1999). All gene therapy to date on humans has been directed at somatic cells, whereas germ- line engineering in humans remains controversial and prohibited in for instance the European Union. Somatic gene therapy can be split into two broad categories: a) Ex vivo: which means exterior (where cells are modified outside the body and then transplanted back in again). In some gene Figure 4: Illustration showing in vivo form therapy clinical trials, cells from the patient’s of somatic gene therapy. Source: blood or bone marrow are removed and grown GURMUNSINGH (2018) in the laboratory. Cells are exposed to the virus that is carrying the desired gene. The virus Viral vectors that are used in somatic gene enters into the cells and inserts the desired therapy experiments include: retroviruses, gene into the cells’ DNA. The cells grow in the adenoviruses, adeno-associated viruses, and laboratory and are then returned to the patient herpes simplex viruses.

Animal Research International (2019) 16(2): 3367 – 3392 Ugwu et al. 33733684 i) Retroviruses: Retroviruses were the first Researchers hope this treatment will one day viruses used as vectors in gene therapy help people with muscular dystrophy (Lehrman, experiments. They are unusual because instead 1999). of using DNA to carry their genetic information to the cell’s protein-making machinery, ii) Adenoviruses: To avoid the problem of retroviruses use a related material called inserting genes at the wrong sites, some ribonucleic acid (RNA) as their primary carrier of researchers have turned to other types of genetic information. When retroviruses invade a viruses, such as the adenoviruses, which cause cell, they use an enzyme called reverse the common cold. Stripped of their disease- transcriptase to make a DNA copy of their causing genes, adenoviruses take healthy genes genes. Other enzymes then incorporate this into the nucleus of cells, where the DNA is DNA copy into the infected cell's DNA (Cheng et located, but do not usually integrate them into a al., 1994). Although retroviruses have been cell's DNA. Researchers thus trade safety for used in most gene therapy experiments so far, impermanence, because the genes persist in the they still present many problems. Retroviruses cell’s DNA only for days to weeks (Mayeux, can invade only cells that are actively dividing, 2005). Adenoviruses can also infect a broader limiting potential targets for therapy to blood variety of cells than retroviruses do, including cells, skin cells, stem cells, and other fast- cells that divide more slowly, such as lung cells. growing tissues. In addition, the viruses have no However, adenoviruses are also more likely to specific targets in the infected cells' be attacked by the patient's immune system, chromosomes. As a result, the genes they carry and the high levels of virus required for are inserted in a haphazard manner (Caplen et treatment often provoke an undesirable al., 1995). Ideally, retroviruses insert genes into inflammatory response. Despite these the middle of a strand of DNA that does not drawbacks, adenoviruses have been used in contain other genes. The genes might, however, attempts to treat cancers of the liver and be inserted smack in the middle of a crucial ovaries (Kato et al., 2005). gene, rendering it defective and blocking key cellular functions, causing more damage than iii) Adeno associated virus: One repair. Retroviruses could also integrate new of the most promising potential gene-delivery genes into a stretch of DNA where they could systems, or vectors, is a recently discovered cause cancer. Despite the presence of virus called the adeno-associated virus, which promoters, moreover, the added genes typically infects a broad range of cells, including both do not produce sufficient amounts of proteins to dividing and non-dividing cells. Researchers effectively treat disease. In addition, the believe that most humans carry adeno- patient’s body generally recognizes retroviruses associated viruses, which do not cause disease as foreign invaders, provoking adverse immune and do not provoke an immune response. responses (Djikmans et al., 2004). Scientists have demonstrated that the adeno- As researchers have grown more associated virus can be used to correct genetic confident, however, they have begun injecting defects in animals. It is now being used in altered retroviruses directly into tissues where preliminary studies to treat hemophilia, a the corrected genes are needed and have, so hereditary blood disease, in humans (Robrish, far, observed few problems. In clinical trials of 1999). The chief drawback of the adeno- patients with cystic fibrosis, a disease in which a associated virus is that it is small, carrying only mutated gene impairs lung function, healthy two genes in its natural state. Its payload is genes are inserted directly into the lining of therefore relatively limited. It can produce bronchial tubes (Kato et al., 2005). In studies of unintended genetic damage because the adeno- animals, researchers have used retroviruses to associated virus inserts its genes directly into inject genes directly into muscle tissue to learn the host cell's DNA. Researchers have also had if the genes will produce normal muscle difficulties manufacturing large quantities of the proteins. altered virus.

Animal Research International (2019) 16(2): 3367 – 3392 Gene therapy, physiological applications, problems and prospects 33693375 iv) Herpes simplex virus: Scientists have destabilization or destruction by exonucleases or found that the herpes simplex virus, the cause cellular helicases (Stephenson, 1999). Finally, of the common cold sore, has a very large the ribose of the RNA is 2'-O-methyl modified to genome compared to other virus vectors. This add protection against cleavage by RNase H large genome enables scientists to insert more activities (Cole-Strauss et al., 1996). than one therapeutic gene into a single virus, When designing a chimeric oligonucleotide, paving the way for the treatment of disorders there is a replication of a short portion of the caused by more than one gene defect. The virus base sequence of the target gene surrounding makes an ideal vector because it can infect a the base pair mutation so that it aligns perfectly wide variety of tissues, including muscle, tumor, with the gene, except for the one base pair liver, pancreas, nerve, and lung cells (Sails, where the mutation occurs (Jaroslav et al., 2004). One problem with using herpes simplex 2002). In this location, the correct base is virus is that the virus is cytopathic—that is, it substituted into the oligonucleotide. Therefore, kills the cells that it infects. In addition, the when the chimera inserts between the strands virus can cause encephalitis (inflammation of of target DNA, the mismatched base pair is the brain) if it replicates freely in the brain. recognized by the endogenous repair system, Scientists are developing a form of herpes and the sequence is changed in either the simplex virus in which the genes that direct the chimeraplast (using the target DNA as a virus’s replication and cell-killing abilities have template), or in the target DNA (using the been removed (Saiki et al., 1985). chimera as a template) (Robrish, 1999).In its mechanism of repair, chimeric oligonucleotides 3. Chimeraplasty: Chimeraplasty, also known probably work by pairing with a plasmid target as targeted gene correction, is a technique in based on homology using DNA pairing enzymes which a synthetically created molecule and complexes. After pairing, endogenous repair consisting of both RNA and DNA is used to machinery recognizes the mismatch between repair single base pair mutations, deletions, or the gene and the chimera, and uses mismatch insertions in DNA (Alberts et al., 2002). The repair to correct the "spelling mistake" by using technique of chimeraplasty was developed in the chimera as the template sequence. Then, the lab of Drs. Eric B. Kmiec, Kyonggeun Yoon the chimera decays, leaving only the corrected and Allyson Cole-Strauss at Thomas Jefferson target DNA (Smaglik, 2006). University in Philadelphia, Pennsylvania. Kmiec For in vivo repair, the oligonucleotides studies homologous recombination and he can be attached to organ-specific ligands. realized that the rate of recombination is Liposomes and synthetic polymers are also used increased for active genes being transcribed into to deliver chimeric molecules to the appropriate mRNAs. This led him to question whether cells or tissue. In plants, microscopic gold synthetic RNA could be used in gene repair by particles are coated with the chimeric molecules tricking the cell to incorporate good DNA into and fired into cells (Sails, 2004). In all cases, mutated sites (Cole-Strauss et al., 1999). the oligonucleotides that enter the nucleus can A chimeraplast consists of a paper-clip repair point mutations within the cell after shaped, double-stranded stretch of DNA pairing with their sequence-specific target DNA interspersed with short strands of RNA. The by causing the cell's repair machinery to design of the chimeraplast is a result of the perform mismatch repair on the point mutation. discovery that hybrids of RNA and DNA are very After correction, the chimera decays, leaving the active in homologous pairing reactions (Yoon et corrected gene (Smaglik, 2006). al., 1996). Furthermore, it was found that the hairpin caps at the ends of the molecules do not Techniques/Protocols in Gene Therapy impede base pairing with target genes. The purpose of the short RNA strands is to activate 1. Polymerase Chain Reaction: Polymerase the oligonucleotide ("oligo") for recombination, chain reaction (PCR) is a common and often and the hairpin caps protect the molecule from indispensible technique used in medical and

