Crispr Design Tool Chop Chop

Genetic Engineering is a process of recombinant DNA technology that involves direct manipulation of genomes for altering the genetic makeup of organisms. Previously it was limited to more straightforward procedures such as cloning of DNA fragments and their growth within bacterial species. Controlled breeding and selection of progeny with desired characteristics was the basis until the field of genetic engineering expanded to include cell-to-cell variations, whole-genome alterations, and manipulation of heritable as well as nonheritable DNA constructs of organisms [1]. Genes can be transferred to and from higher organisms for several desired benefits. The use of genetic engineering is not limited to humans, as many agronomically improved crops have also been generated. Genetic material from different organisms can be cut up and combined to create a hybrid in another organism for newer heritable genetic combinations with enhanced features [2].

The general process of genetic modification includes isolation of DNA from cells, cutting the DNA into smaller fragments using restriction endonucleases, mixing up and combining the DNA molecules via DNA ligase, introducing the hybrid DNA into cells, and ultimately the selection of transfected cells [3]. The focus of genetic manipulation has been to treat the human diseases by introducing animal models of disease for detailed study and addition of functional genes into the organisms having heritable genetic disorders and their genetic manipulation to prevent the spread of diseases across generations. Gene therapy is the introduction of genetic material into the cells to recompense the missing functionality of a specific gene or to make a required protein in larger amounts. Different vectors are used to transfer the engineered gene into the cells for proper functioning, such as the use of viruses as vectors [4].

CRISPR is the cutting-edge gene-editing technology, which is one of the trending topics in biomedical research nowadays. These are the DNA sequences within bacterial genomes that can be used to edit genomes of nearly any organism. CRISPR can identify mutated genes, chop them out, and replace them with repaired sequences of DNA [5].

The use of genetic engineering or gene therapy for the treatment of human disease has been a long-sought dream of scientists across the world. Diseases caused by mutations in single genes are easier to be treated using gene-editing such as sickle cell anemia or cystic fibrosis. However, complex disorders like heart diseases or cancers are still difficult to be cured using gene therapy as they result from an interplay of genes and the environment.

Recombinant DNA technology improves the health condition by the development of improved pharmaceuticals. Recombinant vaccines are the chief example of the benefits of recombinant genetic techniques for the improvement of life for humans. Many of the drugs, as well as the vaccines that have been approved by the FDA and are currently used clinically, have been developed from genetic engineering technologies. Various therapeutic products such as growth hormones, recombinant proteins, anticancer drugs, and antibodies have been produced using genetic manipulation for improved health economics [6].

In 2017, the FDA approved the first genetic engineering-based treatment introduced by Novartis for leukemia patients in which genetic alteration of patients' cells is carried out to enable them to fight cancer cells. Hence it boosts the immune system to put an end to the disease [7].

gene editing

Table of Contents

Gene knock-out strategies
- Cancer
- Blood Disorders
- AIDS
- Cystic Fibrosis
- Muscular Dystrophy
- Huntington's Disease
Recent Advances
References

Gene knock-out strategies

Genetic engineering can be employed to treat the monogenic disorders in which there is a mutated gene causing the disease. Many such studies are being carried out, and several alternative strategies have been proposed. One such disease is Amyloid transthyretin (ATTR) amyloidosis, which occurs as a result of a mutation in the TTR gene and leads to amyloid protein build up in the nerves connecting the spinal cord and brain. Research is going on to utilize the CRISPR technology to knock-out the mutated gene responsible for this. This involves gene deactivation or deletion of a gene sequence [8].

Leber congenital amaurosis (LCA) is an inherited childhood blindness disease resulting from a mutation in around 14 genes, but the most common form (LCA10) is due to CEP290 gene mutation. This gene encodes a protein present in various cell types, including the photoreceptor cells of the eyes. Genetic mutation from this gene is cut using the gene-editing tool CRISPR/Cas9 via sub-retinal injection for delivering it in the patient's eyes [9]. As the eye is an immune-privileged part of the body, there are fewer chances of immune response induction by the CRISPR system, which is a major issue blocking the wide range success of this technology. But these concerns are laid to rest when the treatment is to be provided at an immune-privileged site such as the eye in the case of LCA [10].

