shRNA-The Stealthy Silencers-Unleashing Their Power in Gene Regulation and Disease Therapy

Introduction of shRNA

Short hairpin RNA (shRNA) is a term referring to RNA molecules that employ the process of RNA interference (RNAi) to inhibit the expression of specific genes. It utilizes a natural mechanism of gene silencing by inhibiting expression of genes through RNAi, with a natural length of approximately 20–29 base pairs. It is generated from DNA vectors transcribed to form siRNAs that then guides the RNA-induced silencing complex (RISC) to target the mRNA. shRNA is an RNA with a stem-loop structure that induces RNAi for gene silencing. shRNA has a stem of a 19-29 base pair region of double-strand RNA that is linked by a loop of single-strand RNA with a dinucleotide 3' overhang. The stem and loop configuration is critical to its processing and function in gene silencing. It is characterized by its stable expression from DNA vectors that provide long-term silencing effects. It is thus utilized for suppressing expression of target genes over longer periods of time in applications like cancer treatment or disease studies.

Fig. 1 Cellular processing of shRNAs and sources of by-products; alternative processing of shRNAs and its impact on target specificity^1,5.

Design Principles of shRNA

• Target Selection

The most important factor to consider when selecting an effective target site for an mRNA molecule is to choose a site within the ORF, since the ORF has a lower chance of having secondary structures which can hinder the binding of shRNA to the mRNA. In addition, it is desirable to choose a target site that has low GC content. Low GC content can lead to stable secondary structures that hinder shRNA binding. Another desirable property of the target site is that it does not overlap with any SNP or be unique to any other gene. There are many bioinformatics tools and algorithms to find good target sites for shRNA knockdown. The tools take into account factors such as nucleotide composition, secondary structure, and off-target effects of shRNA. For example, there are DSIR and siDirect algorithms which can be used to find good target sequences for shRNA knockdown.

• shRNA Sequence Design

Stem length ranges from 19 to 29 base pairs (bp), while the loop length is approximately 5–9 nucleotides. The optimum stem length is 19 nucleotides (nt) in most cases. For the loop sequence, a commonly used sequence is 5′-UUCAAGAGA-3′, which is derived from a naturally occurring miRNA sequence. The GC content of the shRNA sequence is important for the stability and functionality of the construct. In general, the GC content should be between 30–50% to allow for optimal hybridization and avoid problems with secondary structure formation. Additionally, runs of four or more consecutive As or Us are to be avoided, as these sequences can serve as termination signals for RNA polymerase III.

• Vector Design for shRNA Expression

Usually, shRNA is transcribed from DNA vectors by RNA polymerase III (Pol III) promoters like U6 or H1. These promoters efficiently transcribe shRNA and they are commonly used because of their high level of expression in a wide variety of cell types. The promoter used can affect the amount of shRNA expressed and the stability of the shRNA. Some studies choose to use the U6 promoter. Selection markers and reporter genes are sometimes added to the expression vector for selecting and monitoring cells that express shRNA. Selection markers, like antibiotic resistance genes, can select for cells that have taken up the vector. Reporter genes, like GFP, can be used to monitor shRNA expression levels and check that gene silencing has occurred.

Methods for generation of shRNA constructs

• The first method

The majority of studies use the most commonly used method (74% of studies) to create shRNA constructs. This involves synthesizing, annealing and ligating two oligonucleotides (oligos) into an expression vector. Although this is a quick method to create shRNA constructs, the cost of synthesizing the oligos is almost double than other methods, and the false positive rate found by sequencing is high (typically 20 – 40%). One reason for the unreliability of this method is the difficulty in synthesizing longer oligos (length > 35 bases). Because this method uses two long oligos, the chance of mutation caused by synthesis error is doubled.

