GENOME EDITING IN MEDICINE

ABSTRACT

After the discovery of the CRISPR-Cas9^[1] technique in 2012 the field of genome editing has grown vastly in all directions, it has intrigued many scientists and clinical industries to invest capital and time into it leading to discoveries of many other effective techniques in genome editing. It has various applications like human gene therapy, crop improvement, disease modeling, neuroscience, and drug discovery. Biomedicine and genome therapy being the most promising and valuable genres of all, in this article we try to summarize the current knowledge and applications of gene editing and how it will shape the future of medicine and therapy.

KEYWORDS – Gene editing techniques, CRISPR, therapeutic gene editing, immunotherapy.

INTRODUCTION

The human genome project was completed in 2003 but only left us with the basic knowledge of a healthy individual’s genomic data, often faults and errors are observed in the genetic mapping of certain individuals; Gene aberrations have a huge impact on the livelihood of a population, it causes abnormalities and incurable diseases like cystic fibrosis, sickle cell disease, and genetic syndromes like down’s and turner’s. These abnormalities can be due to numerous reasons like exposure to UVR, carcinogens, and even random mutations. Genome editing is the technique that is being used and researched over the past decade to help tackle these obstacles. The existing genome of a diseased individual is altered to eliminate abnormalities or add missing pieces to the gene sequence.

GENE EDITING TECHNIQUES

Although CRISPR is an excellent tool for editing many newer and more effective technologies have been discovered since its discovery. Base editing^[2][3] is a more recent cutting-edge technology that operates by chemically altering the gene sequence rather than double-strand break repairs.

The most recent tool that even eliminates the limitations of base editing is Prime editing^[4] which works just like CRISPR tech but without the use of double-strand break repairs making it more

efficient and reliable.

FIGURE ^[5] – Mechanisms of different genome editing tools.

THERAPEUTIC ASPECT OF GENOME EDITING

Gene editing therapy has shown effective responses for the treatment of various conditions like thalassemia, sickle cell disease, and AIDS. It is being used now in numerous fields like cancer research, cardiovascular diseases, hematological diseases, metabolic conditions, neurodegenerative syndromes, and hereditary diseases.

TABLE ^[6] – List of some of the recent therapeutic gene editing studies in in vivo preclinical and clinical models

Disease	Target organ	Gene editing tool	Delivery system	Therapeutic modality
Hemophilia A and B	Mouse liver	ZFN	Systemic injection of AAV8	HDR- and HITI- dependent gene insertion
Hunter’s syndrome	Mouse liver	ZFN	Systemic injection of AAV2/AAV8	NHEJ- or HDR- mediated integration into albumin locus
Congenital muscular dystrophy type1A	Mouse muscle	CRISPR/dCas 9	Intramuscular or systemic injection of AAV9	CRISPR activator- based gene upregulation
Retinitis pigmentosa	Transgenic mouse model with human Rhodopsin ge ne	CRISPR/Cas9	Electroporation of Cas9 and dual gRNAs in mouse retina	NHEJ-based gene knockdown
Retinitis pigmentosa	Mouse retina	CRISPR/Cas9	Subretinal injection of AAV8	NHEJ-mediated targeted Nre inactivati on

 

Disease	Target organ	Gene editing tool	Delivery system	Therapeutic modality
Oxygen- induced retinopathy	Mouse eye	CRISPR/Cas9	Intravitreal injection of rAAV1	NHEJ-based mutant gene disruption
Primary open- angle glaucoma	Mouse eye	CRISPR/Cas9	Intravitreal injection of Adenovirus (Ad5)	NHEJ-based mutant gene disruption
Huntington disease	Mouse brain	CRISPR/Cas9	Stereotactic injection of AAV1	SNP-based allele- specific editing of Htt gene
Rett syndrome	Mouse brain	CRISPR/Cas9	Stereotactic injection of AAV1/2	NHEJ-based disruption of multiple genes
Cardiac syndrome	Mouse heart	CRISPR/Cas9	Systemic injection of AAV9	NHEJ-based mutant gene knockdown
Dystrophic cardiomyopat hy	Mouse heart	CRISPR/Cas9	Retro-orbital and intraperitoneal injection of AAV rh74	NHEJ-based mutant Dmd exon 23 excision
Cancer	Programmed death1 ligand (PD-L1) tumor xenograft	CRISPR/Cas9	Lentiviral delivery	PD-1-deficient CAR-T cells
HIV	HIV-infected humanized mouse	CRISPR/Cas9	Intravenous injection of AAV- DJ/8	HIV-1 proviral DNA excision

 

Disease	Target organ	Gene editing tool	Delivery system	Therapeutic modality
	spleen, brain, heart, lungs, and so on
β-Thalassemia	Rhesus macaques	Transposase	Intravenous injection of HDAd5/35++ vect or	Transposase-based gene integration

FUTURE OF GENOME EDITING

Genome editing in cancer immunotherapy

One potential application of immunotherapy is using genetically engineered T cells, which are known as chimeric antigen receptor (CAR) T cells. These cells can target tumor-associated antigens and

potentially lead to an enhanced response to therapy.^[7]

Figure ^[8] – Production of CAR T cell products.