In the past few years, gene therapy has made vital progress in the field of medical advances, and many clinical studies based on genetic engineering, for treatment of disease, are under development at this time. Some of the major disease currently being targeted using genetic engineering include the following:

Cancer

One of the most discussed topics of medical research is cancer therapeutics due to the complexity of this disease. Two-third of all the gene therapy trials are for cancers. Among the recent advancements to treat the different cancer types is the suicide gene therapy , in which suicide-inducing genes are introduced into the cancer cells. Various receptors of different cells have been used as targets such as vascular endothelial growth factor receptor VEGFR, cluster of differentiation (CD) 44 and 133, EGFR, PSA, or Transferrin receptor (TfR). Other strategies include anti-angiogenesis and oncolytic therapy [11].

Many vectors are being exploited for the delivery of cancer therapy, such as the non-viral vectors, including nanoparticles, liposomes, or synthetic viruses. For gene transfer, viruses like adeno-associated virus, parvovirus, and lentiviruses have also been used as vectors [12].

Genetic engineering is not limited to the use of gene therapy vectors, but immunomodulating strategies have also been utilized for the treatment of cancer. Active immunomodulation involves the use of vaccines produced against the tumor cells such as genetically modified tumor cell vaccine using the poxvirus, recombinant fowlpox virus, vaccinia virus, or a combination named TRICOM. Passive immunotherapy is based on the use of antibodies against cancer cells such as Pertuzumab, Trastuzumab, and Ado-Trastuzumab against the HER/2 receptor in breast cancer. Other antibodies include Rituximab, Ibritumomab, and Tositumomab for CD20 Protein on lymphoma cells and Panitumumab against the EGFR on colorectal cancer [13].

Blood Disorders

Gene therapy holds great promise in terms of treatment of blood disorders. Cell-based microparticles (MP) such as naturally occurring megakaryocytic microparticles have been used to deliver the small RNAs and plasmid DNA into the hematopoietic cells in a study published in Science Advances . This technique could be utilized for therapeutic purposes in treating blood diseases such as sickle cell anemia. It can also be exploited for the delivery of personalized medicine [14].

Several approaches to treat hemophilia have been used. CRISPR/Cas9 mediated correction of the mutated gene has been achieved in hemophilia B patients via induced pluripotent stem cells (iPSCs). A deletion in the exon 2 of the factor IX (F9) gene has been repaired by Morishige et al. using Cas9 nuclease and guide RNA vector of expression [15].

A mutation in the HBB gene causes beta-thalassemia in which there is reduced production of hemoglobin. This leads to anemia and an increased risk of blood clots formation. Gene-editing technology is being used to edit the DNA sequence of the HBB gene by removing the cells from patients, editing of these cells, and inserting back the modified cells into the patient [16].

AIDS

HIV/AIDS has been one of the most difficult diseases to counter despite medical advances. Conventional therapies have limitations. Therefore, alternative approaches are being considered to produce genetically modified cells that secrete antiviral proteins. These proteins interfere with the entry of HIV and prevent its progression. T cell-based therapies have been in clinical trials in which the CCR5 of CD4+ T cells has been modified using the zinc finger protein (ZFN) and infused in patients receiving antiretroviral treatment [17]. Although this therapy needs improvement but shows promising potential.

The potential of transgenes to elicit immune response also needs to be studied further for using hematopoietic stem cells for HIV therapies. As reported in the Scientific American , using genetic engineering tools, T cells can be modified to have an HIV-specific CAR receptor by modification of the stem cells. These not only identify the virus but also binds to it interfering with the ability of HIV to enter the T cell [18].

Cystic Fibrosis

Gene therapy was used to target the disease resulting from an abnormality in a single cell. Cystic fibrosis was treated via the transfer of a usual copy of the CFTR gene into relevant epithelial cells. Although it seems to be a simpler approach as it is single-gene disorder and the airways are accessible, it has encountered many limitations such as inflammatory response when the body takes plasmid DNA as foreign, reduced gene expression after multiple doses [19].