Animal Research International (2019) 16(2): 3367 – 3392 Ugwu et al. 33683376 biological research laboratories for a variety of (kb), although some techniques allow for applications. These include DNA cloning for amplification of fragments up to 40 kb in size. sequencing, DNA-based phylogeny, or functional The reaction produces limited amount of final analysis of genes; the diagnosis of hereditary amplified product that is governed by the diseases; the identification of genetic available reagents in the reaction and the fingerprints (used in forensic sciences and feedback-inhibition of the reaction products paternity testing); and the detection and (Chien et al., 1976). diagnosis of infectious diseases (Lawyer et al., A basic PCR set up requires several 1993). The method/protocol relies on thermal components and reagents. These components cycling, consisting of cycles of repeated heating are: (i) DNA template that contains the DNA and cooling of the reaction for DNA melting and region (target) to be amplified, (ii) two primers enzymatic replication of the DNA. Primers that are complementary to the 3' (three prime) containing sequences complementary to the ends of each of the sense and anti-sense strand target region along with a DNA polymerase of the DNA target, (iii) taq polymerase or (after which the method is named) are key another DNA polymerase with a temperature components to enable selective and repeated optimum at around 70 °C, (iv) deoxynucleoside amplification (Park, 2004). As PCR progresses, triphosphates (dNTPs; nucleotides containing the DNA generated is itself used as a template triphosphate groups), the building blocks for replication, setting in motion a chain reaction through which the DNA polymerase synthesizes in which the DNA template is exponentially a new DNA strand, (v) buffer solution, providing amplified. PCR can be extensively modified to a suitable chemical environment for optimum perform a wide array of genetic manipulations activity and stability of the DNA polymerase, (vi) (Pierce and Wangh, 2007). divalent cations, magnesium or manganese Almost all PCR applications employ a ions; generally Mg2+ is used, but Mn2+ can be heat-stable DNA polymerase, such as Taq utilized for PCR-mediated DNA mutagenesis, as polymerase, an enzyme originally isolated from higher Mn2+ concentration increases the error the bacterium Thermus aquaticus. The DNA rate during DNA synthesis and (vii) monovalent polymerase enzymatically assembles a new DNA cation potassium ions (Innis et al., 1988; strand from DNA building blocks, the Isenbarger et al., 2008). nucleotides, by using single stranded DNA as a The PCR is commonly carried out in a template and DNA oligonucleotides (also called reaction volume of 10 – 200 μl in small reaction DNA primers), which are required for initiation tubes (0.2 – 0.5 ml volumes) in a thermal of DNA synthesis (Sarkar et al., 1990). Majority cycler. The thermal cycler heats and cools the of PCR methods use thermal cycling, i.e., reaction tubes to achieve the required alternately heating and cooling the PCR sample temperatures at each step of the reaction. Many to a defined series of temperature steps. These modern thermal cyclers make use of the Peltier thermal cycling steps are necessary first to effect, which permits both heating and cooling physically separate the two strands in a DNA of the block holding the PCR tubes simply by double helix at a high temperature in a process reversing the electric current. Thin-walled called DNA melting. At lower temperature, each reaction tubes are favorable thermal conductors strand is then used as the template in DNA that allow for rapid thermal equilibration. Most synthesis by the DNA polymerase to selectively thermal cyclers have heated lids to prevent amplify the target DNA. The selectivity of PCR condensation at the top of the reaction tube. results from the use of primers that are Older thermocyclers lacking a heated lid require complementary to the DNA region targeted for a layer of oil on top of the reaction mixture or a amplification under specific thermal cycling ball of wax inside the tube (Lawyer et al., conditions (Arena et al., 2011). PCR is used to 1993). amplify specific region of DNA strand (the DNA Typically, PCR consists of a series of 20 target). Most PCR methods typically amplify – 40 repeated temperature changes, called DNA fragments of up to ~10 kilo base pairs cycles, with each cycle commonly consisting of

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2 – 3 discrete temperature steps. The cycling is adding dNTPs that are complementary to the often preceded by a single temperature step template in 5' to 3' direction, condensing the 5'- (called hold) at a high temperature (about phosphate group of dNTPs with the 3'-hydroxyl 90°C), and followed by one hold at the end for group at the end of the nascent (extending) final product extension or brief storage (Park, DNA strand. The extension time depends both 2004). The temperatures used and the length of on the DNA polymerase used and on the length time they are applied in each cycle depend on a of the DNA fragment to be amplified. Usually, at number of parameters. These include the its optimum temperature, the DNA polymerase enzyme used for DNA synthesis, the will polymerize a thousand bases per minute. concentration of divalent ions and dNTPs in the Under optimum conditions, i.e., if there are no reaction, and the melting temperature (Tm) of limitations due to limiting substrates or the primers. reagents, at each extension step, the amount of DNA target is doubled, leading to exponential Initialization step: This step consists of (geometric) amplification of the specific DNA heating the reaction to a temperature of fragment. between 94 – 96 °C (or 98 °C if extremely thermostable polymerases are used), which is Final elongation: This step is occasionally held for 1 – 9 minutes. It is only required for performed at a temperature between 70 – 74 °C DNA polymerases that require heat activation by for 5 – 15 minutes after the last PCR cycle to hot-start PCR (Isenbarger et al., 2008). ensure that any remaining single-stranded DNA is fully extended. Denaturation step: This step is the first regular cycling event and consists of heating the Final hold: This step at 4–15 °C for an reaction to temperature between 94 – 98 °C for indefinite time may be employed for short-term 20 – 30 seconds. It causes DNA melting of the storage of the reaction (Park, 2004). DNA template by disrupting the hydrogen bonds To check whether the PCR generated between complementary bases, yielding single- the anticipated DNA fragment (sometimes stranded DNA molecules (Lawyer et al., 1993; referred to as the amplimer or amplicon). Isenbarger et al., 2008). Agarose gel electrophoresis is employed for size separation of the PCR products. The size(s) of Annealing step: The reaction temperature is PCR products is determined by comparison with lowered to 50 – 65 °C for 20 – 40 seconds a DNA ladder (a molecular weight marker), allowing annealing of the primers to the single- which contains DNA fragments of known size, stranded DNA template. Typically the annealing run on the gel alongside the PCR products. The temperature is about 3 - 5 oC below the Tm of PCR process can be divided into three stages: the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer Exponential amplification: At every cycle, sequence closely matches the template the quantity of product is doubled (assuming sequence. The polymerase binds to the primer- 100% reaction efficiency). The reaction is very template hybrid and begins DNA formation sensitive: only minute quantities of DNA need to (Pierce and Wangh, 2007). be present.

Extension/elongation step: The temperature Leveling off stage: The reaction slows down at this step is determined by the type of DNA as the DNA polymerase loses activity and as polymerase used; Taq polymerase has its consumption of reagents such as dNTPs and optimum activity temperature at 75 – 80 °C, primers causes them to become limiting. and commonly a temperature of 72 °C is used with this enzyme. At this step the DNA Plateau: No more product accumulates due to polymerase synthesizes a new DNA strand exhaustion of reagents and enzyme (Pierce and complementary to the DNA template strand by Wangh, 2007; Isenbarger et al., 2008).

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PCR Optimization involving the insertion of a DNA sequence into a plasmid or the genetic material of another In practice, PCR can fail due to various reasons. organism (Sanger et al., 1977). Escherichia coli In most cases due to its sensitivity to colonies can be rapidly screened by PCR for contamination causing amplification of spurious correct DNA vector constructs. PCR may also be DNA products. Because of this, a number of used for genetic fingerprinting; a forensic techniques and procedures have been technique used to identify a person or organism developed for optimizing PCR conditions. (forensic entomology) by comparing Contamination with extraneous DNA is experimental DNAs through different PCR-based addressed with laboratory protocols and methods (Cheng et al., 1994). procedures that separate pre-PCR mixtures from potential DNA contaminants (Chien et al., b. Amplification and quantification of 1976). This usually involves spatial separation of DNA: Because the regions of DNA that it PCR-setup areas from areas for analysis or targeted are amplified by PCR; PCR can be used purification of PCR products, use of disposable to analyze extremely small amounts of sample. plastic ware, and thoroughly cleaning the work This is often critical for forensic analysis, when surface between reaction setups (Rychlik et al., only a trace amount of DNA is available as 1990). Primer design techniques are important evidence. PCR may also be used in the analysis in improving PCR product yield and in avoiding of ancient DNA that is tens of thousands of the formation of spurious DNA products, and years old (Mullis, 1990; Park, 2004). the usage of alternate buffer components or Quantitative PCR methods allow the estimation polymerase enzymes can help with amplification of the amount of a given sequence present in a of long or otherwise problematic regions of DNA sample a technique often applied to (Park, 2004). Addition of reagents, such as quantitatively determine levels of gene formamide, in buffer systems may increase the expression. Real-time PCR is an established tool specificity and yield of PCR. Computer for DNA quantification that measures the simulations of theoretical PCR results (Electronic accumulation of DNA product after each round PCR) may be performed to assist in primer of PCR amplification (Pavlov et al., 2006). design (Pavlov et al., 2004; Isenbarger et al., 2008). c. PCR in diagnosis of diseases: PCR permits early diagnosis of malignant diseases such as Applications of PCR leukemia and lymphomas, which is currently the highest-developed in cancer research and is a. Selective DNA isolation: PCR allows already being used routinely. PCR assays can be isolation of DNA fragments from genomic DNA performed directly on genomic DNA samples to by selective amplification of a specific region of detect translocation-specific malignant cells at a DNA. This use of PCR augments many methods, sensitivity that is at least 10,000-fold higher such as generating hybridization probes and than that of other methods (Pavlov et al., DNA cloning, which require larger amounts of 2004). DNA, representing a specific DNA region (Park, PCR aids the identification of non- 2004). PCR supplies these techniques with high cultivatable or slow-growing microorganisms amounts of pure DNA, enabling analysis of DNA such as anaerobic bacteria, mycobacteria, or samples even from very small amounts of viruses from tissue culture assays and animal starting materials for gene therapy protocols. models. The basis for PCR diagnostic Other applications of PCR include (i) applications in microbiology is the detection of DNA sequencing to determine unknown PCR- infectious agents and the discrimination of non- amplified sequences in which one of the pathogenic from pathogenic strains by virtue of amplification primers may be used in Sanger specific genes (Pavlov et al., 2006; Pierce and sequencing, and (ii) isolation of a DNA sequence Wangh, 2007). to expedite recombinant DNA technologies

