Magoosh GRE

Gene Therapy and Monogenic Disorders

| March 18, 2015

1. Introduction

The Human Genome Project has greatly facilitated research into the genetic understanding of monogenic inherited diseases. Gene therapy has been used as an impressive and innovative tool over the past few decades with an explosion of clinical trials taking place worldwide. This paper aims to discuss monogenic disorders, highlighting several in particular to display the vast differences as well as the common lack of available treatment. Gene therapy shall be investigated thoroughly, its uses, implications and how as a tool it can improve the treatment of monogenic diseases, throughout critical analysis shall be made for surrounding issues. This paper hopes to have touched upon a few key issues discussing some of the successes to- date and the directions gene therapy is heading towards as we enter a new decade.

2. Monogenetic Disorders

A plethora of human diseases have been identified as having a genetic origin and whilst some of these arise as result of mutations occurring at multiple locations, a substantial proportion manifest as result of a single gene mutation, synonymous with the common term of “monogenic disorders” [1].

Monogenic disorders have been sub classified further, each corresponding to their patterns of inheritance. Firstly, autosomal dominant, of which includes some of the following; familial combined hyperlipidaemia, familial hypercholesterolaemia, dominant otosclerosis, adult polycystic kidney disease, multiple exostoses, Marfan syndrome, Huntington’s disease, neurofibromatosis, myotonic dystrophy, congenital spherocytosis and polposis coli. The second group encompasses autosomal recessive disorders some of which are; cystic fibrosis, alpha-1-antitrypsin deficiency, phenyketonuria, congenital adrenal hyperplasia, spinal muscular atrophy, Roberts syndrome, Niemann-Pick syndrome, cell anaemia, adenosine deaminase severe combined immune deficiency, and beta-Thalassaemia. The final group consists of sex-linked disorders (X-linked recessive), which include following; fragile X syndrome, Becker muscular dystrophy, Duchenne muscular dystrophy, X-linked ichithyosis, X-linked severe combined immune deficiency syndrome, Lesch-Nyhan syndrome, Haemophilia A, and Haemophilia B [2].

An online database known as the “Online Mendelian Inheritance in Man” (OMIM) created in the mid 1980’s and at present in 2010 exposes over 2500 disease phenotypes, which have been connected to specific genes [3]. Contrary to the rare nature of monogenic diseases a significant number of the population are affected, with the World Health Organisation (WHO) reporting approximately 10 in every 1000 births to be affected [4].

This paper shall focus and elaborate further on three monogenic disorders, an autosomal dominant (Huntington’s Disease), autosomal recessive (Adenosine deaminase deficiency of the Severe Combined Immunodeficiency’s) and finally one from the third group, the X-linked diseases (Fragile X- syndrome).
Huntington’s disease.

Huntington’s disease (HD) first described by George Huntington in 1872 after which it was named. Since its discovery knowledge surrounding it has evolved with the aide of clinicians, scientists, patients and family members [5][6]. Huntington’s disease is n autosomal dominant disorder classified as a progressive neurodenegeneraive disorder, which results in the substantial loss and functioning of neuronal tissue [7][8].

Huntingtin is a protein expressed in all human and mammalian cells, highly concentrated in neural tissue and the testes with small amounts present in the heart, liver and lungs. Numerous theories on the mechanisms by which HD causes ill health have been put forth, including haploinsufficiency, the mutant HD gene causing a toxic gain of function amongst others. However, the underlying pathogeneses of HD is yet to be fully elucidated [6]. Purine models have indicated an age-dependent expansion, which as yet hasn’t been exploited in humans but may be an area for future research. The use of transgenic mice has been invaluable as a tool in gene therapy, with the earliest mouse models being used in the 1970’s. Post mortem brain tissue from patients who had suffered Huntingtons disease isn’t ideal for research and animal models, including mice, Drosophil.a spp and Caenorhabditis. elegans are championed as they provide histopathological and biological studies reflecting the early stages of disease, and the crucial intercellular interactions otherwise unattainable from human sources [7].

Symptoms for Huntingtons disease can appear anywhere from between the ages of 1 to 80 years of age, before which individuals lead a healthy and unsuspecting life with clinical abnormalities escaping detection [6]. Huntington’s disease is amongst one of the eight autosomal dominant inherited and progressive neurodegenerative diseases [7]. The prediagnostic phase is an important signal, where individuals become increasing irritated, forgetful and disinherited which are amongst the signs. The phenotype expresses itself typically during the middle age with cognitive decline, in coordination, chorea, dystonia and behaviour difficulties being the prominent signs. Eventually merging with the diagnostic stage, the differential diagnosis for HD includes imaging techniques such as magnetic resonance imaging (MRI) and computerised tomography (CT) scans and positive emission tomography (PET) which reveal losses in neural tissue and increased frontal horns of the lateral ventricles [6].