A single dose to treat cystic fibrosis is set to be developed by the collaboration of the UK Cystic Fibrosis Gene Therapy Consortium . The treatment is based on the delivery of the healthy copy of the CFTR gene, which is generally mutated in cystic fibrosis. Previously, repeated doses of fat droplets-based gene therapy were proved to be effective for the treatment of this disease [20].

Researchers have proved that the mutation behind cystic fibrosis can be treated using CRISPR in human lung cells from the patients. This disease can develop from multiple different mutations. Hence the challenge for researchers would be to develop multiple CRISPR based therapies besides the most common mutations being targeted [10].

Muscular Dystrophy

Novel gene therapy has shown promising potential to treat Duchene Muscular Dystrophy (DMD) that safely stopped the deterioration of muscles. In this disease, there is a mutation in the DMD gene that leads to little or no production of dystrophin protein, causing muscle weakness and cell death [21]. A group of researchers developed an innovative approach to counter the disease by cutting out the mutation hotspot in which most of the mutations occur, leading to DMD. Further research is being carried on, which focuses on the removal of the whole mutated protein instead of fixing the mutations.

The study published in Nature Medicine reports that a synthetic transgene encoding a utrophin (µUtro), which is a dystrophin related protein, has been developed that can be delivered using the adeno-associated virus vectors and led to the prevention of myonecrosis [22]. It is also reported that no immune responses have been triggered that prevented the successful application of previous therapies.

Huntington's Disease

FDA has approved a drug that targets Huntington Disease in which nerve cells of the brain breakdown and the person's ability to move and think are affected. The main goal of genetic engineering in the case of Huntington disease is to limit the production of the toxic huntingtin protein. This is achieved by blocking the HTT gene utilizing microRNAs. Antisense oligonucleotide therapy has also been in consideration for curing this disease [23].

Recent Advances

Various genetic alteration strategies to treat Parkinson's disease have been identified and proposed by the researchers at the University of Pittsburgh and Sechenov University recently. It is a neurodegenerative disorder causing impaired cognitive and motor functions. CRISPR/Cas9 technology has been thought for direct manipulation of the genetic material and development of drugs for improved iron homeostasis. Parkinson's disease models have been generated using this technology, and various neuroinflammatory pathways have been dissected [24].

The application of genetic engineering for treatment or diagnosis of diseases has enormous potential. Gene therapy for immune genetic disorders like Severe Combined Immune Deficiency (SCID) and Chronic Granulomatous Disorder (CGD) have also been developed, and clinical trials are underway.

Despite all the advantages, numerous ethical concerns have been raised upon the use of genetic engineering, as some also regard it as meddling with life in unnatural ways. Various other challenges also need to be overcome to treat diseases using genetic modification widely.

