Short hairpin RNA

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Lentiviral delivery of shRNA and the mechanism of RNA interference in mammalian cells. ShRNA Lentivirus.svg
Lentiviral delivery of shRNA and the mechanism of RNA interference in mammalian cells.

A short hairpin RNA or small hairpin RNA (shRNA/Hairpin Vector) is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). [1] [2] Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors. shRNA is an advantageous mediator of RNAi in that it has a relatively low rate of degradation and turnover. However, it requires use of an expression vector, which has the potential to cause side effects in medicinal applications. [3]

Contents

The promoter choice is essential to achieve robust shRNA expression. At first, polymerase III promoters such as U6 and H1 were used; however, these promoters lack spatial and temporal control. [3] As such, there has been a shift to using polymerase II promoters, which are inducible, to regulate shRNA expression.

Delivery

Expression of shRNA in cells can be obtained by delivery of plasmids or through viral or bacterial vectors.

Delivery of plasmids to cells through transfection to obtain shRNA expression can be accomplished using commercially available reagents in vitro. However, this method is not applicable in vivo and thus has limited utility.

Use of a bacterial vector to obtain shRNA expression in cells is a relatively recent approach. It builds off research showing that recombinant Escherichia coli , containing a plasmid with shRNA, fed to mice can knock-down target gene expression in the intestinal epithelium. [4] This approach was used in 2012 in clinical trials to try to treat patients with Familial Adenomatous Polyposis. [5]

A variety of viral vectors can be used to obtain shRNA expression in cells including adeno-associated viruses (AAVs), adenoviruses, and lentiviruses. With adeno-associated viruses and adenoviruses, the genomes remain episomal. This is advantageous as insertional mutagenesis is avoided. It is disadvantageous in that the progeny of the cell will lose the virus quickly through cell division unless the cell divides very slowly. AAVs differ from adenoviruses in that the viral genes have been removed and they have diminished packing capacity. Lentiviruses integrate into sections of transcriptionally active chromatin and are thus passed on to progeny cells. With this approach there is increased risk of insertional mutagenesis; however, the risk can be reduced by using an integrase-deficient lentivirus. [6]

The dicer protein from Giardia intestinalis. 2ffl-by-domain.png
The dicer protein from Giardia intestinalis .

Mechanism of action

Once the vector has integrated into the host genome, the shRNA is then transcribed in the nucleus by polymerase II or polymerase III depending on the promoter choice. The product mimics pri-microRNA (pri-miRNA) and is processed by Drosha. The resulting pre-shRNA is exported from the nucleus by Exportin 5. This product is then processed by Dicer and loaded into the RNA-induced silencing complex (RISC). The sense (passenger) strand is degraded. The antisense (guide) strand directs RISC to mRNA that has a complementary sequence. In the case of perfect complementarity, RISC cleaves the mRNA. In the case of imperfect complementarity, RISC represses translation of the mRNA. In both of these cases, the shRNA leads to target gene silencing.

Applications in gene therapy

Due to the ability of shRNA to provide specific, long-lasting, gene silencing there has been great interest in using shRNA for gene therapy applications. Three examples of shRNA-based therapies are discussed below.

Gradalis, Inc. developed the FANG vaccine, which is used in treatment of advanced cancers. FANG relies on a bifunctional shRNA (bi-shRNA) against the immunosuppressive transforming growth factors (TGF) β1 and β2. [8] Autologous tumor cells were harvested from patients and a plasmid encoding the bifunctional shRNA and granulocyte-macrophage colony-stimulating factor (GMCSF) was introduced ex vivo through electroporation. These cells were later irradiated and injected back into the patient.

Marina Biotech developed CEQ508 which is used to treat Familial Adenomatous Polyposis. CEQ508 uses a bacterial vector to deliver shRNA against β-catenin.

Gradalis, Inc. developed bifunctional shRNA-STMN1 (pbi-shRNA STMN1), which is used to treat advanced and/or metastatic cancers. This pbi-shRNA STMN1 is against stathmin 1 and is delivered intratumorally through bilamellar invaginated vesicle (BIV) lipoplex (LP) technology.