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Viral DNA can likewise be detected by different DNA sequences. By targeting multiple PCR. The primers used must be specific to the genes at once, additional information may be targeted sequences in the DNA of a virus, and gained from a single test-run that otherwise the PCR can be used for diagnostic analyses or would require several times the reagents and DNA sequencing of the viral genome. The high more time to perform. Annealing temperatures sensitivity of PCR permits virus detection soon for each of the primer sets must be fully after infection and even before the onset of optimized to work correctly within a single disease. Such early detection may give reaction and amplicon sizes. That is, their base physicians a significant lead in treatment. The pair length should be different enough to form quantity or amount of virus ("viral load") in a distinct bands when visualized by gel patient can also be quantified by PCR-based electrophoresis (Pierce and Wangh, 2007; DNA quantitation techniques (Sails, 2004). Isenbarger et al., 2008).

Variations of the PCR Technique in Gene Nested PCR: increases the specificity of DNA Therapy amplification, by reducing background arising from non-specific amplification of DNA. Two sets Allele-specific PCR: a diagnostic or cloning of primers are used in two successive PCRs. In technique based on single-nucleotide the first reaction, one pair of primers is used to polymorphisms (SNP) (single-base differences in generate DNA products, which besides the DNA). SNP requires prior knowledge of a DNA intended target, may still consist of non- sequence, including differences between alleles, specifically amplified DNA fragments. The and uses primers whose 3' ends encompass the product(s) are then use in a second PCR with a SNP. PCR amplification under stringent set of primers whose binding sites are conditions is much less efficient in the presence completely or partially different from and of a mismatch between template and primer, so located 3' of each of the primers used in the successful amplification with an SNP-specific first reaction. Nested PCR is often more primer signals presence of the specific SNP in a successful in specifically amplifying long DNA sequence (Lawyer et al., 1993). fragments than conventional PCR, but it requires more detailed knowledge of the target Asymmetric PCR: preferentially amplifies one sequences (Sarker et al., 1990; Pierce and DNA strand in a double-stranded DNA template. Wangh, 2007). It is used in sequencing and hybridization probing where amplification of only one of the Nanoparticles two complementary strands is required. PCR is carried out normally, but with excess of the Transgenic technology has been widely used in primer for the strand targeted for amplification. breeding new plant and animal varieties. Because of the slow (arithmetic) amplification Effective gene delivery into the target cells is an later in the reaction after the limiting primer has essential step in these practices (Jaroslav et al., been used up, extra cycles of PCR are required. 2002). Both viral and nonviral vectors have A recent modification of this process, called been used for gene delivery especially with Linear-After-The-Exponential-PCR (LATE-PCR) regards to gene therapy. Nanoparticles as gene uses a limiting primer with a higher melting vectors, due to their reduced immunogenicity, temperature (Tm) than the excess primer to improved safety and the ability to carry larger maintain reaction efficiency as the limiting DNA loads has become an attractive alternative primer concentration decreases mid-reaction in gene delivery. Cationic liposomes have been (Pierce and Wangh, 2007). widely used as non-viral gene vectors, but, because of its cell-specific, easily inactivation of Multiplex PCR: is the use of multiple primer serum protein, and other reasons, its scope of sets within a single PCR mixture to produce application is also limited (Mehrdad et al., amplicons of varying sizes that are specific to 2008).

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Because of their unique physical and chemical by PEI-modified magnetic nanoparticles (Kamau properties, nanoparticles have been widely used et al., 2006). It is found that the efficiency of in the field of drug and gene delivery to target gene delivery to the target sites is/are not only cells. As a novel kind of gene vectors, affected by the mass ratio of nanoparticle/DNA, nanoparticles have many advantages (Pathak et but also the amount of the nanoparticle/DNA al., 2009). DNA molecules can be protected by complexes. Based on the analysis of the PEI- nanoparticles against nuclease degradation by modified magnetic nanoparticles, the wrapping and condensing nucleic acids, realize combination of nanoparticles has a good ability the specificity of gene delivery by conjugating to bind with plasmid DNA (Kumari et al., 2010). with special target molecules, and achieve Moreover, the formation of nanoparticle/DNA controlled release of DNA effectively extending complexes can protect DNA against enzyme the duration of action (Veiseh et al., 2009). degradation. Such advantages of the PEI- Besides the advantages of ordinary modified magnetic nanoparticles make it have a nanoparticles, other nanoparticles like magnetic potential strength as vector for gene delivery nanoparticles are superparamagnetic. They can (Kato et al., 2005; Cui et al., 2012). induce the DNA moving and condensing to the The nanoparticles are used to deliver target cells in the presence of a magnetic field, green fluorescent protein expression carrier which can significantly improve their efficiency (pEGFP-N1) into PK-15 cells as gene vectors. as gene carriers (Vijayanathan et al., 2002). The expression of green fluorescent protein Although the application of magnetic gene (GFP) is detected by fluorescence nanoparticles in gene diagnosis and therapy has microscope. The efficiency of GFP expression been studied extensively, the gene transfer can be enhanced significantly in the presence of systems based on magnetic nanoparticles for a magnetic field (Kaneda, 2003). The reason is gene delivery into mammalian cells are few that the plasmid DNA can be directionally studied (Plank et al., 2003a). Effective gene delivered to the receptor cells by magnetic delivery into mammalian cells is an essential nanoparticle under the magnetic field, which step in reproductive cloning using somatic cell condensed the concentration of nuclear transfer technique. New advances in nanoparticle/DNA complexes on the surface of this field report a new approach to deliver genes the cells, resulting in the increased cellular to porcine somatic cells using PEI-modified endocytosis of the complexes (Cui et al., 2012). magnetic nanoparticles as gene vector (Plank et The delivery of genes of interest into al., 2003b). To access the potential of magnetic PK-15 and PEF cells using magnetic nanoparticles as gene transfer vectors in nanoparticles provides an important transgenic animal production, PEI-modified experimental basis for the application of the

Fe3O4 magnetic nanoparticles were employed to magnetic nanoparticles as gene transfer vector transfer reporter gene into somatic cells for for somatic cells, thereby providing the gene delivery efficiency (Yang et al., 2008; Cui foundation study for its application in gene et al., 2012). therapy as well as an important technique in The PEI-modified magnetic breeding new transgenic cloned plants and nanoparticles are spherical in shape with an animals (Scherer et al., 2002; Kaneda, 2003). average diameter of about 150 nm. The surface of the nanoparticle becomes coarse and rough, Sonoporation and the average diameter increases to 200 nm after conjugated with the plasmid DNA. The Sonoporation, or cellular sonication, is the use zeta potential of nanoparticle/DNA complexes of sound (typically ultrasonic frequencies) for drops down from +29.4 mV to +23.1 mV after modifying the permeability of the cell plasma the conjugation. Agarose gel electrophoresis membrane. This technique is usually used in experiments show that DNA plasmids can be molecular biology and non-viral gene therapy in loaded and protected effectively against order to allow uptake of large molecules such as degradation of exonuclease and endonuclease DNA into the cell, in a cell disruption process

Animal Research International (2019) 16(2): 3367 – 3392 Gene therapy, physiological applications, problems and prospects 33336981 called cell transfection or cell transformation Measurement of the acoustics used in (Arena et al., 2011). Sonoporation employs the sonoporation is listed in terms of mechanical acoustic cavitation of microbubbles to enhance index, which quantifies the likelihood that delivery of these large molecules (Figure 5). exposure to diagnostic ultrasound will produce an adverse biological effect by a non-thermal action based on pressure. Sonoporation uses microbubbles for significantly enhancing cell transfection, and in some cases is required for DNA (Jaroslav et al., 2002).