Genetic testing of these individuals would also reveal the gene responsible for Huntingtons disease was first identified in 1993 and is located on the short arm of chromosome four at loci 4p16.13 and is linked to an expansion of the trinucleotide repeat sequence CAG (cytosine (C) –adenosine (A) –guanine (G)). The normal range falls between 28-35 CAG repeats, whilst with an individual with full penetrance this disease would on average display 41 or more and incomplete penetrance would reach between 36-40 repeats [6][8].

The CAG triplet is a code for the amino acid glutamine, when this CAG then increases in repetition; the expanding polyglutamine tract generates a HD gene product, the aforementioned “Huntingtin” protein, the accumulation of which results in death of nervous tissue, adversely effects the control of ones movement, memory and behaviour [7]

At present there is no cure for treating HD, regardless of over two decades researching this field, medical treatment concerning this involved management through genetic counselling, help trough support groups and drugs to treat aspects of the condition, antichoretic drugs such as tetrabenezine or neuroleptics for example may help to alleviate constant involuntary movements. Aside from these, there is no other treatment to combat this progressive neurodegenerative disorder [6][7].

Adenosine deaminase deficiency of the Severe Combined Immunodeficiency’s (ADA-SCID)

Severe combined immunodeficiency’s emanate from a variety of molecular defects, which have severe implications on lymphocyte development and function, common λ chain, JAK-3 and Interleukin (IL)-3 are lymphocyte-specific signalling molecules which are some of those effected. Molecules such as DNA ligase, Artemis, RAG-1/2 which control the rearrangement of the T-cell receptor and immunoglobulin genes as well as those which control signalling through the pre-T-cell receptor such as CD3δ and CD3ε can also have adverse effects when defected [10].

The enzyme, Adenosine deaminase is expressed in all tissues of the body, part of the purine salvage pathway it catalzes the deamination of the deoxydenosine (dAdo) and adenosine (Ado) to deoxyionosine inosine. The accumulation of the former substrate is principally cytotoxic for T cells, and B cell to a lesser extent, this cytotoxicity equates to an absence of adequate numbers of functioning T- and B- cells resulting to a severe combined immunodeficiency disease (SCID) [11].

Individuals who possess this defect display cognitive and behaviour dysfunction, frequent continued infections, which ultimately often are the cause of death within these patients, fatality usually taking place within a 2-year period [10][11].

The current major treatment for ADA-SCID is HLA-identical allogenic bone marrow transplant (BMT) also known as haematopoietic stem cell transplantation (HSCT), however this is only applicable for 25-30% of patients. The alternative to BMT/HSCT therapy has fatal consequences, as is reflected with high mortality and morbidity rates. Other treatments include enzyme replacement therapy (ERT) and autologous haematopoietic stem cell gene therapy (GT) of which shall be considered more in depth in the following sections [10][11].

Fragile X Syndrome

A site located on the long arm of the X-chromosome at Xq27.3, also known as the “fragile site” is the cause of origin for a genetic disorder known as “Fragile X syndrome” or “fragile site mental retardation” and in the majority of cases comes about as result of the meiotic instability of specific alleles [12]. Although this site is not a protein coding region (exon) it does however result in the loss of gene activity. This monogenetic autosomal recessive disorder reflects itself via a complex array of behavioural and cognitive phenotypes, of which causative pathological mechanisms are still yet to be attained [4]. Fragile X syndrome is the most common cause of inherited mental retardation and is displayed in a variety of clinical phenotypes in both sexes on average effecting 1 in every 1000 males and 1 in every 4-6000 females [4][12]. Similar to all other recessive X-linked traits it is males who predominantly phenotypically express this type of retardation. A repeated 3-pair base sequence of CGG, in the FMR-1 (fragile X mental retardation-1) locate don the fragile X is the cause [13]. The normal range for this repeat sequence falls between 6 to 54, whereas phenotypically normal transmitting males along with their daughter, in addition to carrier females possess a substantially higher number ranging from 55 to 200 copies and are asymptomatic. This triplet repeats amplification as it has come to be known has various outlooks, and those males and females who have fragile X syndrome, their range of this CGG triplet repeat on average falls between 200 to 1,3000 copies and thus considered “full mutations” [13].