Several challenges typically confront shRNA-based therapeutics. The most significant challenge is delivery. shRNA is typically delivered through use of a vector, and although they are generally efficient, they pose significant safety concerns. In particular, viral based gene therapy approaches have proved dangerous in past clinical trials. In the first generation of retro viral gene therapy, some patients treated with viral vectors for Wiskott–Aldrich syndrome developed acute T-cell leukaemia. This was determined to have been caused by viral vector insertion location. [9] Potential oversaturation of RISC is also a problem. If the shRNA is expressed at levels that are too high, the cell might not be able to correctly process the endogenous RNA which could cause significant problems. Another challenge is the possibility that the patient might mount an immune response against the therapy. [10] Finally, there might be off-target effects and the shRNA could silence other unintended genes. In developing successful new shRNA-based therapeutics, all of these challenges must be taken into account.

See also

Related Research Articles

Gene silencing is the regulation of gene expression in a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation and is often used in research. In particular, methods used to silence genes are being increasingly used to produce therapeutics to combat cancer and other diseases, such as infectious diseases and neurodegenerative disorders.

<span class="mw-page-title-main">Small interfering RNA</span> Biomolecule

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA at first non-coding RNA molecules, typically 20–24 base pairs in length, similar to miRNA, and operating within the RNA interference (RNAi) pathway. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation.

<span class="mw-page-title-main">Dicer</span> Enzyme that cleaves double-stranded RNA (dsRNA) into short dsRNA fragments

Dicer, also known as endoribonuclease Dicer or helicase with RNase motif, is an enzyme that in humans is encoded by the DICER1 gene. Being part of the RNase III family, Dicer cleaves double-stranded RNA (dsRNA) and pre-microRNA (pre-miRNA) into short double-stranded RNA fragments called small interfering RNA and microRNA, respectively. These fragments are approximately 20–25 base pairs long with a two-base overhang on the 3′-end. Dicer facilitates the activation of the RNA-induced silencing complex (RISC), which is essential for RNA interference. RISC has a catalytic component Argonaute, which is an endonuclease capable of degrading messenger RNA (mRNA).

Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: "transformation" is typically used to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells, including plant cells. In animal cells, transfection is the preferred term as transformation is also used to refer to progression to a cancerous state (carcinogenesis) in these cells. Transduction is often used to describe virus-mediated gene transfer into eukaryotic cells.

<span class="mw-page-title-main">Antisense RNA</span>

Antisense RNA (asRNA), also referred to as antisense transcript, natural antisense transcript (NAT) or antisense oligonucleotide, is a single stranded RNA that is complementary to a protein coding messenger RNA (mRNA) with which it hybridizes, and thereby blocks its translation into protein. The asRNAs have been found in both prokaryotes and eukaryotes, and can be classified into short and long non-coding RNAs (ncRNAs). The primary function of asRNA is regulating gene expression. asRNAs may also be produced synthetically and have found wide spread use as research tools for gene knockdown. They may also have therapeutic applications.

The RNA-induced silencing complex, or RISC, is a multiprotein complex, specifically a ribonucleoprotein, which functions in gene silencing via a variety of pathways at the transcriptional and translational levels. Using single-stranded RNA (ssRNA) fragments, such as microRNA (miRNA), or double-stranded small interfering RNA (siRNA), the complex functions as a key tool in gene regulation. The single strand of RNA acts as a template for RISC to recognize complementary messenger RNA (mRNA) transcript. Once found, one of the proteins in RISC, Argonaute, activates and cleaves the mRNA. This process is called RNA interference (RNAi) and it is found in many eukaryotes; it is a key process in defense against viral infections, as it is triggered by the presence of double-stranded RNA (dsRNA).

A DNA construct is an artificially-designed segment of DNA borne on a vector that can be used to incorporate genetic material into a target tissue or cell. A DNA construct contains a DNA insert, called a transgene, delivered via a transformation vector which allows the insert sequence to be replicated and/or expressed in the target cell. This gene can be cloned from a naturally occurring gene, or synthetically constructed. The vector can be delivered using physical, chemical or viral methods. Typically, the vectors used in DNA constructs contain an origin of replication, a multiple cloning site, and a selectable marker. Certain vectors can carry additional regulatory elements based on the expression system involved.

Lentivirus is a genus of retroviruses that cause chronic and deadly diseases characterized by long incubation periods, in humans and other mammalian species. The genus includes the human immunodeficiency virus (HIV), which causes AIDS. Lentiviruses are distributed worldwide, and are known to be hosted in apes, cows, goats, horses, cats, and sheep as well as several other mammals.

