In the middle of the 20th century and withthe discovery of DNA by Watson and Crick, this discovery led to a revolution inthe world of biology and medicine because of the great importance of thiscompound. DNA is a molecule that carry genetic information found in all livingorganisms and viruses, have important role in growth, development, reproductionand regulate of many biological processes. DNA in living organisms composedfrom two strands, each one made up of four chemical bases: Adenine, Thymine,Cytosine and Guanine, and each one of the strand is complementary to the otherby base pairing which show DNA as double helix. A mutation is any change that occurs in DNAsequence that happen during DNA replication or as result of exposure tochemical and physical factors ,this change might happen in somatic or germcells during replication and it’s related with many hereditary diseases ( e.g.
sickle cell anemia) or non-hereditary diseases (e.g. cancer). The term of gene therapy emerged during the1960s .In 1972, Theodore Friedmann and Richard Roblin published a paper inScience called “Gene therapy for human genetic disease?” which citedStanfield Roger’s proposal in 1970 that “good DNA” could be used toreplace defective DNA in people with genetic disorders. A 4-year-old girl wasthe first to be treated for congenital disease called adenosine deaminase (ADA)deficiency using a gene therapy technique. Gene therapy is a process to repair thischange in the DNA sequence in an attempt to restore the normal function of themutant genes, by inserting the normal gene into the cells or disrupting thegene in which the mutation occurred to overcome these diseases, by viral ornon-viral vectors.
This article will focus on viral vectors due to theirefficiency advantages in gene delivery. What is The GeneTherapy? Gene therapy is defined by insertionnew DNA into a patients with hereditary or non-hereditary diseases to treatthese disease. This is done by viral or non-viral vectors in order to replacingmutated genes, inactivation mutated genes or insert new DNA fragment usuallycontains a functioning gene to correct the effects of a disease that resultfrom mutation in genes1. Viruses could be used as vectors to transfer’good’ genes into a human cells in vitro and in vivo. First, it must removeviral genes which cause the diseases.
Then it should replace those genes withgenes encoding the desired effect (e.g. insulin production in the case ofdiabetics), the insertion of foreign DNA into viral vectors done by manytechniques, one of them done by using restriction enzymes to cut the viralgenome at specific sites and insert the foreign DNA into viral genome andligate it by using DNA ligase which produce recombinant DNA (viral genes andinsertion genes). This procedure must be done in such a way that the geneswhich allow the virus to insert its genome into its host’s genome 1. Target cells like the patient’s nerve or germcells are infected with the vectors. The vectors then transfer its genomecontaining the normal gene into the target cells. The production of afunctional protein product from the normal gene restores the target cell to anormal state 1. There are two types of gene therapy accordingto target cells treatment:1.
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Somatic genetherapy: transferDNA fragments into all cells body except germ cells which produce eggs andsperms and it is effect confined to the patient and didn’t pass to children, manygenes are transferred to somatic cells including hematopoietic cells, livercells, cancer cells and muscle cells. 2. Germline genetherapy: transferDNA fragments into germ cell and it is effect pass to children patients, thistype of gene therapy gives great hope to treat some rare genetic diseases .There are noclinical trials done in this area, because they are related to ethical issues.
ViralVectors A viruses is an infectious particlesthat don’t reproduces only within host body, contain genetic material (DNA orRNA genome) inside a protein shell called a capsid, genetic material can besingle or double strands. Some viruses have an internal or external membraneenvelope. Viruses don’t have organelles and that means they can’t make ATP orenergy for themselves 1. Viral replication starts when the virusesenter to host and attach to receptors integrated in host cell membrane, thenviruses inject genetic material into host cell.
When viruses enter into hostcells, it must transfer its genetic material into the nucleus of the host. Thevirus can enter to the nucleus directly, or inject its genetic material intothe nuclear envelop without entrance into the nucleus. The virus then uses theown cell materials (enzymes) and organelles to produce capsid proteins andreplicate the viral genome.
These parts then self-assemble into new virusparticles, which can exit the cell and infect healthier host cells 1. There are types of viruses that classifiedaccording to genetic material, size, shape or mode of replication. Many ofviruses’ type used as vectors in gene therapy but There are four types ofviruses used mainly as vectors in gene therapy – Retroviruses, Adenoviruses,Herpes-simplex virus and Adeno-associated virus 2. 1. Retroviruses Group of viruses that belong to Retroviridaefamily and contained single strand RNA genome. Retroviruses have unique enzyme,called reverse transcriptase that help them to transcript their RNA into DNAafter invade host cells, then retroviralDNA can insert and integrate into the chromosomal DNA of the host cell, to beexpressed there. One of the most studied species ofretroviruses is Lentiviruses and widely used in gene delivers, humanimmunodeficiency virus (HIV) which cause AIDS is most important example oflentiviruses.