Diagnostic tools for Fragile X syndrome typically involve chromosome analysis, as over 99% of individuals possess the full mutant FMR1 gene and numerous protein investigations. Presently there is no treatment available to cure this disease; the benefits of diagnosis early however may elicit interventions improving the quality of life, such as speech therapy, and aiding psychological aspects of the patient and family lives [4].

3. Gene Therapy

The term “gene therapy” is used to describe the introduction of genetic material into specific cells or tissues in order to generate a therapeutic response. One simple example of gene therapy is the deliver of a normal cystic fibrosis transmembrane conductance regulator gene (CFTR) gene using the vector in the form of a liposome carrier (vectors shall be discussed in further detail in later sections), copies of the normal CFTR gene are produced through recombinant DNA technology in vitro and the ionic changed (negatively) copies of DNA will be combined with cationic (positive) liposome’s to generate DNA-liposome complexes. The desired cell through cell surface receptors will then incorporate these complexes and the newly introduced DNA should reach the cell nucleus and begin to express the normal cystic fibrosis transmembrane receptor [14].
Since 1989 when the first human gene therapy trial was carried out over 1000 clinical trials underwent completion, are ongoing, or have been amended for improvements a figure that will continue to increase. This gene therapy research expands beyond 30 countries worldwide, with mainly Western countries such as the United States, United Kingdom and Germany predominating and involves over a hundred genes [15]. The practical uses of this therapy concerns two cell types; stem cells which are accessible and terminally differentiated, postmitotic, long-lived cells [16].

Gene Transfer

The transfer of the genetic material can be as result of two approaches ex vivo that takes place outside of the patient’s body as is seen when haematopoietic stem cells (HSCs) are extracted and purified from patients blood or bone marrow to allow for the gene delivery to be carried out in vitro which leads onto an optional pre-selection for which cells have fully incorporated the genes. For ex vivo gene therapy a guideline has to be adhered to so cells that undergo treatment can be conveniently and readily extracted, if they can survive the environment in culture, undergo various manipulations and be re-induced into the patient. In vivo approach is one where gene delivery must be targeted towards a specific tissue (e.g. nasal tissue in patients suffering form cystic fibrosis) and as these are difficult to obtain for in vitro culturing this alternative in vivo approach must be adopted although is greatly reduces efficacy of the gene transfer and the alleviated option of pre-selection of cells which have fully incorporated the gene modification [17].

The most favourable approach of gene therapy is genetic augmentation, this involves implementing an exogenous copy of the wild-type allele into cells, of which the expression will go onto produce a functional protein used to rescue the cell from the previous mutated disease phenotype. This however is only applicable for diseases transpiring as result of autosomal recessive mutation (s) in monogenic diseases such as cystic fibrosis (CF), Fabry disease and sickle cell anaemia. Unfortunately integrating an extra copy of the wild-type allele into the genome cannot rectify genetic disorders such as Huntington’s disease, which occur as result of a dominant mutation [14].

To address the prospect of correcting an inherited defect via insertion of a normal copy of the gene in question and restoring cells affected to normality for the patient seems ideal in theory, however, many stages need to be carefully considered. Initially the gene in question needs to be identified and cloned, once removed and isolated from the other genetic material its function has to be fully researched and investigated prior to engineering a method of delivery into specific target cells. Once these initial stages have been met, the next step would require all aspects of gene deliver to be considered such as the time specific-delivery, expression of the gene at a certain location/locations within the cell, tightly regulated expression of the newly inserted gene with its surrounding genetic material within the cell. In addition, it has to be carefully synchronised to the cells cycle and to adequately response to crucial intracellular messenger molecules. Further to all of this is must have high efficacy in gene transference so the residual mutated cells do not compromise or limit the effective of the corrected cells. Another consideration that arises is the need to “shut off” the production of any abnormal products from the mutated gene to coincide with the induction of the correct gene product [18].

Gene Therapy- Methods of Delivery

A variety of methods have been engineered to deliver the gene to its desired location and include the use of a carrier known as a “vector”, these are commonly retro-and lentiviruses, adenoviruses, adeno-associated viruses. Integrative vectors are necessary when dividing cells, haematopoietic stem cells (HSCs) are utilised to ensure replication of the transgene; these include ribonucleic acid (RNA) viruses such as retro-and lentiviruses. Non- integrative vectors are used when gene transfer takes place in post-mitotis long-lived cells, of which included the deoxyribonucleic acid (DNA) viruses; adenovirus (Ad) and the most non-pathological viral vehicle available – the adeno-associated virus (AAV) [15][16].

