How Does Gene Therapy Work?

Gene Therapy Techniques

Just how does gene therapy work? In this part of genehome is more specific information about gene therapy techniques. These techniques help researchers and healthcare professionals to find ways to treat diseases at the genetic level.

Gene therapy techniques

What is gene addition?
Gene addition is a common method being explored for monogenic diseases. This gene therapy technique usually involves the insertion of functional (or healthy) copies of a gene into a person’s cells by way of a vector. Vectors deliver the functional gene to the patient’s cells, either in vivo or ex vivo.
in vivo
ex vivo

Gene addition is a common method being explored for monogenic diseases. This gene therapy technique usually involves the insertion of functional (or healthy) copies of a gene into a person’s cells by way of a vector.1,2 Vectors deliver the functional gene to the patient’s cells, either in vivo or ex vivo.3

 Next Level Knowledge

The goal of gene addition is for the newly inserted gene, known as the Transgenea portion of DNA from one organism inserted into the genome of another organism

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, to perform an essential function to modify the effects of the mutated gene (which causes a genetic disease).4 This usually involves the transgene producing a specific protein that is essential to the body’s overall health. Depending on the disease and target cells, the new genetic material may be inserted into the nucleus of the cell or integrated into the cell’s DNA.5

  • In Complex genetic diseasesdiseases in which changes occur in two or more genes

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    and Non-genetic diseasesdiseases that are not attributed to genetics

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    diseases, ongoing gene addition research is focused on adding a functional copy of a mutated gene in order to compensate for (or circumvent) the dysfunctional disease pathway. Gene addition therapy is being explored for diseases including hemoglobinopathies, neurological diseases, metabolic diseases, and central nervous system diseases.6-9
Image of gene addition gene therapy technique

What is gene editing?
Gene editing involves the creation of targeted double-stranded breaks in DNA, with or without instructions to repair them. The goal of gene editing is to either:
  • disrupt or inactivate genetic material OR
  • correct or insert genetic material
In other words, nucleases cause breaks in genetic material that is not working properly so that it can come back together in a way that allows the genetic material to repair itself.

Gene editing involves the creation of targeted double-stranded breaks in DNA, with or without instructions to repair them. The goal of gene editing is to either 10, 11:

  • disrupt or inactivate genetic material OR
  • correct or insert genetic material

In other words, nucleases cause breaks in genetic material that is not working properly so that it can come back together in a way that allows the genetic material to repair itself.10, 11

 Next Level Knowledge

Nucleases being explored for clinical development include Meganucleasesnatural proteins that recognize long DNA sequences. They target a specific site on a gene to make a break or dent to transfer their coding sequence to the gene

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Zinc Finger Nucleasesartificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain; ZFNs are using gene editing applications

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(ZFNs), transcription activator-like effector-based nucleases Transcription activator-like effector-based nucleases (TALEN)an array of single-protein modules, or nuclease, where each module recognizes a single DNA base pair. These nucleases are derived from transcription activator-like effectors, which means they are derived from plant bacteria

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, MegaTALScombination of meganucleases and transcription activator-like effector nuclease (TALEN)

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, and the clustered regularly interspaced short palindromic repeats Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9a laboratory tool that uses a specially designed RNA molecule to guide a Cas9 enzyme to a specific sequence of DNA so it can change or edit pieces of it

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system.11

What is gene editing through gene disruption or inactivation?
Another area of gene therapy under development is gene inactivation, sometimes referred to as gene silencing, knockdown, or knockout. This is when scientists turn off (or down) an existing gene to help address a genetically related health issue. This technique may be used to reduce expression of a protein that is overexpressed. For example, gene inactivation may be used to stop activity from a specific gene in order to allow for activation of the desired therapeutic effect.

Another area of gene therapy under development is Gene inactivationan approach in gene therapy that turns off or reduces the function of a gene in order to have a therapeutic effect

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, sometimes referred to as gene silencing, knockdown, or knockout. This is when scientists turn off (or down) an existing gene to help address a genetically related health issue. This technique may be used to reduce expression of a protein that is overexpressed. For example, gene inactivation may be used to stop activity from a specific gene in order to allow for activation of the desired therapeutic effect.12

 Next Level Knowledge

In gene inactivation, a specially engineered molecule is inserted into the cell that can effectively prevent the target gene from expressing itself. Research is ongoing to create gene inactivation therapy that can combat cancer, blood diseases, infectious diseases, and neurodegenerative diseases. Gene inactivation also holds potential for silencing a damaging gene so that a recessive trait, or Phenotypeobservable characteristics or traits (like blood type or eye color) that come from genotype or environment

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, can be expressed instead. 12-15

Image of gene knockout, also known as gene inactivation, gene therapy technique

What is gene editing through gene correction or repair?
Gene correction is a technique currently in preclinical studies. It is used to recognize and form a break in the DNA, then insert new genetic material to override the faulty gene. The success of this technique is contingent on directing a gene to the correct cells. Delivery models and techniques for gene correction are currently being explored.