Electroporation

Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field (Becker and Kuznetov, 2007). It is usually used in molecular biology as a way of introducing some substance into a cell, such as loading it with a molecular probe, a drug that can change the cell's function, or a piece of coding DNA Figure 5: Schematic illustration showing (Djikmans et al., 2004). sonoporation. Source: Kim et al. (2008) Electroporation is a dynamic phenomenon that depends on the local The bioactivity of sonoporation is similar to, and transmembrane voltage at each point on the cell in some cases found superior to, membrane. It is generally accepted that for a electroporation. Extended exposure to low- given pulse duration and shape, a specific frequency (

Sonoporation is under active study for electroporation (Eth). That is, only the cells the introduction of foreign genes in tissue within areas where E≧Eth are electroporated. If culture cells, especially mammalian cells. a second threshold (Eir) is reached or surpassed, Sonoporation is also being studied for use in electroporation will compromise the viability of targeted gene therapy in vivo, in a medical the cells, i.e., irreversible electroporation (Arena treatment scenario whereby a patient is given et al., 2011). modified DNA, and an ultrasonic transducer In molecular biology, the process of might target this modified DNA into specific electroporation is often used for the regions of the patient's body (Song et al., transformation of bacteria, yeast, and plant 2007). protoplasts. In addition to the lipid membranes, Sonoporation is performed with a bacteria also have cell walls which are different dedicated sonoporator. Sonoporation may also from the lipid membranes and are made of be performed with custom-built piezoelectric peptidoglycan and its derivatives (Pathak et al., transducers connected to bench-top function 2009). However, the walls are naturally porous generators and acoustic amplifiers. Standard and only act as stiff shells that protect bacteria ultrasound medical devices may also be used in from severe environmental impacts. If bacteria some applications (Church, 2005). and plasmids are mixed together, the plasmids can be transferred into the cell after

Animal Research International (2019) 16(2): 3367 – 3392 Ugwu et al. 33683382 electroporation. Several hundred volts across a the suspension, and is inoperable with isolated distance of several millimeters are typically used electrodes, obviously the process involves in this process. Afterwards, the cells have to be certain electrolytic effects, due to small currents handled carefully until they have had a chance and not only fields (Garcia et al., 2010a). to divide producing new cells that contain reproduced plasmids. This process is Gene Gun approximately ten times as effective as chemical transformation (Neumann et al., 1982; Sugar A gene gun or a biolistic particle delivery and Neumann, 1984). system, originally designed for plant This procedure is also highly efficient transformation, a device for injecting cells with for the introduction of foreign genes in tissue genetic information. The payload is an culture cells, especially mammalian cells. For elemental particle of a heavy metal coated with example, it is used in the process of producing plasmid DNA. This technique is often simply knockout mice, as well as in tumor treatment, referred to as bioballistics or biolistics. The gene gene therapy, and cell-based therapy. The gun is able to transform almost any type of cell, process of introducing foreign DNAs into including plants, and is not limited to genetic eukaryotic cells is known as transfection. material of the nucleus: it can also transform Electroporation is done with electroporators, organelles, including plastids (Wolf et al., 2002). appliances that create an electro-magnetic field It was invented by John C Sanford, Ed Wolf and in the cell solution. The cell suspension is Nelson Allen at Cornell, and Ted Klein of pipetted into a glass or plastic cuvette which DuPont, between 1983 and 1986. The original has two aluminum electrodes on its sides target was onions (chosen because of their (Garcia et al., 2010). large cell size) and it was used to deliver For bacterial electroporation, typically a particles coated with a marker gene. Genetic suspension of around 50 microliters is used. transformation was then proven when the onion Prior to electroporation it is mixed with the tissue expressed the gene. Gene guns are so far plasmid to be transformed. The mixture is mostly applied to plant cells. However, there is pipetted into the cuvette, the voltage and much potential use in animals and humans as capacitance are set, and the cuvette is inserted well. Gene guns have also been used to deliver into the electroporator. Immediately after DNA vaccines (Yao et al., 2006). The delivery of electroporation, one milliliter of liquid medium is plasmids into rat neurons through the use of a added to the bacteria (in the cuvette or in an gene gun, specifically DRG neurons, is also used eppendorf tube), and the tube is incubated at as a pharmacological precursor in studying the the bacteria's optimal temperature for an hour effects of neurodegenerative diseases such as or more to allow recovery of the cells and Alzheimer's disease (Gan, 1989). expression of antibiotic resistance, followed by Plant transformation using particle spreading on agar plates (Garcia et al., 2011). bombardment/gene gun follows the same The success of the electroporation depends outline as Agrobacterium-mediated method greatly on the purity of the plasmid solution, (Figure 6). The steps taken include: i) isolate most especially on its salt content. Solutions the genes of interest from the source organism; with high salt concentrations might cause an ii) develop a functional transgenic construct electrical discharge (known as arcing), which including the gene of interest; promoters to often reduces the viability of the bacteria drive expression; codon modification, if needed (Thomson et al., 2011). to increase successful production of protein; and For further detailed investigation of the marker genes to facilitate tracking of the process, more attention should be paid to the introduced genes in the host plant; iii) output impedance of the porator device and the incorporate into a useful plasmid; iv) introduce input impedance of the cells suspension (e.g. the transgenes into plant cells; v) regenerate salt content). As the process needs direct the plants cells; and vi) test trait performance or electrical contact between the electrodes and

Animal Research International (2019) 16(2): 3367 – 3392 Gene therapy, physiological applications, problems and prospects 33693383 gene expression at lab, greenhouse and field at multiple insertion sites. These multiple copies level (Gan, 1989). can be linked to silencing of the transgene in subsequent progeny (Gan, 1989; Yao et al., 2006).

Applications of Gene Therapy

Genetic engineering is an indispensible tool for natural scientists. Genes and other genetic information from a wide range of organisms are transformed into bacteria for storage and modification, creating genetically modified bacteria in the process. Bacteria are cheap, easy to grow, multiply quickly, relatively easy to transform and can be stored at -80°C almost indefinitely. Once a gene is isolated it can be stored inside the bacteria providing an unlimited supply for research (Lodish et al., 2000). Figure 6: Schematic illustration of biolistic Gene therapy finds applications in particle delivery system in plants. Source: medicine, agriculture and in the production of Hingra (2018) genetically engineered organisms in order to discover the functions of certain genes (Mayeux, The particle bombardment method begins with 2005). This could be the effect on the coating tungsten or gold particles (micro- phenotype of the organism, where the gene is projectiles) with plasmid DNA. The coated expressed or what other genes it interacts with. particles are coated on a macro-projectile, which is accelerated with air pressure and shot 1. Physiological applications: One promising into plant tissue on a petri plate. A perforated application of gene therapy is in treating type I plate is used to stop the macro-projectile, while diabetes. Current research used an adenovirus allowing the micro-projectiles to pass through to as a vector to deliver the gene for hepatocyte the cells on the other side. As the micro- growth factor (HGF) to pancreatic islet cells projectiles enter the cells, the transgenes are removed from rats. They injected the altered released from the particle surface and may cells into diabetic rats and, within a day, the incorporate into the chromosomal DNA of the rats were controlling their blood glucose levels cells. Selectable markers are used to identify the better than the control rats. This model mimics cells that take-up the transgene. The the transplantation of islet cells in humans and transformed plant cells are then regenerated shows that the addition of the HGF gene greatly into whole plants using tissue culture techniques enhances the islet cells' function and survival. (Yao et al., 2006). Particle bombardment also In May 2006, a team of scientists plays an important role in the transformation of reported a breakthrough for gene therapy, in organelles such as chloroplasts, which enables which they developed a way to prevent the engineering of organelle-encoded herbicide or immune system from rejecting a newly delivered pesticide resistances in crop plants and to study gene. Delivery of 'normal' gene has been photosynthetic processes (Wolf et al., 2002). difficult because the immune system recognizes Limitations to the particle bombardment method the new gene as foreign and rejects the cells with regards to Agrobacterium-mediated carrying it. This problem was overcome utilizing transformation include (i) frequent integration of a newly uncovered network of genes regulated multiple copies of the transgene at a single by molecules known as microRNAs. The use of insertion site, (ii) rearrangement of the inserted this natural function of microRNA is to genes, and (iii) incorporation of the transgene selectively turn off the identity of the