<span class="mw-page-title-main">Viral vector</span> Biotechnology to deliver genetic material into a cell

Viral vectors are tools commonly used by molecular biologists to deliver genetic material into cells. This process can be performed inside a living organism or in cell culture. Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect. Delivery of genes or other genetic material by a vector is termed transduction and the infected cells are described as transduced. Molecular biologists first harnessed this machinery in the 1970s. Paul Berg used a modified SV40 virus containing DNA from the bacteriophage λ to infect monkey kidney cell maintained in culture.

<span class="mw-page-title-main">Gene delivery</span> Introduction of foreign genetic material into host cells

Gene delivery is the process of introducing foreign genetic material, such as DNA or RNA, into host cells. Gene delivery must reach the genome of the host cell to induce gene expression. Successful gene delivery requires the foreign gene delivery to remain stable within the host cell and can either integrate into the genome or replicate independently of it. This requires foreign DNA to be synthesized as part of a vector, which is designed to enter the desired host cell and deliver the transgene to that cell's genome. Vectors utilized as the method for gene delivery can be divided into two categories, recombinant viruses and synthetic vectors.

RNA silencing or RNA interference refers to a family of gene silencing effects by which gene expression is negatively regulated by non-coding RNAs such as microRNAs. RNA silencing may also be defined as sequence-specific regulation of gene expression triggered by double-stranded RNA (dsRNA). RNA silencing mechanisms are conserved among most eukaryotes. The most common and well-studied example is RNA interference (RNAi), in which endogenously expressed microRNA (miRNA) or exogenously derived small interfering RNA (siRNA) induces the degradation of complementary messenger RNA. Other classes of small RNA have been identified, including piwi-interacting RNA (piRNA) and its subspecies repeat associated small interfering RNA (rasiRNA).

In molecular cloning, a vector is any particle used as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.

<span class="mw-page-title-main">Stable nucleic acid lipid particle</span>

Stable nucleic acid lipid particles (SNALPs) are microscopic particles approximately 120 nanometers in diameter, smaller than the wavelengths of visible light. They have been used to deliver siRNAs therapeutically to mammals in vivo. In SNALPs, the siRNA is surrounded by a lipid bilayer containing a mixture of cationic and fusogenic lipids, coated with diffusible polyethylene glycol.

<span class="mw-page-title-main">RNA interference</span> Biological process of gene regulation

RNA interference (RNAi) is a biological process in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA, through translational or transcriptional repression. Historically, RNAi was known by other names, including co-suppression, post-transcriptional gene silencing (PTGS), and quelling. The detailed study of each of these seemingly different processes elucidated that the identity of these phenomena were all actually RNAi. Andrew Fire and Craig C. Mello shared the 2006 Nobel Prize in Physiology or Medicine for their work on RNAi in the nematode worm Caenorhabditis elegans, which they published in 1998. Since the discovery of RNAi and its regulatory potentials, it has become evident that RNAi has immense potential in suppression of desired genes. RNAi is now known as precise, efficient, stable and better than antisense therapy for gene suppression. Antisense RNA produced intracellularly by an expression vector may be developed and find utility as novel therapeutic agents.

<span class="mw-page-title-main">Vectors in gene therapy</span>

Gene therapy utilizes the delivery of DNA into cells, which can be accomplished by several methods, summarized below. The two major classes of methods are those that use recombinant viruses and those that use naked DNA or DNA complexes.

<span class="mw-page-title-main">Reverse genetics</span> Method in molecular genetics

Reverse genetics is a method in molecular genetics that is used to help understand the function(s) of a gene by analysing the phenotypic effects caused by genetically engineering specific nucleic acid sequences within the gene. The process proceeds in the opposite direction to forward genetic screens of classical genetics. While forward genetics seeks to find the genetic basis of a phenotype or trait, reverse genetics seeks to find what phenotypes are controlled by particular genetic sequences.

<span class="mw-page-title-main">Cas9</span> Microbial protein found in Streptococcus pyogenes M1 GAS

Cas9 is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in genetic engineering applications. Its main function is to cut DNA and thereby alter a cell's genome. The CRISPR-Cas9 genome editing technique was a significant contributor to the Nobel Prize in Chemistry in 2020 being awarded to Emmanuelle Charpentier and Jennifer Doudna.

