Components of Gene
Inhibition Therapy

An overview of the components of gene inhibition therapies

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shRNA and miRNA

Gene inhibition is a therapeutic approach that involves deactivating or “silencing” the expression of a mutated gene that is not functioning properly1–4

Clinical applications can include myocardial ischemia, HIV, cancer, amyotrophic lateral sclerosis, facioscapulohumeral muscular dystrophy, and Huntington’s disease5–9

Gene inhibition can be achieved using short hairpin RNAs (shRNAs)10 and artificial microRNAs (miRNAs)11 that can express short double-stranded RNAs, which utilize the endogenous cellular pathway to inhibit gene expression10–12

- shRNAs express small interfering RNAs (siRNAs)11,12
- Artificial miRNAs express miRNAs11

Both shRNAs and artificial miRNAs consist of a hairpin structure that contains two complementary RNA sequences, 19–25 base pairs in length, linked by a short loop of 4–11 nucleotides12,13

inhibition-shrna.svg

miRNA, microRNA; shRNA, short hairpin RNA; siRNA, small interfering RNA.

Mechanism of Action of shRNA and Artificial miRNA

  • Although shRNAs and artificial miRNAs have similar physicochemical properties, they have distinct mechanisms of action10,12

The shRNA product, siRNA, has perfect complementarity with its target mRNA. It generally induces gene inhibition via direct cleavage of the target mRNA at the site of complementarity10,12

Artificial miRNAs have partial complementarity with their target mRNAs and induce gene inhibition via translational repression or mRNA degradation or cleavage10,12,14

Figures adapted from Figure 2 in Lam JKW, et al. 201510.
miRNA, microRNA; mRNA, messenger RNA; shRNA, short hairpin RNA; siRNA, small interfering RNA.

Delivery of shRNA / Artificial miRNA

Exogenous shRNAs or artificial miRNAs can be introduced into cells via an shRNA/artificial miRNA expression cassette in a delivery vector11,15-17

The expression cassette contains three main components: a promoter, the shRNA or artificial miRNA cDNA, and a transcription terminal signal15,18

inhibition-vectorcas@2x

The promoter sequence initiates transcription of shRNA or artificial miRNA and allows for efficient expression11,19

It is upstream of the transcriptional start site19

This DNA sequence serves as the transcription template that gives rise to the shRNA or artificial miRNA15,16,18

This termination signal at or near the 3’ end of the DNA sequence encoding the shRNA/artificial miRNA functions to end gene transcription15,18,20

Promoter

Polymerase III (pol III) promoters (e.g. H1 and U6) are most commonly used for transcription of shRNAs due to their compact size and ubiquitous expression19

Polymerase II (pol II) promoters (e.g. CMV) are frequently used for transcription of artificial miRNAs11

An advantage of pol II promoters over pol III promoters is the ability for regulated and tissue-specific expression11

shRNA / Artificial miRNA cDNA

The transcribed shRNA from the cDNA construct consists of a 21- to 23-nucleotide sense sequence that is identical to the target mRNA sequence, a 9-base pair loop, and a 21- to 23-nucleotide antisense sequence that is complementary to the target mRNA19

The artificial miRNA transcribed from the cDNA construct is a primary miRNA transcript that gets processed to a ~22-nucleotide long double-stranded miRNA5

Termination signal

The sequence of the termination signal depends on the promoter used to drive the shRNA/artificial miRNA expression15

-For promoters directing pol III activity, the DNA sequence should be 5–6 thymidines (T)15

-For promoters directing pol II activity, the DNA sequence should be AATAAA15

Figure created based on content from Tiscornia G, et al. Chapter 3 – Development of lentiviral vectors expressing siRNA. In: Friedmann T, Ross J (eds). Gene Transfer: Delivery and Expression of DNA and RNA, A Laboratory Manual. 1st edn. New York: Cold Spring Harbor Laboratory Press, 2006:23–34 and Singer O, Verma IM. Curr Gene Ther 2008;8(6):483–488.
cDNA, complementary DNA; miRNA, microRNA; shRNA, short hairpin RNA.

A, adenosine; cDNA, complementary DNA; CMV, cytomegalovirus; miRNA, microRNA; mRNA, messenger RNA; shRNA, short hairpin RNA.