Animal Research International (2019) 16(2): 3367 – 3392 Ugwu et al. 33683384 therapeutic gene in cells of the immune system biological and biomedical research studies. Gene and prevent the gene from being found and therapy focuses its application on GMOs by destroyed. The output of the study has trying to find out more about these organisms important implications for the treatment of using the following means: hemophilia and other genetic diseases by gene therapy. i. Loss of function experiments: This occurs RNA interference or gene silencing may in gene knockout experiment, in which an be a new way to treat Huntington's disease. organism is engineered to lack the activity of Short pieces of double-stranded RNA and/or one or more genes. A knockout experiment short interfering RNAs (siRNAs) are used by involves the creation and manipulation of a DNA cells to degrade RNA of a particular sequence. If construct in vitro, which, in a simple knockout, a siRNA is designed to match the RNA copied consists of a copy of the desired gene, which from a faulty gene, then the abnormal protein has been altered such that it is non-functional. product of that gene will not be produced. Embryonic stem cells incorporate the altered Gene therapy have been successfully gene, which replaces the already present used to treat metastatic melanoma using killer functional copy. These stem cells are injected T-cells genetically retargeted to attack the into blastocysts, which are implanted into cancer cells. This study constitutes one of the surrogate mothers. This allows the experimenter first demonstrations that gene therapy can and to analyze the defects caused by this mutation will be effective in treating cancer. Gene therapy and thereby determine the role of particular has been used to treat Leber's Congenital genes. It is used especially frequently in Amaurosis (LCA), a rare inherited retinal developmental biology. Another method useful degenerative disorder that causes blindness in in organisms such as Drosophila, is to induce children. The patients had a defect in the RPE65 mutations in a large population and then screen gene, which was replaced with a functional copy the progeny for the desired mutation. A similar using adeno-associated virus. In all the clinical process can be used in both plants and trials, patients recovered functional vision prokaryotes (Pavlov et al., 2004). without apparent side-effects. These studies, which used adeno-associated virus, have ii. Gain of function experiments: This is the spawned a number of new studies investigating logical counterpart of knockouts. Gains of gene therapy for human retinal disease. function experiments are sometimes performed in conjunction with knockout experiments to 2. Agricultural applications: Gene therapy more finely establish the function of the desired has seen basic applications in the field of gene. The process is much the same as that in agriculture and most especially in animal health knockout engineering, except that the construct and production. This has helped to enhance the is designed to increase the function of the gene, genetic capabilities of farm animals. Applications usually by providing extra copies of the gene or of gene therapy in animal production have led inducing synthesis of the protein more to the creation of farm animals with rapid frequently (Thomson et al., 2011). growth and maturity rates, increased meat/beef production and increased immunity of farm iii. Tracking experiments: This seeks to gain animals with respect to common diseases which information about the localization and these animals are normally exposed to etc. interaction of the desired protein. Tracking experiment is done by replacing the wild-type 3. Production of genetically modified gene with a 'fusion' gene, which is a organisms (GMOs): GMOs are organisms juxtaposition of the wild-type gene with a whose genetic materials have been altered as a reporting element such as green fluorescent result of the incorporation of DNA molecules protein (GFP) that will allow easy visualization of from different sources, leading to the creation of the products of the genetic modification (Alberts organisms with novel genes. GMOs are useful in et al., 2002). While this is a useful technique,

Animal Research International (2019) 16(2): 3367 – 3392 Gene therapy, physiological applications, problems and prospects 33693385 the manipulation can destroy the function of the One major advantage of using N-TIRE is gene, thus creating secondary effects and that, when done correctly according to careful possibly calling into question the outcome of the calculations and protocols, it only affects the experiment. More sophisticated techniques are target tissue. Proteins, the extracellular matrix, now in development that can track protein and critical structures such as blood vessels and products without mitigating their function, such nerves are all unaffected and left healthy by this as the addition of small sequences that will treatment. This allows for quicker recovery and serve as binding motifs to enhance the activities facilitates rapid replacement of dead tumor cells of monoclonal antibodies. with healthy cells (Garcia et al., 2011). Although medical scientists are not iv. Expression studies: This aims to discover ready to routinely use irreversible where and when specific proteins are produced. electroporation to treat complicated tumors in In these experiments, the DNA sequence before humans, N-TIRE is more commonly used to the DNA that codes for a protein, known as a treat simple cutaneous or subcutaneous tumors gene's promoter, is reintroduced into an in humans. In addition to treating cutaneous organism with the protein coding region and subcutaneous tumors, there have been replaced by a reporter gene such as GFP or an attempts at using this technology to treat enzyme that catalyzes the production of a dye prostate, lung, kidney and liver cancer in (Caplen et al., 1995). Thus the time and place humans. However, scientists are still in the where a particular protein is produced can be process of understanding this technology and its observed. Expression studies can be taken a effect on animals and humans (Garcia et al., step further by changing the promoter to find 2010b). A recent study tested the safety of N- which pieces are crucial for the proper TIRE on humans suffering from lung, kidney or expression of the gene and are actually bound liver tumors. Of the 69 tumors treated, 49 of by transcription factor proteins; this process is them achieved complete tumor ablation. The known as promoter bashing (Dijkmans et al., most successful treatment rate occurred in liver 2004). tumors. This study provides encouraging A technique called the non-thermal evidence for the future of the use of N-TIRE as irreversible electroporation (N-TIRE) has been a method of treating cancer in humans, hence successfully used in treating many different becoming a vital tool in gene therapy (Pathak et types of tumors and other unwanted tissue. This al., 2009). procedure is done using small electrodes (about Physiologically, electroporation as a 1mm in diameter), placed either inside or protocol in gene therapy can also be used to surrounding the target tissue to apply short, help deliver drugs or genes into the cell by repetitive bursts of electricity at a applying short and intense electric pulses that predetermined voltage and frequency (Kaneda, transiently permeabilize cell membrane, thus 2003). The bursts of electricity increases the allowing transport of molecules otherwise not resting transmembrane potential (TMP), so that transported through a cellular membrane. This nanopores are formed in the plasma membrane. procedure is referred to as electrochemotherapy When the electricity applied to the tissue is when the molecule to be transported is a above the electric field threshold of the target chemotherapeutic agent or gene electrotransfer tissue, the cells become permanently permeable when the molecule to be transported is DNA from the formation of nanopores. As a result, (Thomson et al., 2011). the cells are unable to repair the damage and Electroporation allows cellular die due to a loss of homeostasis. N-TIRE is introduction of large highly charged molecules unique to other tumor ablation techniques in such as DNA which would never passively that it does not create thermal damage to the diffuse across the hydrophobic bilayer core. This tissues around it (Pathak et al., 2009; Miklavcic phenomenon indicates that the mechanism is et al., 2010). the creation of nm-scale water-filled holes in the membrane (Vijayanathan et al., 2002). Although

Animal Research International (2019) 16(2): 3367 – 3392 3386 Ugwu et al. 3368 electroporation and dielectric breakdown both effectiveness is always a possibility. result from application of an electric field, the Furthermore, the immune system's enhanced mechanisms involved are fundamentally response to invaders that it has seen before different. In dielectric breakdown the barrier makes it much more difficult for gene therapy to material is ionized, creating a conductive be repeated in patients. pathway. The material alteration is thus chemical in nature. In contrast, during iii. Problems with viral vectors: Viruses, the electroporation the lipid molecules are not carrier of choice in most gene therapy studies, chemically altered but simply shift position, present a variety of potential problems to the opening up a pore which acts as the conductive patient: toxicity, immune and inflammatory pathway through the bilayer as it is filled with responses, and gene control and targeting water (Miklavcic et al., 2010). issues. In addition, there is always the fear that the viral vector, once inside the patient, may Problems and Prospects of Gene Therapy: recover its ability to cause disease. Since the first clinical gene-therapy trial was conducted, much attention and considerable iv. Multigene disorders: Conditions or promise has been given to the field. There has disorders that arise from mutations in a single been substantial public- and private-sector gene are the best candidates for gene therapy. investment, as well as increasingly higher levels Unfortunately, some of the most commonly of research activity. Numerous preclinical occurring disorders, such as heart disease, high animal-model studies have provided proofs of blood pressure, Alzheimer's disease, arthritis, concept for multiple potential clinical and diabetes, are caused by the combined applications. Also, major advances have been effects of variations in many genes. Multigene made in understanding vector biology and or multifactorial disorders such as those improving vector design and production with mentioned above would be especially difficult to regards to gene therapy (Sakaguchi et al., effectively treat using gene therapy. 2008). v. Chance of inducing a tumor (insertional Problems Associated with Gene Therapy: mutagenesis): If the DNA is integrated in the Some of the problems associated with gene wrong place in the genome, for example in a therapy include: tumor suppressor gene, it could induce a tumor. This has occurred in clinical trials for X-linked i. Short-lived nature of gene therapy: severe combined immunodeficiency (X-SCID) Before gene therapy can produce a permanent patients, in which hematopoietic stem cells were cure for any condition, the therapeutic DNA transduced with a corrective transgene using a introduced into targeted cells must remain retrovirus, and this led to the development of T functional and the cells containing the cell leukemia in 3 of 20 patients. One possible therapeutic DNA must be long-lived and stable. solution for this is to add a functional tumor The problem with integrating therapeutic DNA suppressor gene onto the DNA to be integrated; into the genome and the rapidly dividing nature however, this poses its own problems, since the of many cells prevent gene therapy from longer the DNA is, the harder it is to integrate it achieving any long-term benefits. Patients will efficiently into cell genomes (Lehrman, 1999; have to undergo multiple rounds of gene Pierce and Wangh, 2007; Khan et al., 2008) therapy. Prospects of Gene Therapy ii. Immune response: Anytime a foreign object is introduced into human system, the There are a lot of prospects pertaining to gene immune system has evolved to attack the therapy. Gene therapy as we all know it is not a invader. The risk of stimulating the immune ‘cure all’ option. It is not always successful but it system in a way that reduces gene therapy means that a disease can be eradicated for a