Adenovirus varieties have been explored extensively as a viral vector for gene therapy and also as an oncolytic virus.

<span class="mw-page-title-main">Lentiviral vector in gene therapy</span>

Lentiviral vectors in gene therapy is a method by which genes can be inserted, modified, or deleted in organisms using lentiviruses.

DNA-directed RNA interference (ddRNAi) is a gene-silencing technique that utilizes DNA constructs to activate an animal cell's endogenous RNA interference (RNAi) pathways. DNA constructs are designed to express self-complementary double-stranded RNAs, typically short-hairpin RNAs, that bring about the silencing of a target gene or genes once processed. Any RNA, including endogenous messenger RNA (mRNAs) or viral RNAs, can be silenced by designing constructs to express double-stranded RNA complementary to the desired mRNA target.

References

  1. ↑ Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS (April 2002). "Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells". Genes & Development. 16 (8): 948–58. doi:10.1101/gad.981002. PMC   152352 . PMID   11959843.
  2. ↑ Brummelkamp TR, Bernards R, Agami R (April 2002). "A system for stable expression of short interfering RNAs in mammalian cells". Science. 296 (5567): 550–3. Bibcode:2002Sci...296..550B. doi:10.1126/science.1068999. hdl: 1874/15573 . PMID   11910072.
  3. 1 2 Wang Z, Rao DD, Senzer N, Nemunaitis J (December 2011). "RNA interference and cancer therapy". Pharmaceutical Research. 28 (12): 2983–95. doi:10.1007/s11095-011-0604-5. PMID   22009588. S2CID   36738846.
  4. ↑ Xiang S, Fruehauf J, Li CJ (June 2006). "Short hairpin RNA-expressing bacteria elicit RNA interference in mammals". Nature Biotechnology. 24 (6): 697–702. doi:10.1038/nbt1211. PMID   16699500. S2CID   17700388.
  5. ↑ Burnett JC, Rossi JJ, Tiemann K (September 2011). "Current progress of siRNA/shRNA therapeutics in clinical trials". Biotechnology Journal. 6 (9): 1130–46. doi:10.1002/biot.201100054. PMC   3388104 . PMID   21744502.
  6. ↑ Lombardo A, Genovese P, Beausejour CM, Colleoni S, Lee YL, Kim KA, Ando D, Urnov FD, Galli C, Gregory PD, Holmes MC, Naldini L (November 2007). "Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery". Nature Biotechnology. 25 (11): 1298–306. doi:10.1038/nbt1353. PMID   17965707. S2CID   16008349.
  7. ↑ Macrae IJ, Zhou K, Li F, Repic A, Brooks AN, Cande WZ, Adams PD, Doudna JA (January 2006). "Structural basis for double-stranded RNA processing by Dicer". Science. 311 (5758): 195–8. Bibcode:2006Sci...311..195M. doi:10.1126/science.1121638. PMID   16410517. S2CID   23785494.
  8. ↑ Senzer N, Barve M, Kuhn J, Melnyk A, Beitsch P, Lazar M, Lifshitz S, Magee M, Oh J, Mill SW, Bedell C, Higgs C, Kumar P, Yu Y, Norvell F, Phalon C, Taquet N, Rao DD, Wang Z, Jay CM, Pappen BO, Wallraven G, Brunicardi FC, Shanahan DM, Maples PB, Nemunaitis J (March 2012). "Phase I trial of "bi-shRNAi(furin)/GMCSF DNA/autologous tumor cell" vaccine (FANG) in advanced cancer". Molecular Therapy. 20 (3): 679–86. doi:10.1038/mt.2011.269. PMC   3293620 . PMID   22186789.
  9. ↑ Persons DA, Baum C (February 2011). "Solving the problem of γ-retroviral vectors containing long terminal repeats". Molecular Therapy. 19 (2): 229–31. doi:10.1038/mt.2010.305. PMC   3034864 . PMID   21289636.
  10. ↑ Whitehead KA, Dahlman JE, Langer RS, Anderson DG (2011). "Silencing or stimulation? siRNA delivery and the immune system". Annual Review of Chemical and Biomolecular Engineering. 2: 77–96. doi:10.1146/annurev-chembioeng-061010-114133. PMID   22432611.
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