AAV has already shown sustained efficacy in animal models (>3 yrs) for retinal degeneration due to genetic defects of the photoreceptor and retinal pigment epithelial cells. AAV is also very similar in its proliferative nature to that of Ad in both proliferative and non-proliferating cells, one drawback to this vector is reflected in that it has a low capacity when compared to others [8] [16].

The delivery vehicle has a significant contribution to the outcome of the therapy and the success or failure depends on its efficacy, safety and whether it produces a sustained result with minimal toxic side-effects [14].

To-date viral vectors are accountable for approximately 70% of gene therapy research trials; other methods of delivery include non-viral vectors and the use of mammalian artificial chromosomes [14] [15].
Retroviral vectors where the first to be utilised for gene therapy and originate from murine retroviruses, they still remain the most commonly used vector with over 28% of gene therapy trials using them to-date. These vectors are limited in capacity to withhold therapeutic genes, however target dividing (HSCs) with a high degree of effectiveness and permit the stable transfer of genes as they assimilate to the chromosome of the target cells. The second most frequently used vector (26% of all trials) are Adenoviruses. Although Adenoviruses can contain a larger DNA cargo, this is still insufficient to contain the genes for the testing of all the desired clinical applications. Adenoviruses have sourced as the vector for treating cystic fibrosis and proved to be an effective method in transducing cells both in vitro and in vivo and has also proved advantages in that it is a more reliable viral vector than the retrovirus due to the fact genetic manipulation can occur within a controlled environment and kept within a regulatory range . The remaining viruses used include pox virus, vaccine virus, herpes simplex virus and adeno-associated virus [8][15].

Non viral vectors such as liposomes were designed due to the lack of accommodation on viral vectors for the genes, which hence led to the devilment of synthetic vectors, the most basic non viral vector gene delivery method makes use of “naked DNA”, this naked DNA is injected into specific target tissue/s directly, e.g., muscle tissue to generate elevated levels of gene expression, although this is less than those achieved via virus vectors. This non-viral vector approach has become the most popular amongst researchers in clinical trials [14][15].

Candidate diseases for gene therapy: Cancer and cardiovascular

The objective therapeutic goal for any treatment development for cancer is the effective ridding of most (or the majority) of cancer cells whilst still avoiding simultaneous damage to normal healthy cells and tissues. Gene therapy has therapy had originally been a prime target to meet these expectation and although theoretically very feasible the inability of this therapy to reach these abnormally dividing cells was disappointing. Numerous gene therapy clinical trials have reached beyond Phase I, utilising both viral and non-viral vectors and using local intratumoral routes to assess target cells and not systematic circulation. Five methods of approach are in the scene which have been directed for cancer therapy and include; inhibition of tumour angiogenesis, immunotherapy, induction of apoptosis, conditional replication of the oncolytic virus and suicide genes [25]. So far the majority of clinical trials are attributed to the treatment of cancer with approximately 65% of all genes therapy trials. Numerous cancers have been the goal including lung, gynaecological, skin, urological, haematological tumours an paediatric tumours being amongst the objectives [20].

Cardiovascular disorders are relatively novel targets for gene therapy closely following cancer and the monogenic disorders, however are unravelling as one of the most promising targets for this therapeutic intervention. Vessels within the cardiovascular system are one of the most accessible for gene transfer and in the majority of cases only transient expression of the transfected gene in some target cells will be needed to produce therapeutic benefits [25]. Gene therapy has been rapidly adopted as the research as it has a major potential to treating atherosclerosis. Adenoviruses have been the vector predominantly used to mediate gene therapy for angiogenesis atherosclerosis along with coronary artery restenosis, cardiac muscle cells in the myocardium. Gene therapy is a crucial future in treating these diseases, as replacing potentially fatal treatments such as angioplasty’s, heart-bypass surgery and could take over as a long-term solution from patients who have to in take a plethora of drug [19][20].

Research into cardiovascular diseases has increased to 9.1% in 2007 from 8.3% in 2004, and is now the second most applied for gene therapy trial. Angiogenesis is at the forefront of this research to elevate blood circulation to ischemia regions, with the aim to stroke prevention. The use of various animal models including that of the mouse and rabbit, have seen the successful gene transfer of vascular endothelial growth factors (VEGFs) and fibroblast growth factors (FGFs) which have enhanced blood performance and collateral development in these models. Further work in this growing area will see improvement in vector choice, delivery pathways and the identification of new genes to precede full clinical trials exhibiting the full potential of gene therapy in treating cardiovascular diseases [20] [25].

