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1 Healing the Fundamental Unit of Heredity (Gene Therapy):

2 Current Perspective and What the Future Holds

4Abstract

5The ability to make precise adjustments to the human genome has been a goal of healing

6gene also introduces as the fundamental unite of heredity, in biomolecular technology in

7genetic diseases have opened new knowledge such as gene therapy. Gene therapy is a

8technique to repair DNA where its usage is to treat malignancy and inherited genetic

9diseases. Gene therapy is a choice to the genetic cloth that goals to remedy a sickness this is

10hard to deal with or perhaps has no treatment. Currently, gene remedy is done in approaches

11to patients, specifically embryonic cells and somatic cells, every in vivo and ex vivo. Moral

12considerations with modification of the difficulty's cells and oversight of regulation and

13reagents want to be taken into consideration within the gene therapy project. Applications

14for using gene remedies have begun to be widely used, which include in case of maximum

15cancers, coronary heart disorder, infectious sicknesses, and others. Gene therapy has spread

16to a wide range of applications then go beyond the modification of genetic disorders.

17Advances in genetic modification of cancer cells and immunity and the use of viruses and

18bacteria to control cancer cells have resulted in many clinical trials and product

19developments for cancer treatment. The miracles and blessings of gene therapy are might

20believe, but even though they are being studied and developed now and, in the future, so that

21the desire for gene therapy may be even better future.

22Keywords: gene therapy, genetic recombination, which, gene therapy application.

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23Introduction

24 Genetics constantly has a role in each ailment, both big and small. Deoxyribose

25Nucleic Acid (DNA) variant and differentiation in how the DNA works also the environment

26in which humans develop has a major contribution in every ailment. The genetic abnormality

27in humans, including gene abnormalities, chromosome instability, epigenetic, most cancers,

28and another incapacity complicated has been issued how the era work in control remedy and

29recuperation. As understood with the aid of ability of genetic improvement thru gene

30correction or specific modification region for a selected target of treatment, Gene remedy is a

31technique of treating or disease remedy therapy by using shifting one or more nucleic acids to

32target cells using repairing broken genes1,2,3.

33 Gene therapy technology never breaks from genetic manipulation in terms of

34producing a Genetically Modified Organism (GMO). Gene therapy is a laboratory experiment

35that including a group of drugs for advanced therapy, and including modified cells and

36modified tissue. The way gene therapy works are by repairing and deactivating or replacing

37malfunctioning genes that can cause disease intending to rebuild normal function. Therefore,

38this gene therapy has the potential to cure diseases that can’t be cured with natural remedies

39and can cure diseases that are classified as difficult to cure. Gene therapy treatment has a

40huge potential in genetic diseases such as cancer, specific viral infection, and recessive

41genetic abnormalities (Cystic fibrosis, hemophilia, muscle dystrophy dan sickle cell

42anemia)1,15,21,22.

43 To date, there are about 2,000 clinical trials for gene therapy in various diseases that

44have occurred or are on going3. Some clinical on 2018 shows regarding gene therapy in β-

45thalassemia patients using transfusion states that the therapy of CD34+ cells transduced in the

46BB305 vector can reduce or even eliminate the need for red blood cell transfusions in 22

47patients with severe β-thalassemia without any adverse side effects22. While in another studies

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482017 stated that a single intravenous infusion of SPK-9001 could consistently increase the

49activation of coagulant factor IX by 30%, which allowed prophylactic discontinuation,

50bleeding and eliminated the need for exogenous in 3 men with hemophilia B. In children with

51associated hypophosphatemia - X treatment with burosumab can increase phosphate

52reabsorption in the renal tubules, serum phosphorus levels, physical function, and reduce the

53pain and severity of rickets10. In some cases, Gene therapies can halt disease progression

54entirely by addressing and correcting its underlying genetic cause, decrease morbidity, and

55increase survival6.