Gene correction is a technique currently in Preclinical Studya required study that measures the safety of a medication in animals before it is allowed to be tested in people

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. It is used to recognize and form a break in the DNA, then insert new genetic material to override the faulty gene. The success of this technique is contingent on directing a gene to the correct cells. Delivery models and techniques for gene correction are currently being explored.16

Image of gene repair, a gene editing technique
Gene-ius Questions

You may have heard about a specific gene editing technique called Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9a laboratory tool that uses a specially designed RNA molecule to guide a Cas9 enzyme to a specific sequence of DNA so it can change or edit pieces of it

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, the clustered regularly interspaced short palindromic repeats system. Utilizing the CRISPR/Cas9 system for gene editing requires a lab to design a small piece of RNA to target a sequence of DNA in a genome. The modified RNA is then bound with the Cas9 enzyme and inserted in the cells ex vivo or in vivo. Inside the cells, the modified RNA is able to recognize the DNA sequence and the Cas9 enzyme can cut the DNA in the targeted location. Once the DNA is cut, cells can use their own DNA repair machinery to add or delete pieces of genetic material, or to replace an existing segment to make changes to the DNA.17 

How is genetic material delivered to cells?
For any type of gene therapy to work, whether making a gene inactive or adding/correcting a gene, the genetic material needs to get inside the cells of the person with the disease. Delivery can be either inside the body or outside—either method can be used in both men and women:
  • Ex vivo gene therapy refers to the process of genetically altering a person’s cells outside of the body and then transplanting them back in
    • Today, ex vivo gene therapy techniques are most frequently applied to hematopoietic stem cells (HSCs), which are relevant to blood and immunological diseases and genetic diseases that affect tissues and organs easily accessible by blood cells
  • In vivo gene therapy refers to direct administration either intravenously, known as systemic administration, or locally to a specific organ of interest (eg, eye, muscle)
    • In vivo delivery has been proven in many areas of research. Some of the currently approved gene therapies deliver genetic material in vivo. Targeted in vivo gene therapy will continue to evolve as scientists continue to experiment with additional methods of gene delivery

For any type of gene therapy to work, whether making a gene inactive or adding/correcting a gene, the genetic material needs to get inside the cells of the person with the disease. Delivery can be either inside the body or outside—either method can be used in both men and women:

  • Ex vivooutside the body

    See glossary for more terms >
    gene therapy refers to the process of genetically altering a person’s cells outside of the body and then transplanting them back in18, 19
    • Today, ex vivo gene therapy techniques are most frequently applied to hematopoietic stem cells (HSCs), which are relevant to blood and immunological diseases and genetic diseases that affect tissues and organs easily accessible by blood cells 
  • In vivoinside the body

    See glossary for more terms >
     gene therapy refers to direct administration either intravenously, known as systemic administration, or locally to a specific organ of interest (eg, eye, muscle)19
    • In vivo delivery has been proven in many areas of research. Some of the currently approved gene therapies deliver genetic material in vivo. Targeted in vivo gene therapy will continue to evolve as scientists continue to experiment with additional methods of gene delivery
Example of ex vivo gene therapy and in vivo gene therapy

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References

1. FDA Commissioner. What is gene therapy? How does it work? US Food and Drug Administration. Accessed March 1, 2020. https://www.fda.gov/consumers/consumer-updates/what-gene-therapy-how-does-it-work 2. Collins M, Thrasher A. Gene therapy: progress and predictions. Proc Biol Sci. 2015;282(1821):20143003. 3. STAT Reports. The STAT guide to viral vectors, the linchpin of gene therapy. STAT News; 2019. 4. Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis, 7th edition. W.H. Freeman; 2000. 5. Mali S. Delivery systems for gene therapy. Indian J Hum Genet. 2013;19(1):3-8. 6. Loera-Valencia R, Piras A, Ismail MAM, et al. Targeting Alzheimer’s disease with gene and cell therapies. J Intern Med. 2018;284(1):2-36. 7. Coune PG, Schneider BL, Aebischer P. Parkinson’s disease: gene therapies. Cold Spring Harb Perspect Med. 2012;2(4):a009431. 8. Chandler RJ, Venditti CP. Gene therapy for metabolic diseases. Transl Sci Rare Dis. 2016;1(1):73-89. 9. Canver MC, Orkin SH. Customizing the genome as therapy for the B-hemoglobinopathies. Blood. 2016;127(21):2536-2545. 10. Yanik M, Müller B, Song F, et al. In vivo genome editing as a potential treatment strategy for inherited retinal dystrophies. Prog Retin Eye Res. 2017;56:1‐18. 11. Guha TK, Wai A, Hausner G. Programmable genome editing tools and their regulation for efficient genome engineering. Comput Struct Biotechnol J. 2017;15:146-160. 12. Koshravi MA, Abbasalipour M, Concordet J-P, et al. Targeted deletion of BCL11A gene by CRISPR-Cas9 system for fetal hemoglobin reactivation: a promising approach for gene therapy of beta thalassemia disease. Eur J Pharmacol. 2019;854:398-405. 13. Choi J. Huntington’s Outreach Project for Education, at Stanford: Gene Silencing. Accessed March 1, 2020. https://hopes.stanford.edu/gene-silencing/ 14. Balasubramanian S, Habegger L, Frankish A, MacArthur DG, et al. Gene inactivation and its implications for annotation in the era of personal genomics. Genes Dev. 2011;25(1):1-10. 15. Grant SG, Campbell, CE, Duff C, Toth SL, Worton RG. Gene inactivation as a mechanism for the expression of recessive phenotypes. Am J Hum Genet. 1989;45(4):619-634. 16. Pandey P, Balekar N. Chapter 4.5.1: Replacement Therapy. In: Grumezescu A, ed. Drug Targeting and Stimuli Sensitive Drug Delivery Systems. 2018. Accessed March 1, 2020. https://www.sciencedirect.com/science/article/pii/B9780128136898000045 17. National Institutes of Health. US National Library of Medicine. Genetics Home Reference. Genomic Research. Accessed May 1, 2020. https://ghr.nlm.nih.gov/primer/genomicresearch/genomeediting 18. Keeler AM, ElMallah MK, Flotte TR. Gene therapy 2017: progress and future directions. Clin Transl Sci. 2017:10(4):242-248. 19. American Society of Gene & Cell therapy (ASGCT). Gene and cell therapy FAQs. Accessed March 1, 2020. https://www.asgct.org/education/more-resources/gene-and-cell-therapy-faqs.

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