Candidate diseases for gene therapy: Inherited monogenic diseases

Cancer, cardiovascular disease and inherited monogenic diseases have been the main targets for gene therapy trials. The prevalence of cancer and cardiovascular disease, their impact on individuals and fatality concerning them are strong magnets attracting them to this type of genetic research. With regards to inherited monogenic diseases, the idea of replacing a mutant abnormal gene with a fully functioning normal adversary is theoretically very appealing. With an average life expectancy of 40 years of age and being the most common inherited disease in the United States and Europe, Cystic fibrosis (CF) accounts for a third of all inherited monogenetic trials [20].

The SCIDs are the second most frequently investigated group of genetic diseases and is one, which over the years has displayed sustained and clinically relevant therapeutic benefits [19]. Adenosine deaminase (ADA)- SCID is a favourable target for gene therapy and with the use of retroviral vectors several clinical studies have researched the efficacy of ADA gene transfer into HSCs [10]. This ADA-SCID in 1990 was the first approved therapeutic gene trial carried out in humans and was carried out in two children suffering from this immunodeficiency [20].
Three forms of therapy are shown to increase effective immune recover for patients with ADA-SCID, which include haematopoietic stem cell transplantation (HSCT), currently the treatment of choice this is most widely available to clinicians. Secondly enzyme therapy (ET) which involves the weekly or twice weekly injection of Polyethylene-glycol (PEG) ADA, this works to get rid of adenosine (Ado) and deoxyadenosine (dAdo), these are by -products of nucleotide and nucleic acid interactions [9]. The gene for ADA deficiency was first isolated in 1983, leading to an explosion in research into function and regulation. Further to this ADA-SCID was a great initial candidate for gene therapy as its housekeeping nature as a gene, which is constitutively expressed in cells and is in need of minimal regulation of its expression [18]

Initially research in the early 90’s, A landmark clinical trial chose to use T cells as opposed to HSCs, which were transduced in vitro, expanded and re-infused into two young patients who were also being treated with the enzyme therapy PEG-ADA utilising retroviral vectors. The result was very promising and led to a substantial elevation in the circulating T cells along with ADA enzyme activity. Due to the combined use of the PEG-ADA enzyme therapy however, it was unclear as to whether gene therapy could be attributed to this [10].

Numerous studies have shown that when gene therapy is combined with drugs combating myelosuppresion such as low -dose bulsafan and withdrawing enzyme therapy of PEG-ADA complements the gene therapy treatment and results in a more positive clinical outcome and normal T cell function with an improved production of immunoglobulin’s. Unfortunately a Japanese trial reported the opposite, the removal of PEG-ADA treatment with ADA-SCID patients who were treated with a retrovirus with the gene transferred into CD34+ bone marrow cells and absence of myeloablation. Thus conflicting research leads to a lack of clarity regarding what is most constructive [10].

Approximately twenty different monogenic disorders aside from CF and SCID have been treated (refer to Appendix 1), however the results obtained from trials involving these reflect a transient gene expression, with detectable protein in cases and have yet to show favourable therapeutic advances [20].

Therapy at a DNA level has also been on the horizon as a possible treatment for the musculoskeletal disorder, Huntingtons Disease an autosomal dominant inherited monogenic disorder. Glutamine is the amino acid that is coded for by this triple repeat and its expansion leads to accumulated polygluatamine tract and the production of the Huntingtin protein [9][14]. The primary objective of HD treatment is to cease triplet expansion of CAG expression; gene therapy may be used to inhibit the Huntington mutant protein from being expressed prior to any toxic side effects from arising [7]. The earliest model for Huntingtons in animal models were designed and took place in the 1970’s and was based on selective vulnerability to neurons in the striatum to excitotoxic aminoacids [6].