56 Based on the success of several studies above, gene therapy is known as one of the

57efforts in scientific breakthroughs. The various methodologies in genetic engineering for

58advances in the development of gene therapy vector systems are being optimized for gene

59transfer. It’s been new and unique approaches to treating previously intractable diseases using

60gene therapy. Gene therapy will address the root causes of genetic diseases by modifying the

61expression of a patient’s genes or by repairing or replacing abnormal genes, rather than

62treating disease symptoms. Many experts believe that gene therapies are “shifting medicine

63away from a chronic disease management approach toward disease interception and

64prevention. However, this gene therapy process remains complex, so there are still many

65techniques that require new developments. Therefore, in this article, the author will discuss

66gene therapy, its history, the methods that have been developed, its current application, and

67the prospects for gene therapy in the future.

68

69How it all starts

70 Gene therapy is first known in the 1980s as they used genetics theory 1. The National

71Institutes of Health (NIH) and The Food and Drug Administration (FDA) have played an

72important role in the emergence of a safe and effective human gene therapy. The first

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73breakthrough made was recombinant DNA technology 7. First experiment issued in National

74Institutes of health in 1989 about tumor infiltration using retroviral vector which reinfuse in

75regards to checking the capacity of tumor horning of the cells. The result of the experiment is

76that the human cell can be genetically modified and returned to the patient as harmless. From

77this research, new experiments emerged around the world until now11.

78 Efforts made to increase understanding of the biological basis of disease such as gene

79transmission, the potential adverse events encountered. Besides, scientific advances can also

80improve safety precautions, efficiency, and delivery of gene transfer. There are three

81conceptual technics in gene therapy as, 1) recombination of DNA technology, which works

82when the healthy gene or the wanted gene put on a vector as a plasmid nanostructured or a

83virus; 2) treatment with a synthetic oligonucleotide which there are couple blocks genomic

84structures called nucleotides; 3) Delivery of messenger Ribonucleic acid (mRNA)7.

85

86How things stands

87 Event there are so many successful experiments and thousands of researches such as

88gene delivery technology, target cell biology, gene therapy, understanding the target disease,

89but always there some complex efforts for the sake of development in technical gene therapy,

90this could be a serious issue for now and upcoming event 12. It is currently reported that the

91use of a form of oligonucleotide-induced exon skipping for the treatment of Duchenne

92muscular dystrophy (DMD) and SMA explains that the promising outcome approach is not

93efficient for gene therapy in humans1,12,14. There are two types of gene therapy, it’s embryonal

94cell (germline gene therapy) and somatic cell (somatic gene therapy)1.

951. Germline Gene Therapy

96 Germline gene therapy is a gene therapy with sperms or ovum that has been modified

97with some functional replacement to the genome. This modification will be passed hereditary

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98to all the next generation. In other words, theoretically, this gene therapy should be hugely

99effective management to prevent genetic disease. However, despite its potential to prevent

100congenital disease, germline therapy is highly controversial and there has been very little

101research in its field for technical or ethical reasons1,17,18,23.

102 In germline gene therapy, stem cells such as sperm and egg are modified in a way, that

103is, inserting the updated genes into the genome. Modifications in this way can be carried out

104from generation to generation to create the next. This is why germline gene therapy is so

105effective in correcting genetic diseases13.

106 On gene therapy using germline, The genes will be transferred into the ovum or zygote

107so that when the ovum is fertilized with sperm to form a zygote, by carrying the previously

108inserted the zygote will develop genes so that the new organism that is formed has genes that

109function in the intended therapy18.

1102. Somatic Gene Therapy

111 Somatic Gene therapy works when the therapeutic gene had transferred to a somatic

112cell of the patient. The functional DNA is transferred to the targeted cell though in vivo or ex

113vivo. In this gene therapy targeted cells wouldn’t be able to pass among the generation. The

114gene is transferred directly intended to the targeted organ, so it will be functional as it wishes

115for. Somatic Gene therapy wouldn’t affect the embryonic cell. This gene therapy is provided

116as a major genetic laboratory worldwide1,22.