Animal Research International (2019) 16(2): 3367 – 3392 Gene therapy, physiological applications, problems and prospects 3369338 7 person and their future offspring, so it is inside the cells within the body. The acquired remedied in not just one generation but also in DNA becomes expressed by the cell machinery subsequent generations (Arena et al., 2011). resulting in the production of a therapeutic Gene therapy also has the potential to protein which in turn treats the patient’s ‘silence’ a gene as in the case of a subject with diseases. The technology of gene therapy is HIV, which had not yet developed into AIDS, based on the effective delivery of the corrective scientists could save them the pain and genes and to do this, scientists have developed suffering of the disease by using gene therapy gene delivery vehicles called vectors. The to ‘silence’ the disease before its onset. I believe vectors encapsulate therapeutic genes for that if the cynics and those who are skeptical to delivery into the target cells. Many of the this technique were ever faced with cancer or a vectors currently in use are based on attenuated child born with a genetic disease, they would or modified versions of viruses. Plasmids, which change their views. These skeptics would almost are circular pieces of DNA extracted from certainly choose gene therapy, especially if it bacteria, are also used as vectors. The was the last hope for them or one of their loved therapeutic gene to be transferred is extracted ones – as is the case for many gene therapy from the cell of a healthy individual. The gene is patients (Khan et al., 2008). In 2000, a team of extracted by cutting the DNA using a restriction medical doctors in Paris described results from a enzyme (restriction enzymes "digest" DNA at study involving two children suffering from a designated nucleotide locations along the DNA severe combined immunodeficiency disorder chain).There are a lot of techniques/protocols (SCID-XI), which had restricted them to life in that are used in gene therapy. These techniques an isolated environment (Kato et al., 2005). include the polymerase chain reaction (PCR), These investigators used a MoMLV vector to electroporation, nanoparticles, sonoporation etc. transfer a curative gene (γc cytokine receptor These protocols rely on different means to subunit) into the patients’ lymphocytes ex vivo, deliver the DNA of interest to the target sites and after amplification of the cells, returned within the genes. PCR utilizes heat-stable DNA them to the patients. Both patients were able to polymerase such as Taq-polymerase, an enzyme leave the hospital and resume normal lives that is originally isolated from the bacterium (Yang, 2007). Thermus aquaticus. Nanoparticles as a gene Gene therapy has the potential to vector have the ability to carry and transfect eliminate and prevent hereditary diseases such larger loads of DNA and this makes it attractive as cystic fibrosis and is a possible cure for heart in gene delivery. Because of their unique disease, AIDS, Alziehmer’s disease and cancer. physical and chemical properties, nanoparticles This potential will go a long way to ensure can protect DNA molecules against nuclease better health for generations unborn, boost degradation. Sonoporation technique is a non- immune health in individuals with immune viral means of DNA delivery in the gene. This deficiency diseases, enhance agricultural technique employs acoustic cavitation of productivity of farm animals, and in the microbubbles so as to enhance delivery of production of genetically modified animals genes. Electroporation is a highly efficient (GMOs) which will further help in medical and technique for introduction of foreign genes in biomedical research. tissue culture in mammalian cells. The success of the electroporation depends greatly on the Conclusion: Gene therapy is the use of DNA purity of the plasmid solution, especially on its (as a pharmaceutical agent) to treat disease. It salt content. Solutions with high salt is also an experimental medical treatment that concentrations might cause an electrical manipulates a gene or genes within cells in discharge (known as arcing), which often order to produce proteins that change the reduces the viability of the bacteria. Gene function of those cells. In gene therapy, DNA therapy finds applications in genetically that encodes a therapeutic protein is packaged engineered organisms in order to discover the within a “vector” which is used to get the DNA functions of certain genes. This could be the

Animal Research International (2019) 16(2): 3367 – 3392 3388 Ugwu et al. 3368 effect on the phenotype of the organism, where muscle contraction. Biomedical the gene is expressed or what other genes it Engineering Online, 10(102): 1 – 20. interacts with. Gene therapy generally finds AUSTRALIAN SCIENCE and MEDIA CENTRE application in loss of function, gain of function, (2010). DNA transfer prevents tracking and expression. Clinical trials of gene mitochondrial disease in humans. therapy experiments have seen advances in https://www.sciencemediacentre.co.nz/ understanding vector biology and improving 2010/04/15/dna-transfer-prevents-mitoc vector designs. Some problems associated with hondrial-disease-in-humans/ Accessed gene therapy include; short-lived nature of the May 16, 2018. therapy, problem of viral vectors, multigene BECKER, S. M. and KUZNETSOV, A. V. (2007). disorders etc. Gene therapy experiments Local temperature rises influence in vivo suffered a major setback as a result of intense electroporation pore development: a criticisms and skepticisms following the death of numerical stratum corneum lipid phase an 18-year old man as result of massive transition model. Journal of Biomechanical immune reaction arising from the vector Engineering, 129(5): 712 – 717. (Adenovirus, Ad5 vector) used in the clinical BERG, J. M., TYMOCZKO, J. L., STRYER, L. and trial. The outcome of this trial led to a much CLARKE, N. D. (2002). DNA, RNA, and tighter regulation of all trials relating to gene the Flow of Genetic Information: Amino therapy. However there are other reports of Acids Are Encoded by Groups of Three successful clinical trials of gene therapy Bases Starting from a Fixed Point. experiments involving children suffering from a Chapter 5, Biochemistry, 5th Edition, W. severe combined immunodeficiency disorder H. Freeman and Company, New York. (SCID-XI), which had restricted them to life in BRIVANLOU, A. H. and DARNELL, J. E. (2002). an isolated environment. These investigators Signal transduction and the control of used a MoMLV vector to transfer a curative gene expression. Science, 295(5556): gene (γc cytokine receptor subunit) into the 813 – 818. patients’ lymphocytes ex vivo, and after CAPLEN, N. J., ALTON, E. W. F. W., amplification of the cells, returned them to the MIDDLETON, P. G., SMITH, S. N., patients. Both patients were able to leave the STEEL, D. M. and MUKONGE, F. M. hospital and resume normal lives. The success (1995). Liposome-mediated CFTR gene of the SCID-XI trial likely reflects the path that transfer to the nasal epithelium of gene therapy will follow during the next 1 to 2 patients with cystic fibrosis. Nature decades: success, but with some complications. Medicine, 1(1): 39 – 46. These experiences add further credence to the CHURCH, C. C. (2005). Frequency, pulse length, general viewpoint that gene therapy is a field in and the mechanical index. Acoustics its infancy, and despite some pitfalls, it is well Research Letters Online, 6(3): 162 – grounded in fundamental scientific principles 168. with real clinical promise for the future. CHENG, S., FOCKLER, C., BARNES, W. M. and HIGUCHI, R. (1994). Effective REFERENCES amplification of long targets from cloned inserts and human genomic DNA. ALBERTS, B., JOHNSON, A., LEWIS, J., RAFF, Proceedings of the National Academy of M., ROBERTS, K. and WALTER, P. Sciences, 91(12): 5695 – 5699. (2002). Molecular Biology of the Cell. 4th CHIEN, A., EDGAR, D. B. and TRELA, J. M. Edition, Garland Science, New York. (1976). Deoxyribonucleic acid polymerase ARENA, C., SANO, M., ROSSMEISL, J., from the extreme thermophile Thermus CALDWELL, J., GARCIA, P., RYLANDER, aquaticus. Journal of Bacteriology, M. and DAVALOS, R. (2011). High- 127(3): 1550 – 1557. frequency irreversible electroporation COGHLAN, A. (1999). Look, no new genes: A (h-fire) for non-thermal ablation without subtle technique tricks plant cells in