The use of antisense gene therapy although applied through use of oligonucleotides with the aim to reduce CAG expression levels hasn’t been as successful as anticipated, with a 50% reduction in protein levels, which may not be a substantial enough percentage for long term treatment. The early stages of antisense and antigene approaches however continue to look like a good prospect for the future in their promise to prevent mutant expansion of the Huntington allele [7]. Trials were conducted in transgenic mice for HD with two mouse lines that took place within 3-18 week duration. The first mouse was exposed to deoxycycline (Dox) in drinking water, the second without Dox drinking water. Deoxycycline a substance derived from the compound tetracycline, binds to the trans activator on tetracycline and removes the tetracycline-response operator. Results from these trials showed that with the absence of Dox, “Gene on” can lead to a number of tumours and the transgenic mice are predisposed to developing inclusion bodies however, results from those treated with the Dox drinking water “Gene on” actually had a positive effect and improved neuropathology. These trials were significant in that they showed the inhabitation of the mutant expansion of the Huntington allele could reverse HD. One condition to this outcome is that the normal gene expression of HD is maintained [7][8][9].

Gene therapy – the negative aspects concerning monogenic disorders.

Setbacks to gene therapy surfaced in 1999 in the form of a drought in recruiting patients into gene therapy trials. In the U.S a child patient who participating in a clinical for ornithine transcarbamylase (OTC) had an adverse reaction to the retrovirus being used and as result died due to an unexpected inflammatory response [15]. This case in addition to many other incidences of negative effects has devastating implication in recruiting or gaining interest in gene therapy trials. Another case concerning X-linked SCID again using a retroviral-mediated delivery to the bone marrow of the wild type gene led to 3 of the 17 patients taking part in that trial developing leukemia due to its insertion [17].

The major obstacle is however, the delivery of vectors to the target site. There is a general lack of a productive efficient gene delivery system; the majority being virus vectors, which doesn’t bring about toxic effects, one way of addressing this has come in the form of synthetic vectors [17]. In addition poor gene transfer, which leads to a reduced protein expression, the risk of mutagenesis as result of integrating vectors, which are virus, based still remains a big safety issue. As with the case with the US death, immunogenicity of the vector and the integrity of the transgene itself can be a cause for concern, so the preclinical assessment for gene safety is inadequate and in need of revision [16].

In addition to the target cell or tissue, one problem with in vivo administration of viral vectors is the surrounding and adjacent cell types of multiple natures that will be exposed to the same virus. This risk of potential infection and possible deleterious results posed to healthy normal cells [21].

The National Institute of Health (US) in 1995, a panel assessed the investment in research for gene therapy, the executive report of which highlighted seven key issues. Some of the issues included in this report were the following: while the expectation of gene therapy are great, definitive clinical efficacy had yet to be demonstrated, significant issues surround all basic aspects of gene therapy, with the major problems concerning gene transfer vectors alongside inadequate knowledge of the biological interaction of these vectors with the host cells/tissue. Further to this it mentioned that the eagerness to go to clinical trials with gene therapy, resulted in the compromise in basic research on disease pathophysiology, although it being critical to the eventual success of the gene therapy. Concerning results of numerous trials to date it pinpointed that protocols had been adversely affected by low frequency of gene transfer, the reliance of qualitative rather than quantitative aspects of the gene transfer and expression, deficiency in suitable controls and poorly defined biochemical or disease end points for the research. Ultimately this panel critiqued the hype concerning gene therapy with results from laboratories and clinical studies be they academic, federal or institutional influencing widespread public perceptions to believing gene therapy is more developed than it is actually is. This over-estimation s though to jeopardize the integrity of this field of research and may inhibit progress in the future [22].

Other considerations concerning gene therapy

In addition to the classical scientific and medical dilemmas religious traditions have a contribution to the ethical meaning and consequences of gene therapy. In 1983 several Roman Bishops positioned a paper in opposition to created an “engineered genetic trait into the germ line of the human species”. The official position concerning most religious communities however is more caution than opposition; concerns include some of the following areas “the status of the human embryo”, “respect for human finitude”, and social justice and germ line transduction which could create a public dislike of advances in gene therapy if it compromises lineage progression within families [21] [23].

Gene therapy: treating monogenic disorder, the future.

The development of quantitative models of cancer risk assessment for gene therapy has been put forth to reduce the risk of cancer in patients taking part in gene therapy trials, this far two models have been put forth, cell-based and murine-based and should ideally be performed on all methods of viral delivery viral and any future developing vehicles of deliverance. A further advance in the future should be geared towards reducing the oncogenic risk of gene therapy by targeting transgene insertion. At present two methods share the platform to target these interactions; using phage C31 integrase and homologous recombination [21].