Retroviruses have many characteristics madethem good vectors in order to gene delivery, some of these characteristics arethe efficient way of these viruses to enter into host cells and its ability tointegration into the host chromosomal DNA without expression of any immunogenicviral particles. The disadvantages of these viruses include that these vectorrequire cell division for stable infection and the limited capacity for thesevectors which prevents the delivery of large DNA fragments 2. 2. AdenovirusesAnothergroup of viruses that used in gene therapy, these viruses belong toAdenoviridae family, nonenveloped viruses and contained double stranded DNAgenome. The adenoviruses genome encodes nearly 35 proteins that are expressedin two phases: early phase and late phase.
The early phase happens before thestart of viral DNA replication it is take about 7 hours post-infection, and thelate phase which follow the DNA replication. About ~20 early proteins haveregulatory functions that allow the virus to take control of the cell and tocarry out viral DNA replication. The late proteins have structural role in thevirus. Adenoviruses responsible about many of respiratory infections. The advantages of adenoviruses as vectors arethat they can infect large numbers of cell types, including nondividing cells,and the genome of the adenoviruses does not integrate into the host cellschromosomes. The ds DNA of adenoviruses keep free inside nucleus of infectedcells. The presence of viral genes is necessary to replication and spreading ofthese viruses and immune system stimulation are Classified as disadvantages ofadenoviruses 2. 3.
Herpes-simplexviruses Herpes-simplex viruses or also known as humanherpesviruses, herpesvirus 1 and 2 (HHV-1 and HHV-2), are two members of the herpesvirusfamily, Herpesviridae, belong to the subfamily of Alphaherpesvirinae. Consistof double stranded DNA genome, contain around 100-200 genes that involved incapsid and lipid bilayer envelop membrane formation and included in infectivityand genome replication of these viruses. Previous studies indicate that thereare correlation between HSV-1 and Alzheimer’s disease. Herpesviruses are currently used in genedelivery due to some special features over other viral vectors. Some of thesefeatures are the high capacity of these vectors to transfer long sequence ofnew DNA into cells and the ability to targets nerve cells. Some ofdisadvantages of herpesviruses are that the large number of their genes whichis required to replication and spreading inside the host and difficult toproduce 2. 4. Adeno-associatedviruses Adeno-associated virus belong toparvovirus family, small viruses contain single-stranded DNA genome.
Adeno-associated viruses don’t associated in causes of diseases, and need toanother types of viruses like Herpes-simplex viruses or adenoviruses to act ashelper viruses, help them to replicate. Adeno-associated virus can integrates atspecific site on chromosomes, these viruses can insert foreign DNA at aspecific site on chromosome 19 with near 100% certainty. In addition, theseviruses don’t not produce immune response. These characteristics made them goodvector for gene delivery.
Conversely, There are a few disadvantages to usingadeno-associated virus as vectors, including the small amount of geneticmaterial it can transfer (low capacity) and the difficult to produce 2. Some GeneTherapy examples Many achievements have beenaccomplished in the field of gene therapy despite many challenges, studies arestill ongoing and the results are promising, many diseases have been treated. 1. Adenosinedeaminase deficiency Adenosine deaminase (ADA) deficiency isan inherited diseases that cause problems in the immune system and lead tocause severe combined immunodeficiency (SCID), Adenosine deaminase responsiblefor convert some toxic compound (deoxyadenosine) that effect immune system intonontoxic compound (deoxyinosine), so any mutation in ADA gene lead into hightoxic level in the body.
SCID that result from ADA deficiency was treated byusing gene therapy, retroviruses vectors carry the intact gene of ADA gene weretransfer into autologous hematopoietic stem cells, these stem cellsreturned into 10 patients afternonmyeloablative chemotherapy these for patients. The sustained expression ofADA gene in multiple hematopoietic-cell lineages allowed for the detoxificationof purine metabolites and improvement in the patients’ physical development3. 2. Cysticfibrosis Cystic Fibrosis is an inherited diseasecaused by mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that passed intochildren, the effect of cystic fibrosis mainly on respiratory system. Viral andnon-viral vectors are used in the treatment of cystic fibrosis with differenttechniques.
Adeno-associated viruses which is small viruses to carry CFTR gene,so researchers have attempted to create a functional CFTR “mini gene”, usingtechniques such as cutting the CFTR in half and using two complementary AAVs.Also, Sendai viruses are used as vectors carry CFTR gene to treat cysticfibrosis because its ability to infect airway epithelial cells due to thepresence of sialic acid and cholesterol receptors on their apical surface.These recombinant SeV vector carrying the CFTR gene, CFTR chloride channelactivity was confirmed in vitro and in vivo 4. 3. Hemophilia Hemophilia is bleeding disease that affectblood clotting in patients, two major types of hemophilia: hemophilia A (factorVIII deficiency) and hemophilia B (factor IX deficiency).