Delivery Methods for Gene Inhibition

Exogenous shRNA and artificial miRNA can be introduced into cells by one of two delivery vectors10-13,16,21:

inhibition-delivery1.svg

Viral vector delivery of gene expression cassettes that express shRNA or artificial miRNA

inhibition-delivery2.svg

Transfection of a plasmid encoding the shRNA or artificial miRNA

miRNA, microRNA; shRNA, short hairpin RNA.

inhibition-delivery1.svg

Viral Vector Delivery of Gene Expression Cassettes

Viral vectors are naturally occurring biological agents that have evolved to deliver their nucleic acid into a host cell for replication22

Viral vectors used in gene therapy have been genetically modified to be replication incompetent; the non-essential viral genes are replaced by the therapeutic gene of interest16,23

The advantage of this delivery system is to provide high transfection efficiency and a high level of constant expression of the shRNA or artificial miRNA24

Viral vectors that are most commonly used to deliver shRNAs or artificial miRNAs include12,16:

Lentiviruses
Lentiviruses
Retroviruses
Retroviruses
Adenoviruses
Adenoviruses
Adeno-associated <br>viruses (AAVs)
Adeno-associated
viruses (AAVs)

miRNA, microRNA; shRNA, short hairpin RNA.

Components Specific to shRNA / Artificial miRNA Viral Vectors

inhibition-vectorca2@2x

Inverted terminal repeats (ITRs) or long terminal repeats (LTRs) flank the silencing cassette. LTRs are present in lentiviral and retroviral vectors and facilitate integration of the viral transfer vector into the host genome. ITRs are T-shaped sequences present in adenoviruses and recombinant adeno-associated viruses providing the viral origins of replication; ITRs contain the packaging signal26-29

Inverted terminal repeats (ITRs) or long terminal repeats (LTRs) flank the silencing cassette. LTRs are present in lentiviral and retroviral vectors and facilitate integration of the viral transfer vector into the host genome. ITRs are T-shaped sequences present in adenoviruses and recombinant adeno-associated viruses providing the viral origins of replication; ITRs contain the packaging signal26-29

cDNA, complementary DNA; miRNA, microRNA; shRNA, short hairpin RNA.

inhibition-delivery2.svg

Transfection of Plasmid for Delivery
of shRNA / Artificial miRNA

Plasmids consist of a circular, double-stranded DNA molecule that can be customized to encode for one or more transcripts that produce RNAi and/or proteins30,31

Plasmids have lower immunogenicity compared with viral vectors making them an attractive alternative for the delivery of shRNAs and artificial miRNAs12,30

However, they demonstrate poor gene transfer efficiency30

Example of a Plasmid-Delivered Gene Inhibition Therapy
transfection plasmid

Figure adapted from Figure 1a in Ghisoli M, et al. 201631.
CMV, cytomegalovirus; huGM-CSF2, human granulocyte-macrophage colony-stimulating factor 2; kan, kanamycin; miRNA, microRNA; Ori, origin of replication; poly A, polyadenylation; RNAi, RNA interference; sh-Fur, furin shRNA; shRNA, short hairpin RNA.

Intracellular Processing of the Expression Cassette

Once inside the cell, the expression cassette gets processed differently depending on the delivery vector16

Retroviral and lentiviral vectors integrate their genetic material into the host genome, whereas genetic material from AAV vectors is generally maintained as an episome in the target cell nucleus22,32

Plasmid DNA can be delivered to the nucleus via methods such as transfection or electroporation16

The integrated sequences, or episomal/plasmid DNA, can then be transcribed by RNA polymerase II or III to begin the process of gene inhibition11,16,19

inhibition-intra.svg

Figure adapted from Figure 1 in Perwitasari O, et al. 201317.
AAV, adeno-associated virus; miRNA, microRNA; shRNA, short hairpin RNA.