In the search for creating better vectors, researchers are combining the best elements of the different virus types to produce “hybrid vectors”. One such example is the assimilation of the site-specific machinery of the wild-type AAV complimenting the efficient internalization and properties of nuclear targeting of the adenovirus
Future aspirations point towards developing site-specific integrating vectors, enhancing vector ability to home in on and infect specific and desired target cells, to understand how to predict responses by patients to inflammatory vectors, one of the major challenges over the next few decades [24][25].

A major contribution to changes in the future will be seen using homologous recombination as mentioned earlier. The HR pathway harnesses cellular pathways, one of the major mechanisms used in the repair of DNA damage, such as double stranded breaks, exposure to radiation and ultraviolet light and chemical damage. The HR pathway makes use of a second copy a the broken gene, which is usually held on the sister chromatid to serve as the template for DNA repair in addition to reparing the DNA in the surrounding vicinity [26]. This therapy removes autologous HSCs makes any correction to the mutant gene in vitro and re-inserts into the patients blood circulation. Homologous recombination has been used as a tool in murine models for many years as a method of genetic manipulation and is an exciting area of research into the treatment of monogenetic disorders. Drawbacks have been encountered concerning hematological-related disorders and barriers to maximizing this treatment also remain as stem cells are not always attainable as HSCs and in general embryonic stem cells (ESCs) are not autologous in addition to ethical issues surrounding the use of ESCs in treating hereditary diseases [1].

Other developments include induced pluripotent stem cells (iPSC), RNA interference (RNAi) and zinc finger nuclease (ZNF) [24][27].

The 21st century saw the discovery that already fully differentiated somatic cells could be reprogrammed through use of several transcription factor-encoding genes to generate induced pluripotent stem cells (iPSCs) led to somatic cells being exploited to attain iPSCs. Four genes out of twenty-four candidates which had the ability to present pluripotent nature in mouse embryonic and adult fibroblasts, which where then transduced using four retroviruses which possessed genes encoding the transcriptions factors, Oct4, Sox2, klf-4 and c-Myc, and the target cells underwent reprogramming displaying an embryonic stem cell (ESC) like nature, which were to be termed “ iPSCs”. The clinical potential of these genetic discoveries are infinite, as they envisage an inexhaustible source of autologous pluripotent stem cells. Research has been carried out for the potential uses in murine rodent models both in vitro and in vivo and has showed promising signs. Some principle drawbacks have been identified concerning this up-coming area of therapeutics include the possible inability to evade immune rejection alongside ethical issues, which shall be discussed in more detail in further sections [1][27].

4. Conclusion

Monogenic diseases incorporate a vast array of diseases. As this paper has investigated many differences occur at every level when reviewing this infinite area of research, from disease prevalence, from the rare cases of Fragile X syndrome to the more common cystic fibrosis, to the effect/s of the single gene mutation, which more often or not are fatal for the individuals who are affected. One aspect that unfortunately is well known presence of the ineffective and lacking treatments available to cure these individually unique diseases, the result of which has led to a robust rush towards turning to a genetic savior in the form of gene therapy. At present, the benchmark is Severe combined imunodiffciency, with ADA- and X-SCID in particular showing a promising future in the combat of the single gene mutations and the investment of much research. Numerous other medical areas have utilized gene therapy and although some of the literature fails to see the benefits, it has been very much championed in other areas of medicine such as cardiology and cancer therapy. With the advent of new theories of gene intervention in the form of hybrid vectors and homologous recombination, future research shall march on as attempts are already underway. Clinical trials, predominantly taking place in the Western world have already begun engineering ideas and manipulate genes in order alleviate the problems from the past. One consideration, which has already been highlighted by the National Institute of Health as, mentioned in this paper is the accountability for such huge investments into gene therapy. Aside from the classic expectation from society to overcome cancer, cardiovascular and the often-rare monogenic diseases, other factors influence this research such as available financial funding. Unfortunately, governments have recently entered an uncertain economic future and s face the need to critically analyze and balance the advantages’ and disvantages to continuing medical research, not exclusively in search for cures to pathological diseases and disorders but to also further elucidate mechanisms of pathogenesis which remain unknown at present. The future still looks positive, as underlying pathological mechanisms are beginning to be elucidated; the next stages of discovering Gold standard treatment in the form of gene therapy look promising, as inherited monogenic diseases are the ideal candidates. With knowledge that society looks upon academic and experts in the field to provide answers and develop treatment basic research at both the molecular, cellular ad animal levels are gradually being accepted and encouraged, coinciding with the long-standing culture of scientific freedom which exists internationally.