References

[1]	D. M. Bodine, "Genetic Engineering," NHGRI, 24 March 2019. [Online]. Available: https://www.genome.gov/genetics-glossary/Genetic-Engineering. [Accessed 21 Dec 2019].
[2]	E. Rosenberg, "Genetic Engineering," in It's in Your DNA, Academic Press, 2017, pp. 81-93.
[3]	S. Brenner and J. H. Miller, Encycolpedia of Genetics, Academic Press, 2001.
[4]	NIH NLM, "How does gene therapy work?," 10 Dec 2019. [Online]. Available: https://ghr.nlm.nih.gov/primer/therapy/procedures. [Accessed 22 Dec 2019].
[5]	C. Pulido-Quetglas, E. Aparicio-Prat, C. Arnan, T. Polidori , T. Hermoso and Palumbo et al., "Scalable Design of Paired CRISPR Guide RNAs for Genomic Deletion," PLoS Comput Biol, vol. 13, no. 3, p. e1005341, 2017.
[6]	S. Khan, M. W. Ullah, R. Siddique, G. Nabi, S. Manan, M. Yousaf and H. Hou, "Role of Recombinant DNA Technology to Improve Life," International Journal of Genomics, vol. 2016, p. 14, 2016.
[7]	FDA, "FDA approval brings first gene therapy to the United States," 30 Aug 2017. [Online]. Available: https://www.fda.gov/news-events/press-announcements/fda-approval-brings-first-gene-therapy-united-states. [Accessed 2019].
[8]	K. Speights, "What Diseases Could Gene Editing Cure?," The Motley Fool, 4 Jun 2018. [Online]. [Accessed 22 Dec 2019].
[9]	M. L. Maeder, S. Shen, E. R. Burnight, S. Gloskowski, R. Mepani and Friedland et al., "Therapeutic Correction of an LCA-Causing Splice Defect in the CEP290 Gene by CRISPR/Cas-Mediated Genome Editing," GENE EDITING AND GENE REGULATION III, vol. 13, no. S1, pp. S273-274, 2015.
[10]	C. R. FERNÁNDEZ, "7 Diseases CRISPR Technology Could Cure," LabioTech.eu, 23 July 2019. [Online]. Available: https://www.labiotech.eu/tops/crispr-technology-cure-disease/. [Accessed Dec 2019].
[11]	M. Malecki, "Frontiers in Suicide Gene Therapy of Cancer," J Genet Syndr Gene Ther., vol. 2012, no. 3, p. e114, 2013.
[12]	S. Collins, B. Guinn, P. Harrison, M. Scallan, G. O'Sullivan and M. Tangney, "Viral vectors in cancer immunotherapy: which vector for which strategy?," Curr Gene Ther. , vol. 8, no. 2, pp. 66-78, 2008.
[13]	M. H. Amer, "Gene therapy for cancer: present status and future perspective," Mol Cell Ther, vol. 2, no. 27, 2014.
[14]	C.-Y. Kao and E. T. Papoutsakis, "Engineering human megakaryocytic microparticles for targeted delivery of nucleic acids to hematopoietic stem and progenitor cells," Science Advances, vol. 4, no. 11, 2018.
[15]	S. Morishige, S. Mizuno, H. Ozawa, T. Nakamura and Mazahery et al., "CRISPR/Cas9-mediated gene correction in hemophilia B patient-derived iPSCs," International Journal of Hematology, pp. 1-9, 2019.
[16]	D. P. Dever, R. O. Bak, A. Reinisch, J. Camarena and Washington et al., "CRISPR/Cas9 Beta-globin Gene Targeting in Human Hematopoietic Stem Cells," Nature, vol. 539, no. 7629, p. 384–389, 2016.
[17]	A. Falkenhagen and S. Joshi, "Genetic Strategies for HIV Treatment and Prevention," Mol Ther Nucleic Acids, vol. 13, pp. 514-533, 2018.
[18]	S. Begley, "Genetically Modified T Cells Might Help Fight HIV," Scientific American, 2 Jan 2018. [Online]. [Accessed 22 Dec 2019].
[19]	T. J. Burney and J. C. Davies, "Gene therapy for the treatment of cystic fibrosis," Appl Clin Genet., vol. 5, pp. 29-36, 2012.
[20]	A. BURIK, "New Gene Therapy Could Treat Cystic Fibrosis With One Dose," LabioTech, 8 June 2018. [Online]. Available: https://www.labiotech.eu/medical/cystic-fibrosis-gene-therapy/. [Accessed 22 Dec 2019].
[21]	GEN, "Novel Gene Therapy for Duchenne Muscular Dystrophy Shows Promise," Genetic Engineering and Biotechnology News, 9 Oct 2019. [Online]. Available: https://www.genengnews.com/news/novel-gene-therapy-for-duchenne-muscular-dystrophy-shows-promise/. [Accessed 22 Dec 2019].
[22]	Y. Song, L. Morales, A. S. Malik, A. F. Mead, C. D. Greer and Mitchell et al., "Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models," Nature Medicine, vol. 25, p. 1505–1511, 2019.
[23]	ASGCT, Huntington's Disease, Milwaukee, WI: American Society of Gene and Cell Therapy, 2019.
[24]	J. Luo, P. Padhi, H. Jin, V. Anantharam, G. Zenitsky, Q. Wang, A. Willette, A. Kanthasamy and A. Kanthasamy, "Utilization of the CRISPR-Cas9 Gene Editing System to Dissect Neuroinflammatory and Neuropharmacological Mechanisms in Parkinson's Disease.," J Neuroimmune Pharmacol, vol. 14, no. 4, pp. 595-607, 2019.

Dr Martin receives his MD from University of Iowa. His expertise includes microbiology, anatomy and clinical psychology. He also dedicates himself to continuous learning in different fields.