117 Somatic cell gene therapy is a technology with a wide range of applications used in

118human health. One example of somatic cell gene therapy is DNA vaccination, which is

119similar in procedure to conventional vaccination. In this therapy, any side effects are limited

120to these patients and are not passed on to the next generation 17. In general, the genetic

121makeup of the individual is not affected by the changes that have been corrected however,

122this therapy can be improved and contribute to the normal function of the defective organ9.

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123 Somatic cell gene therapy is considered as the only therapy that can be accepted by

124the community, this is because this therapy can affect cells, tissues, or organs that are targeted

125to the patient and are not passed on to the next generation. This therapy is a genome

126modification exemplified by CRISPR technology or epigenetic modification with a gene

127therapy approach with the same effect. This somatic cell gene therapy has two categories,

128namely ex vivo and in vivo8.

129

130Non-Viral method

131 Non-viral Vectors usually known as naked DNA are easier to produce in large

132numbers and less immune response of the host. In the past decade, low transfection rates and

133low gene expression were considered to be drawbacks of this method however, recent

134advances in vector technology have resulted in molecular production and techniques with the

135same efficiency as viral techniques. Transfection is a method to overcome the problem of

136inserting negatively charged molecules (DNA and RNA phosphate skeletons) into cells with

137negatively charged membranes. The use of normal (modified organic silica or silicate) is

138another non-viral method. This ease of action makes silica a good choice for gene

139delivery20,24.

140 Naked DNA injection is the simplest non-viral transfer method. This method is

141performed with naked plasmid PCR however, naked DNA retrieval by cells is generally

142inefficient. Naked DNA injection focuses on increasing the efficiency of DNA retrieval by

143developing new methods such as; electroporation, sonoporation, and use of genes, in which

144the DNA coated with gold particles is inserted into the cell with helium24.  

145

146

147

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148Physical Methods 

149 In this physical method, DNA delivery uses a small injection to embryonic stem cells

150to produce transgenic organisms in addition to transferring the antisense RNA into C.

151elegant. Electrophoresis is a high voltage method impulses to transfer DNA from a cell

152membrane. On the surface of the cell membrane, small pores are formed temporarily as a

153result of an electric shock. This electrophoresis can be applied to a wide variety of cell types,

154the high rate of cell death can limit its use in clinical applications20,24. 

155 Another physical method for gene transfer is biolistic particle delivery, also known as

156the particle bomber. This method relies on the high-speed micro-processing of nucleic acids

157to the receiving cell using membrane penetration. This method has been used successfully in

158the delivery of nucleic acids to cultured cells and cells in vivo20. 

159

160Chemical Methods 

161 Gene therapy used synthetic oligonucleotides to deactivate genes involved in the

162disease process. By using the specific antisense for the target gene will impair the

163transcription of the defective gene. Dendrimers are branched macromolecules whose particle

164surfaces can be filled by various methods, and many of the final structural properties of the

165particles are determined by these surfaces. The presence of genetic material such as DNA or

166RNA results in temporary nucleic acid linkages with cationic dendrimers24. 

167

168Challenges in Gene Therapy

169 The capacity of gene therapy to cure human diseases is currently a good reality

170though, the need for supervision, ethical considerations related to the genetic modification of

171the subject's cells, and the availability of suitable skills, infrastructure, and reagents are also

172materials that should be considered with great care11,12. 

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173 Recently there was a debate on genetic ethics between the Japanese, British and

174American ethical committees where experiments on healthy human embryos were approved

175by the Japanese and British ethical committees, however, in America, it was still conservative

176which stated that the experiment was still waiting for improvements in the use of the

177technique. and ethical issues1. 

178

179Gene Therapy Applications

180 Gene therapy can be used as an alternative treatment for diseases for which no cure or

181vaccine has yet been found. Eve Nicholas quoted in Patil et al., 2018 article explained that

182there are several criteria for selecting gene therapy diseases in humans, namely:

1831. An incurable disease.

1842. The affected organs, tissues, and cell types have been identified.

1853. The defective normal gene has been isolated and cloned.

1864. Introduction of normal genes into the target tissue.

1875. Genes can be expressed adequately techniques are available to verify the safety of the

188 procedure.

