Components of Gene
Inhibition Therapy

An overview of the components of gene inhibition therapies

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

shRNA
shRNA part 1
shRNA part 2
siRNA
Artificial miRNA
miRNA part 1
miRNA part 2
miRNA

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 action12,14

target mRNA

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 complementarity12,14

target mRNA 2

Artificial miRNAs have partial complementarity with their target mRNAs and induce gene inhibition via translational repression or mRNA degradation or cleavage12,14,15

Delivery Methods for Gene Inhibition

Exogenous shRNA and artificial miRNA can be introduced into cells by one of two delivery vectors10-13,17,22:

The expression cassette contains three main components: a promoter, the shRNA or artificial miRNA cDNA, and a transcription terminal signal16,19

promoter graphic

Promoter

The promoter sequence initiates transcription of shRNA or artificial miRNA and allows for efficient expression11,20. It is upstream of the transcriptional start site20

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 expression20
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

This DNA sequence serves as the transcription template that gives rise to the shRNA or artificial miRNA16,19

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 mRNA20
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

This termination signal at or near the 3’ end of the DNA sequence encoding the shRNA/artificial miRNA functions to end gene transcription16,19,21

The sequence of the termination signal depends on the promoter used to drive the shRNA/artificial miRNA expression16
-For promoters directing pol III activity, the DNA sequence should be 5–6 thymidines (T)16
-For promoters directing pol II activity, the DNA sequence should be AATAAA16

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

  • Transfection of a plasmid encoding the shRNA or artificial miRNA

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 replication23
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 interest17,24
The advantage of this delivery system is to provide high transfection efficiency and a high level of constant expression of the shRNA or artificial miRNA25
Viral vectors that are most commonly used to deliver shRNAs or artificial miRNAs include12,17:

Components Specific to shRNA / Artificial miRNA 
Viral Vectors

promoter graphic 2 Y

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

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

Intracellular Processing of the Expression Cassette

Once inside the cell, the expression cassette gets processed differently depending on the delivery vector17

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 nucleus23,32

Plasmid DNA can be delivered to the nucleus via methods such as transfection or electroporation17

The integrated sequences, or episomal/plasmid DNA, can then be transcribed by RNA polymerase II or III to begin the process of gene inhibition11,17,20

intracellular processing

Summary / Module Recap

In gene therapy, current research is focused on the use of shRNAs and artificial miRNAs for gene inhibition10,33,34

Delivery of shRNAs or artificial miRNAs can be achieved by either viral vector delivery or transfection of a plasmid10-13,17–22

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 backbone11,16-18

The expression cassette contains three main components: a promoter, the shRNA or artificial miRNA cDNA, and a transcription terminal signal16

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