Adenovirus-associatedvirus vectors carry human factor IX in a peripheral vein in six patients withhemophilia B showed improve in the level of bleeding phenotype without anytoxicity in the patient’s bodies 5. 4. Duchennemuscular dystrophy Duchenne muscular dystrophy (DMD) is aninherited disease one of nine type muscular dystrophy result by miss functionof DMD gene which responsible for dystrophin production, any mutation in DMDgene lead to muscle weakness. Recombinant adeno-associated virus (rAAV) vectorscarry intact copy of DMD gene transferred to skeletal muscle showed decrease inthe muscular dystrophy symptoms 6. 5. CancerStudiesare ongoing in the field of gene therapy on cancer, there are great hopes foreffective treatment of various types of cancer, some studies have shownpositive results, understanding how cancer is produced is very helpful inresearch.
Tumor necrosis factor alpha (TNF?) is type of cytokines haveanti-cancer properties, sarcoma patients are candidates for the TNF treatment.The TNFerade construct represents such an approach. TNFerade was constructed asa second generation adenovector, expressing the human TNF? cDNA. To increasethe effective of this treatment and decrease the toxicity, the researchers usedradiation-inducible immediate response Egr-1 (early growth response) genepromoter which ligated upstream to the transcriptional start site of the humanTNF cDNA.
TNFerade vectors give maximum expression of TNF gene. The activity of TNFerade in combination withradiation has been evaluated in a number of different human models, includinghuman prostate cancer, human malignant glioma, radio resistant human laryngealcarcinoma, and human esophageal adenocarcinoma. This approach of gene therapyshowed acceptable range of the toxicity and the tumor response ranged fromcomplete, partial and simple response by effect this approach affect theapoptosis and cellular necrosis 7.
6. Choroidermia Choroideremia (CHM) is a hereditarydisease that cause progressive vision loss due to CHM genes mutation which iscoding gene for the Rab escort protein 1 (REP1). Adeno-associated viruses carrynormal copy of REP1 gene transferred into the retina of six Choroideremiapatients. Patients were tested showimproved in vision and gradually regained the ability to see 8. Gene Therapy ChallengesThere aremany challenges to gene therapy, which are related to transport, toxicity, andefficacy and gene delivery.
The most important challenge in gene therapy is thelack of a 100% effective way to ensure that the genes reach the nuclei of thetargeted cells to be expressed. This risk is concentrated in that these viralvectors are transferred to abnormal cells and healthy cells alike, and this maylead to problems that may occur in healthy cells 9. Another challenge is that there is also noways ensure to transfer the new genes to target sites in target cellschromosomes and this may lead to other more complex diseases, such as cancer.This risk was observed in clinical trials where hematopoietic stem cells weretransferred to X-linked severe combined immunodeficiency (X-SCID) patients byretroviruses containing intact genes, some of these patients have appeared Tcell leukemia.
There are other concerns that these viral vectors can reach thecells responsible for producing the gametes and cause changes in the DNA theycontain, and the possibility of transferring these changes to the children 9. Other danger of gene therapy based on viralvectors is that the inability to control the amount of the transferred geneexpression, it might be overexpressed, producing large amount of new proteinand this could be harmful, or it might be downexpressed, so the purpose is notto occur. Because viruses stimulate the immune system,viral vectors may encounter a large defense of immune cells in the body.Changes in the genetic material of these viruses may lead to the weakening oftheir resistance and thus to their destruction before the target is achieved. Another challenge is that these viruses maytransported from patients who are undergo to gene therapy into healthy personsor into the environment.
However, gene therapy techniques arecurrently subject to many studies in different fields, and results show manyhopes for patients around the world to overcome these challenges. Experimentsare initially conducted on animals to better understand and overcome theserisks, before any application occurs in clinical trials on humans. Summary Gene therapyis a process to repair any change in the DNA sequence that happen duringreplication or Exposure to physical or chemical factors in an attempt torestore the normal function of the mutant genes, this is done by viral ornon-viral vectors. About 80% of the studies were conducted on viral vectors dueto their efficiency in the gene delivery. Many types of viruses are used ingene therapy, each type has advantages that make it a good vectors and at thesame time have disadvantages that make it impossible to use it in allexperiments. Experiments are still ongoing in the field of gene therapy, someof which have been effective in treating some diseases, and other studies arestill ongoing on some diseases, notably cancer. There are also challenges togene therapy, one of the most prominent of these challenges is the lack of aneffective way to ensure that DNA is transferred to the target location, Currentstudies are trying to find appropriate solutions to these challenges.