189 Cancer, cardiovascular disease, infectious diseases, monogenic diseases, vision-

190related diseases, neurological diseases, and so on are assumed to be curable with gene

191therapy. The disease can be treated if the associated gene is identified and its functional gene

192is found that can substitute the abnormal gene. All of these decades there’s about 1437 trials,

193currently, clinical trials of gene therapy in treating cancer (65.0%) and monogenic diseases

194(11.1%) have achieved great success in current gene therapy, namely 76.1%. Although trials

195targeting cardiovascular disease outnumber trials for the monogenic disease since 2004, this

196trial is now the fourth most common indication (6.9%) as a result of an increasing number of

197trials targeting infectious diseases (7.0 %)12,22.

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198

199Benefits of Gene therapy

200 Gene therapy has many benefits, including 1) gene silencing, where individual cases

201of HIV infection can be suppressed with gene therapy to protect patients from pain and

202suffering before the disease progresses; 2) Gene therapy has the potential to eliminate and

203prevent hereditary diseases such as cystic fibrosis; 3) create a new field of medicine based on

204technology. 4) Regulatory approval for gene therapy licensing is increasing 4,24. A major

205advantage of gene therapy is that continuing expression of the gene(s) allows for a cure

206following a single treatment as opposed to continuous administration of a drug with a

207relatively short half-life5.

208

209Disadvantages of Gene therapy

210 Although gene therapy has the potential to prevent morbidity for years with one

211treatment, this gene therapy has a drawback, namely very expensive cost 19. The renewal of

212gene therapy methods is one of its drawbacks. Stimulation of the immune response in which

213genes are injected by the virus in the body can cause an immune response and pathogenic

214viral vectors. Besides, the generation of genetic disorders due to the presence of multigene

215which are the genetic material being transferred does not necessarily enter the target cells,

216even if they do, it may not be placed in the right place in the genome 24.

217

218What’s the future hold?

219 Gene therapy knowledge is considered an important new approach compared to

220conventional medicine. This raises a lot of hope for the future regarding gene therapy. Gene

221therapy facilitates the continuous, stable, and regular expression of biological agents by

222targeted delivery of genes containing genetic information 16,24. 

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223 Gene therapy using the cell division potential and transplantation of the immunity cell

224to removing the targeted cell in a certain case. Another important challenge is before the

225effect that should be handle before the therapeutic approach has been fully aware. As the

226number of reported successes of gene therapy increases, other factors associated with

227processing need to be considered such as; target disease from gene therapy. Gene therapy

228also currently offers the potential for a single treatment that produces a lifelong cure, leading

229to a discussion of how much therapy is valued and how expensive it is12,24. 

230 The gene therapy capacity is to cure human disease now an established reality, but for

231now, many disease phenotypes and the pathophysiological process will potentially to this

232exciting therapeutic approach lie beyond the reach of existing technology. ‘Achilles heel of

233gene therapy’ was once described as the ability to achieve efficient gene delivery. Even

234though there are several successful protocols, the process of gene therapy remains complex,

235and many techniques need new developments. The specific body cells that need treatment

236should be identified and accessible. The effective way to distribute the gene copies to the

237cells must be available, and the diseases and their strict genetic bonds need to be completely

238understood1,12.

239

240Conclusion

241 The involvement of molecular biology has made it easier for researchers to

242manipulate genes, one of which is by utilizing the development of gene therapy. Gene

243therapy is a treatment method by transferring certain functional genes so that they can replace

244the function of abnormal genes related to the disease. The advantages and disadvantages of

245gene therapy, challenges in the pros and cons of gene therapy methods certainly exist,

246however, this is what makes the basis for gene therapy to be better in the future. 

247

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