Summary / Module Recap

In gene therapy, current research is focused on the use of shRNAs and artificial miRNAs for gene inhibition14,32,33

Delivery of shRNAs or artificial miRNAs can be achieved by either viral vector delivery or transfection of a plasmid10-13,16,18

The main components of the viral vectors and plasmids used in gene inhibition include an shRNA /artificial miRNA expression cassette and the viral vector or plasmid backbone11,15-17

The expression cassette contains three main components: a promoter, the shRNA or artificial miRNA cDNA, and a transcription terminal signal15

References

  1. Wang D, Gao G. Discov Med 2014;18(98):151–161.
  2. Strachan T, Read AP. Genetic approaches to treating disease: In: Human Molecular Genetics. 5th ed. Florida: CRC Press, 2018:696–699.
  3. NCBI. Gene silencing. Available at: https://www.ncbi.nlm.nih.gov/probe/docs/applsilencing/. Accessed March 11, 2020.
  4. Sathyajith D. News Medical Life Sciences. Short Hairpin RNA (shRNA) Interference: Therapeutic Applications. Available at: https://www.news-medical.net/life-sciences/Short-Hairpin-RNA-(shRNA)-Interference-Therapeutic-Applications.aspx#Therapeutic%20applications%20of%20shRNA. Accessed March 11, 2020.
  5. Borel F, et al. Mol Ther 2014;22(4):692–701.
  6. Wolstein O, et al. Mol Ther Methods Clin Dev 2014;1:11.
  7. Stoica L, et al. Ann Neurol 2016;79(4):687–700.
  8. Wallace LM, et al. Mol Ther 2012;20(7):1417–1423.
  9. Spronck EA, et al. Mol Ther Methods Clin Dev 2019;13:334–343.
  10. Sliva K, Schnierle BS. Virol J 2010;7:248.
  11. Herrera-Carrillo E, et al. Hum Gene Ther Methods 2017;28(4):177–190.
  12. Lam JKW, et al. Mol Ther Nucleic Acids 2015;4:e252.
  13. Moore CB, et al. Methods Mol Biol 2010;629:141–158.
  14. Merlin S, Follenzi AT. Mol Ther Methods Clin Dev 2019;12:223–232.
  15. Davidson BL, Harper SQ. Methods Enzymol 2005;392:145–173.
  16. Perwitasari O, et al. Pharmaceuticals (Basel) 2013;6(2):124–160.
  17. Addgene. Molecular biology reference. Available at: https://www.addgene.org/mol-bio-reference/#origins. Accessed March 11, 2020.
  18. Fan J, et al. Cancer Gene Ther 2019. doi: 10.1038/s41417-019-0113-y [Epub ahead of print].
  19. Tiscornia G, et al. Chapter 3 – Development of lentiviral vectors expressing siRNA. In: Friedmann T, Ross J (eds). Gene Transfer: Delivery and Expression of DNA and RNA, A Laboratory Manual. 1st edn. New York: Cold Spring Harbor Laboratory Press, 2006:23–34.
  20. Hutson TH, et al. Hum Gene Ther Methods 2014;25(1):14–32.
  21. Jin HY, et al. Front Genet 2015;6:340.
  22. Naso MF, et al. BioDrugs 2017;31:317–334.
  23. Bouard D, et al. Br J Pharmacol 2009;157:153–165.
  24. Yang N. Int J Pharm Investig 2015;5(4):179–181.
  25. Addgene. γ-Retrovirus Guide. Available at: https://www.addgene.org/viral-vectors/retrovirus/retro-guide/. Accessed March 11, 2020.
  26. Addgene. Lentiviral Guide. Available at: https://www.addgene.org/viral-vectors/lentivirus/lenti-guide/. Accessed March 11, 2020.
  27. Wang D, et al. Nat Rev Drug Discov 2019;18(5):358–378.
  28. Addgene. Adenoviral Guide. Available at: https://www.addgene.org/guides/adenovirus/. Accessed March 11, 2020.
  29. Williams PD, Kingston PA. Cardiovasc Res 2011;91:565–576.
  30. Ghisoli M, et al. Mol Ther 2016;24(8):1478–1483.
  31. Lukashev AN, Zamyatnin Jr AA. Biochem (Moscow) 2016;81(7):700–708.
  32. Wang J, et al. AAPS J 2010;12(4):492–503.
  33. U.S. FDA. Approved Cellular and Gene Therapy Products. Available at: https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/approved-cellular-and-gene-therapy-products. Accessed March 11, 2020.