Animal Research International (2019) 16(2): 3367 – 3392 3389 Gene therapy, physiological applications, problems and prospects 3369

doing their own genetic engineering. Medicine and Biology Society, Boston, New Scientist, 163(2197): 4. Massachusetts. COLE-STRAUSS, A., GAMPER, H., HOLLOMAN, GARCIA, P., ROSSMEISL, J., NEAL, R., ELLIS, T., W. K., MUNOZ, M., CHENG N. and OLSON, J., HENAO-GUERRERO, N., KMIEC, E. B. (1999). Targeted gene ROBERTSON, J. and DAVALOS, R. repair directed by the chimeric (2010b). Intracranial non-thermal RNA/DNA oligonucleotide in a irreversible electroporation in vivo mammalian cell-free extract. Nucleic analysis. The Journal of Membrane Acids Research, 27(5): 1323 – 1330. Biology, 236(1): 127 – 136. COLE-STRAUSS, A., YOON, K., XIANG, Y., GRIFFITHS, J. H., MILLER, J. H., SUZUKI, D. T., BYRNE, B. C., RICE, M. C., GRYN, J., LEWONTIN, R. C. and GELBART, W. M. HOLLOMAN, W. K. and KMIEC, E. B. (2000). An Introduction to Genetic (1996). Correction of the mutation Analysis. 7th Edition, W. H. Freeman, responsible for sickle cell anemia by an New York. RNA-DNA oligonucleotide. Science, GURA, T. (1999). Repairing the genome's 273(5280): 1386 – 1389. spelling mistakes. Science, 285(5426): CUI, J., CUI, H., WANG, Y., SUN, C., LI, K., 316 – 318. REN, H. and DU, W. (2012). Application GURMUNSINGH (2018). Gene Therapy: A of PEI-modified magnetic nanoparticles Medical Revolution. http://gurmunsingh. as gene transfer vector for the genetic wixsite.com/genetherapy/about Accessed modification of animals. Advances in May 16, 2018. Materials Science and Engineering, 2012: HERSHEY, A. D. and CHASE, M. (1952). doi:10.1155/2012/764521 Independent functions of viral protein DIJKMANS, P. A., JUFFERMANS, L. J. M., and nucleic acid in growth of MUSTERS, R. J. P., VAN WAMEL, A., TEN bacteriophage. Journal of General CATE, F. J., VAN GILST, W., VISSER, C. Physiology, 36(1): 39 – 56. A., DE JONG, N. and KAMP, O. (2004). HINGRA, D. (2018). Gene transfer techniques. Microbubbles and ultrasound: from PhD Seminar Presentation. Department diagnosis to therapy. European Journal of Plant Breeding and Genetics, of Echocardiography, 5(4): 245 – 246. Maharana Pratap University of FELGNER, P. L. and RINGOLD, G. M. (1989). Agriculture and Technology, Udaipur. Cationic liposome-mediated transfection. https://www.slideshare.net/devhingra/g Nature, 337(6205): 387 – 388. ene-transfer-techniques-60176818/16 GAN, C. (1989). Gene gun accelerates DNA- Accessed May 16, 2018 coated particles to transform intact IMES, D. L., GEARY, L. A., GRAHN, R. A. and cells. The Scientist, 3(18): 25. LYONS, L. A. (2006). Albinism in the GARCIA, P., NEAL, R., ROSSMEISL, J. and domestic cat (Felis catus) is associated DAVALOS, R. (2010). Non-thermal with a tyrosinase (TYR) mutation. irreversible electroporation for deep Animal Genetics, 37(2): 175 – 178. intracranial disorders. Paper presented INNIS, M. A., MYAMBO, K. B., GELFAND, D. H. at 32nd Annual International and BROW, M. A. (1988). DNA Conference of the IEEE Engineering in sequencing with Thermus aquaticus Medicine and Biology Society, Buenos DNA polymerase and direct sequencing Aires, Argentina. of polymerase chain reaction-amplified GARCIA, P., ROSSMEISL, J. and DAVALOS, R. DNA. Proceedings of the National (2011). Electrical conductivity changes Academy of Sciences, 85(24): 9436 – during irreversible electroporation 9440. treatment of brain cancer. Paper ISENBARGER, T. A., FINNEY, M., RÍOS- presented at 33nd Annual International VELÁZQUEZ, C., HANDELSMAN, J. and Conference of the IEEE Engineering in RUVKUN, G. (2008). Miniprimer PCR, a

Animal Research International (2019) 16(2): 3367 – 3392 3390 Ugwu et al. 3368

new lens for viewing the microbial KUMARI, A., YADAV, S. K. and YADAV, S. C. world. Applied and Environmental (2010). Biodegradable polymeric Microbiology, 74(3): 840 – 849. nanoparticles based drug delivery JAENISCH, R. and BIRD, A. (2003). Epigenetic systems. Colloids and Surfaces B: regulation of gene expression: how the Biointerfaces, 75(1): 1 – 18. genome integrates intrinsic and LAWYER, F. C., STOFFEL, S., SAIKI, R. K., environmental signals. Nature Genetics, CHANG, S. Y., LANDRE, P. A., ABRAMSON, 33: 245 – 254. R. D. and GELFAND, D. H. (1993). High- JAROSLAV, P., MARKUS, G. E., ADAM, G., level expression, purification, and ALEXANDRA, F., SORIN, A., MASUMI, enzymatic characterization of full-length S., CHOUKRI, M., PIERRE, H. R. and Thermus aquaticus DNA polymerase SIGRID, N. (2002). Optimization of and a truncated form deficient in 5'to nonviral transfection: variables 3'exonuclease activity. Genome influencing liposome-mediated gene Research, 2(4): 275 – 287. transfer in proliferating versus quiescent LEHRMAN, S. (1999). Virus treatment questioned cells in culture and in vivo using a after gene therapy death. Nature, porcine restenosis model. Journal of 401(6753): 517–518. Molecular Medicine, 80(11): 724 – 736. LODISH, H., BERK, A., ZIPURSKY, L. S., KAMAU, S. W., HASSA, P. O., STEITZ, B., MATSUDAIRA, P., BALTIMORE, D. and ALKE, P., HEINRICH, H., MARGARETHE, DARNELL, J. (2000). Molecular Cell th H. A., BRIGITTE, R. and MICHAEL, O. Biology. 4 Edition, Scientific American H. (2006). Enhancement of the Books, New York. efficiency of non-viral gene delivery by MAYEUX, R. (2005). Mapping the new frontier: application of pulsed magnetic field complex genetic disorders. The Journal Nucleic Acids Research, 34(5): 40 – 45. of Clinical Investigation, 115(6): 1404 – KANEDA, Y. (2003). New vector innovation for 1407. drug delivery: development of fusigenic MEHRDAD, H., AMIR, A. and PEDRAM, R. non-viral particles. Current Drug (2008). Hydrogel nanoparticles in drug Targets, 4(8): 599 – 602. delivery. Advanced Drug Delivery KATO, N., NEMOTO, K., NAKANISHI, K., Reviews, 60(15): 1638 – 1649. MORISHITA, R., KANEDA, Y., UENOYAMA, MIKLAVCIC, D., MIR, L. and VERNIER, P. (2010). M., IKEDA, T. and FUJIKAWA, K. (2005). Electroporation-based technologies and Non-viral HVJ (hemagglutinating virus treatments. Journal of Membrane of Japan) liposome-mediated retrograde Biology, 236(1): 1 – 2. gene transfer of human hepatocyte MULLIS, K. (1990). The unusual origin of the growth factor into rat nervous system polymerase chain reaction. Scientific promotes functional and histological American, 262(4): 64 – 65. recovery of the crushed nerve. NABEL, E. G., GORDON, D., YANG, Z. Y., WU, B. Neuroscience Research, 52(4): 299 – Y., GAO, X., HUANG, L, XU, L., SAN, H., 310. PLANTZ, G. E. and NABEL, G. J. (1992). KHAN, Z., POETTER, K. and PARK, D. J. (2008). Gene transfer in vivo with DNA- Enhanced solid phase PCR: mechanisms liposome complexes: lack of to increase priming by solid support autoimmunity and gonadal localization. primers. Analytical Biochemistry, Human Gene Therapy, 3(6): 649 – 656. 375(2): 391 – 393. NEUMANN, E., SCHAEFER-RIDDER, M., WANG, KIM, Y. S., RHIM, H., CHOI, M. J., LIM, H. K. Y. and HOFSCHNEIDER, P. H. (1982). and CHOI, D. (2008). High-intensity Gene transfer into mouse lyoma cells by focused ultrasound therapy: an overview electroporation in high electric fields. for radiologists. Korean Journal of European Molecular Biology Organization Radiology, 9(4): 291 – 302. Journal, 1(7):841 – 845.