Medical applications

The earliest trials used ZFNs to knock out the CCR5 co-receptor gene in T cells of HIV-positive patients, thereby making the T cells resistant to the virus. The results were encouraging, and an extension to earlier hematopoietic precursors is planned. TALENs have been used to enhance the efficacy of therapeutic CAR T cells, and at least two trials using CRISPR-Cas9 for this purpose have been approved. ^[9]

Viral diseases

Genome editing has the potential to become a powerful tool in antiviral therapy. It works by modifying genes that are necessary for viral invasion and replication in host cells. By editing these genes, we can produce virus-resistant immune or stem/progenitor cells that can prevent or alleviate viral diseases.^[10]

CONCLUSION AND DISCUSSION

CRISPR-Cas systems can screen disease-causing mutations and detect viral nucleic acids like SARS- CoV-2, aiding in the diagnosis of rare genetic diseases. Gene editing has the potential to be a rapid and accurate diagnostic tool for diseases. It is most inspiring in the field of gene or cell therapy.

Despite significant progress in clinical applications, the field of gene-editing therapeutics needs to address several issues before we hit the ultimate goal of curing all genetic disorders. Scientists should continue to increase the accuracy and efficiency of existing gene-editing agents in parallel with innovations and developments of novel technologies.

ABBREVIATIONS

CRISPR – Clustered regularly interspaced short palindromic repeats UVR – Ultraviolet radiations

ZFN – Zinc finger nucleases

NHEJ      nonhomologous end joining

TALEN – Transcription activator-like effector nucleases

REFERENCES

• Xu Y, Li CRISPR-Cas systems: Overview, innovations and applications in human disease research and gene therapy. Comput Struct Biotechnol J. 2020 Sep 8;18:2401-2415. doi: 10.1016/j.csbj.2020.08.031. PMID: 33005303; PMCID: PMC7508700.
• Komor AC, Kim YB, Packer MS, Zuris JA, Liu Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016 May 19;533(7603):420-4. doi: 10.1038/nature17946. Epub 2016 Apr 20. PMID: 27096365; PMCID: PMC4873371.
• Rees HA, Liu DR. Base editing: precision chemistry on the genome and transcriptome of living Nat Rev Genet. 2018 Dec;19(12):770-788. doi: 10.1038/s41576-018-0059-1. Erratum in: Nat Rev Genet. 2018 Oct 19;: PMID: 30323312; PMCID: PMC6535181.
• Anzalone, A.V., Randolph, P.B., Davis, J.R. et al. Search-and-replace genome editing without double-strand breaks or donor Nature 576, 149–157 (2019). https://doi.org/10.1038/s41586- 019-1711-4
• Matsoukas IG (2020) Prime Editing: Genome Editing for Rare Genetic Diseases Without Double-Strand Breaks or Donor DNA. Front. Genet. 11:528. doi: 10.3389/fgene.2020.00528

• Dasgupta I, Flotte TR, Keeler CRISPR/Cas-Dependent and Nuclease-Free In Vivo Therapeutic Gene Editing. Hum Gene Ther. 2021 Mar;32(5-6):275-293. doi: 10.1089/hum.2021.013. PMID: 33750221; PMCID: PMC7987363.
• June CH, Sadelain Chimeric Antigen Receptor Therapy. N Engl J Med. 2018 Jul 5;379(1):64-73. doi: 10.1056/NEJMra1706169. PMID: 29972754; PMCID: PMC7433347.
• Li H, Yang Y, Hong W, Huang M, Wu M, Zhao Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects. Signal Transduct Target Ther. 2020 Jan 3;5(1):1. doi: 10.1038/s41392-019-0089-y. PMID: 32296011; PMCID: PMC6946647
• Carroll Genome Editing: Past, Present, and Future. Yale J Biol Med. 2017 Dec 19;90(4):653-659. PMID: 29259529; PMCID: PMC5733845.
• Chen S, Yu X, Guo CRISPR-Cas targeting of host genes as an antiviral strategy. Viruses. 2018;10(1):40.