5. References

[1] Wong. GKY, Chui. AT. Gene therapy, gene targeting and induced pluripotent stem cells: Applications in monogenetic disease treatment. Biotechnology Advances, Volume 28, pp 715-724, 2010.




[5] World congress on Huntington’s disease 2005. Lisa J. Bain.

[6] Walker. F.O. Huntingtons disease. Lancet 2007: 369:218-28.

[7] McMurray. C.T. Huntingtons disease:new hope for therapeutics. TINS Vol.24, No.11 (Suppls), November 2001.

[8] Peter Sudbery and Ian Sudbery. Gene Therapy. In Human Molecular Genetics. 3ed, London, pp 321-341. 2010.

[9] Epplen. JT, Gencik. M, Epplen. A, Aldrich, Saft. C, Przuntek. H. Morbus Huntington – a human genetic model disease. Cytogenetics cell Genetics. Volume. 91, (1-4), pp 90-96, 2000.

[10] Gasper. BH, Auiti. A, Porta. F, Candotti. F, Hershfield. MS, Notarangelo. LD. How I treat ADA deficienicy. Blood. Volume. 111, Number, 17, October 2009.

[11] Hoogerbrugge. PM, van Besechem. VW, Kaptein. LCM, Einerhand. MPW and Valerio.D. Gene therapy for adenosine deaminase deficiency. British Medical Bulletin, Vol 51, No.1, pp. 72-81, 1995.

[12] Garber. KB, Visootsak. J, Warren. ST. Fragile X Syndrome. European Journal of Human Genetics. Vol. 16, pp 666-672, 2008.

[13] iGenetics A Molecular Approach. Peter J. Russell. International Edition, Second Edition 2006.
[14] Understanding disease, Pathology and prevention. Mera. S.L. 1997.

[15] Edelstein. M.L, Edelstein.R,Abei, M.R, Wixon. J. Gene therapy clinical trials worldwide to 1989-2004 – an overview. The Journal of Gene Medicine. Vol.6, pp597-602, 2004.

[16] Cavazanna-Calvo. M and Fischer. A. Gene therapy for severe combined immunodeficiency: are we there yet? The Journal of clinical investigation. Volume. 117, Number. 6, June 2007.

[17] Young. L.S, Searle. PF, Onion. D and Mautner. V. Viral gene therapy strategies: from basic science to clinical application. Journal Pathology, Volumer.208, pp 299-318, 2006.

[18] Blease. M.R. Development of Gene Therapy for Immunodeficiency: Adenosine Deaminase Deficiency. Paediatric Research, Vol. 33(Suppl), No.1, 1993.

[19] Schneider. MD and French. BA. The advent of adenovirus. Gene therapy for cardiovascular disease. Circulation, volume.88, pp 1937-1942, 1993.

[20]Edelstein. M.L, Abedi. M.R, Wixon. J. Gene therapy clinical trials worldwide to 2007 – an update. The Journal of Gene Medicine. Volume.9, pp 833-842, 2007.

[21] Porteus. M.H, Connelly. J.P, Pruett. S.M. A look to future directions in gene therapy research for monogenic disorders. Plosgenetics, Volume. 2, Issue.9, September 2006.

[22] Report and recommendations of the panel to assess the NIH investment in research on gene therapy. Executive summary of findings and recommendation. December 7th 1995. Stuart H. Orkin.

[23] Human Inheritable Genetic Modifications. Assessing Scientific, Ethical, Religious, and Policy Issues. Prepared by the American Association for the Advancement of Science. Mark S. Frankel and Audrey R. Chapman. September 2000. Link:

[24] Thomas. C.E, Ehrhardt. A and Kay. MA. Progress and problems with the use of viral vectors for gene therapy. Nature reviews Genetics. Volume, 4, pp 346-358, May 2003.

[25] Rubanyi. G.M. The future of human gene therapy. Molecular aspects of medicine, volume.22, pp. 113-142, 2001.

[26] Kohn. D.B. Update on gene therapy for immunodeficiencies. Clinical immunology, volume. 135, pp. 247-254, 2010.

[27] Meirong. L, Meixia. C, Weidong. H and Xiaobing. F. How far are induced pluripotent stem cells form the clinical. Ageing researcj reviews. Volume.9, Issure.3, pp 257-264, 2010.


Category: Essay & Dissertation Samples, Health Essay Examples

Comments (1)

Trackback URL | Comments RSS Feed

Sites That Link to this Post

  1. Gene Therapy and Monogenic Disorders Essay - | August 31, 2019