Animal Research International (2019) 16(2): 3367 – 3392 Gene therapy, physiological applications, problems and prospects 33693391

NEWTON, C. R., GRAHAM, A., HEPTINSTALL, L. Expert Opinion on Biological Therapy, E., POWELL, S. J., SUMMERS, C., 3(5): 745 – 758. KALSHEKER, N., SMITH, J. C. and PLANK, C., SCHILLINGER, U., SCHERER, F., MARKHAM, A. F. (1989). Analysis of any BERGEMANN, C., ANTON, M., LAUSIER, point mutation in DNA. The J., KROTZ, F., REMY, J. S. and amplification refractory mutation system ROSENECKER, J. (2003b). The (ARMS). Nucleic Acids Research, 17(7): magnetofection method: using magnetic 2503 – 2516. force to enhance gene delivery. ONGSIMEI (2016). Types of Gene Therapy. Biological Chemistry, 384(5): 737 – 747. http://fbme.utm.my/ongsimei/ Accessed ROBRISH, D. (1999). New technique permits May 16, 2018. faster, more accurate gene tailoring. PARK, D. J. (2004). 3'RACE LaNe: a simple and CNEWS Science. Accessed July 2012. Biotechniques, 36(4): 586 – 588. RYCHLIK, W., SPENCER, W. J. and RHOADS, R. PATHAK, A., PATNAIK, S. and GUPTA, K. C. E. (1990). Optimization of the annealing (2009). Recent trends in non-viral vector- temperature for DNA amplification in mediated gene delivery. Biotechnology vitro. Nucleic Acids Research, 18(21): Journal, 4(11): 1559 – 1572. 6409 – 6412. PAVLOV, A. R., PAVLOVA, N. V., KOZYAVKIN, S. SAIKI, R., GELFAND, D., STOFFEL, S., SCHARF, A. and SLESAREV, A. I. (2004). Recent S., HIGUCHI, R. HORN, G., MULLIS, K. developments in the optimization of and ERLICH, H. (1988). Primer-directed thermostable DNA polymerases for enzymatic amplification of DNA with a efficient applications. Trends in thermostable DNA polymerase. Science, Biotechnology, 22(5): 253 – 260. 239(4839): 487 – 491. PAVLOV, A. R., PAVLOVA, N. V., KOZYAVKIN, S. SAIKI, R., SCHARF, S., FALOONA, F., MULLIS, A. and SLESAREV, A. I. (2006). K., HORN, G., ERLICH, H. and Thermostable DNA polymerases for a ARNHEIM, N. (1985). Enzymatic wide spectrum of applications: amplification of beta-globin genomic comparison of a robust hybrid Topo Taq sequences and restriction site analysis to other enzymes. Pages 241 – 257. In: for diagnosis of sickle cell anemia. KIELECZAWA, J. (Ed.). DNA Sequencing Science, 230(4732): 1350 – 1354. II: Optimizing Preparation and Cleanup. SAKAGUCHI, N., KOJIMA, C., HARADA, A., Jones and Bartlett Learning, Burlington, KOIWAI, K., SHIMIZU, K., EMI, N. and Massachusetts, United States. KONO, K. (2008). Generation of highly PIERCE, K. E. and WANGH, L. J. (2007). Linear- potent nonviral gene vectors by after-the-exponential polymerase chain complexation of lipoplexes and reaction and allied technologies Real- transferrin-bearing fusogenic polymer- time detection strategies for rapid, modified liposomes in aqueous glucose reliable diagnosis from single cells. solution. Biomaterials, 29(9): 1262 – Pages 65 – 85. In: THORNHILL, A. 1272. (Ed.). Methods in Molecular Medicine, SAILS, A. D. (2004). Applications of Real-Time Single Cell Diagnostics, Methods and PCR in Clinical Microbiology. Real-time Protocols. Humana Press Incorporated, PCR: an Essential Guide. Horizon Totowa, New Jersey, USA. Bioscience, Norfolk, Caister Academic PLANK, C., ANTON, M., RUDOLPH, C., Press, United Kingdom. ROSENECKER, J. and KRÖTZ, F. SANGER, F., NICKLEN, S. and COULSON, A. R. (2003a). Enhancing and targeting (1977). DNA sequencing with chain- nucleic acid delivery by magnetic force. terminating inhibitors. Proceedings of the National Academy of Sciences of the

Animal Research International (2019) 16(2): 3367 – 3392 Ugwu et al. 33683392

United States of America, 74(12): 5463 Interventional Radiology, 2(5): 611 – – 5467. 621. SARKAR, G., KAPELNER, S. and SOMMER, S. VEISEH, O., KIEVIT, F. M., GUNN, J. W., (1990). Formamide can dramatically RATNER, B. D. and ZHANG, M. (2009). improve the specificity of PCR. Nucleic A ligand-mediated nanovector for Acids Research, 18(24): 7465 – 7470. targeted gene delivery and transfection SCHERER, F., ANTON, M., SCHILLINGER, U., in cancer cells. Biomaterials, 30(4): 649 HENKE, J., BERGEMANN, C., KRUGER, – 657. A, GAUBACHER, B. and PLANK, C. VIJAYANATHAN, V., THOMAS, T. and THOMAS, (2002). Magnetofection: enhancing and T. J. (2002). DNA nanoparticles and targeting gene delivery by magnetic development of DNA delivery vehicles force in vitro and in vivo. Gene Therapy, for gene therapy. Biochemistry, 41(48): 9(2): 102 – 109. 14085 – 14094. SMAGLIK, P. (2006). Chimeraplasty potential: as WOLF, Y. I., ROGOZIN, I. B., GRISHIN, N. V. research advances, hopes rise, but and KOONIN, E. V. (2002). Genome efficiency and safety are still concerns. trees and the tree of life. Trends in Crigler-Najjar Syndrome, International Genetics, 18(9): 472 – 479. Crigler-Najjar Meeting, Bertinoro (Forlì- YANG, S. (2007). New medical technique Cesena, Italy), 23 – 25 April, 2006. punches holes in cells, could treat http://criglernajjar.altervista.org/fromth tumors. http://www.berkeley.edu/news esc.htm Accessed May 16, 2018. /media/releases/2007/02/12_IRE.shtml. SONG, Y., GARAVENTA, G., BOTTERO, S., Accessed July 23, 2012. LEPRINI, A. E., PALLESTRINI, E., YANG, Y. S., SUN, S. J., LIU, C. H., TSUANG, Y. CASTELLO, E., PALLESTRINI, E. A. and H., CHEN, L. T., HONG, C. Y., YANG, H. HUANG, W. E. (2007). Ultrasound- C. and HORNG, H. E. (2008). Ex vivo mediated DNA transfer for bacteria. magnetofection with magnetic Nucleic Acids Research, 35(19): 1073 – nanoparticles: a novel platform for 1080. nonviral tissue engineering. Artificial STEPHENSON, J. (1999). New method to repair Organs, 32(3): 195 – 204. faulty genes stirs interest in YAO, Q., CONG, L., CHANG, J. L., LI, K. X., chimeraplasty technique. Journal of YANG, G. X. and HE, G. Y. (2006). Low American Medical Association, 281(2): copy number gene transfer and stable 119 – 121. expression in a commercial wheat SUGAR, I. P. and NEUMANN, E. (1984). cultivar via particle bombardment. Stochastic model for electric field-induced Journal of Experimental Botany, 57(14): membrane pores electroporation. 3737 – 3746. Biophysical Chemistry, 19(3): 211 – YOON, K., COLE-STRAUSS, A. and KMIEC, E. B. 225. (1996). Targeted gene correction of THOMSON, K., CHEUNG, W., ELLIS, S., episomal DNA in mammalian cells FEDERMAN, D., KAVNOUDIAS, H., mediated by chimeric RNA-DNA LOADER-OLIVER, D., ROBERTS, S. and oligonucleotide. Proceedings of the EVANS, P. (2011). Investigation of the National Academy of Science, 93(5): safety of irreversible electroporation in 2071 – 2076. humans. Journal of Vascular and

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