• The second method

The second method (22% of the studies) uses a PCR strategy. A promoter is used as a template. The hairpin sequence is part of the reverse primer, and PCR generates a cloning cassette containing promoter and hairpin. Correct amplicon formation is sensitive to the sequence of the reverse primer, so this approach necessitates expensive purification (e.g. PAGE) of the long reverse primer to remove truncated oligos that are generated during manufacturing. While it is an advantage that only one long oligo is needed, the predicted secondary structure of this primer can cause the amplification of unwanted products. This can be overcome by substituting the long reverse primer for two shorter primers. The reaction is converted to a two-round nested PCR, with each primer adding half of the hairpin in each round. But repeated cycling causes increased chances of introducing polymerase induced mutations.

• The third method

The third technique (employed in 4 % of the studies) includes several primer extension-based techniques. All rely on a polymerase extending the 3' end of overlapping oligos. One example is a technique where the shRNA template is made from two long (up to 50 nucleotides) partially complementary oligos of similar length, overlapping at the 3' ends [8, 9]. Both oligos serve as template (to extend the opposite oligo) and primer (to copy the opposite oligo). By extension and repeating the extension, a double-stranded product is created, which is similar to the annealed oligo method. A variation of this method uses a long oligo as template and a shorter oligo (generic) as the primer for extension. This is amplified by PCR using a primer that binds to the extended strand. This technique is the least expensive of all the construction methods as it halves the cost of unique oligos (compared to the annealed oligo method) and avoids the cost of oligo purification (compared to the promoter-based PCR method). However, this cost saving may be counteracted by a high polymerase-induced mutation rate either in the initial extension step or by the cycling.

Delivery of shRNA

• Retrovirus vectors

Retro- and lentiviruses express a reverse transcriptase that enables their integration into the host genomes during their life cycle. Consequently, recombinant viral vectors of these types can support continuous expression of the introduced shRNA. Non-replicating viruses are most often used for the purpose of expressing the transgene for safety reasons, by providing minimal components of the viral genome in three or four plasmid vectors. This enables a single round of viral infection and then after stable integration of the recombinant viral genome, the shRNA is continuously expressed without producing an infectious virus. Shortly after the first report of plasmid vector delivered RNAi, a self-inactivating pMSCV retroviral vector was used to stably deliver shRNAs in human tumor cells to knock down the mutant K-RASv12 oncogene, and in HeLa cells human nuclear Dbf2-related (NDR) kinase was knocked down. A replication-competent retrovirus for shRNA delivery soon followed using the modified Rous sarcoma virus-derived vector RCANBP.

• Lentiviral vectors

Modified replication incompetent retroviral vectors are unable to transduce nondividing cells. Unlike retroviruses that can only transduce actively dividing cells in a resting state or at early stages of cell division, lentiviruses are capable of transducing actively dividing cells in both resting and differentiated states. Lentiviruses are a subgroup of retroviruses and the best known is the HIV subtype-1 (HIV-1). SIN lentiviral vector was the first demonstration of lentiviruses being used for RNAi purposes in HEK293 cells. Soon after, Rubinson and colleagues developed a lentiviral system to deliver shRNAs into mammalian cells, stem cells and transgenic mice for the purpose of stable gene silencing. Subsequently, lentiviruses like HIV-based vectors for RNAi-based gene therapy has been undergoing and promises to be a great tool in treating many disorders. The first report of a successful lentiviral vectors for gene therapy of hematopoietic stem cells has been reported and given that lentiviruses are effective transducers of the major target cells for HIV-AIDS therapy, CD34⁺ blood stem cells and CD4⁺ T cells, the virus is being targeted.

• Other viral vectors

Other than the viruses most commonly used for shRNA delivery, several other virus-types, such as herpesviruses and baculoviruses, have been adapted for shRNA delivery. A herpes simplex virus (HSV) amplicon expressing shRNAs targeting epidermal growth factor receptor was constructed, and a replication-deficient HSV vector was used for shRNA transcription in the hippocampus of mice. The replication competent vaccine herpesvirus of Turkey (HVT) was adapted to express shRNAs from a U6 promoter targeting Marek's disease virus replication in chickens. Baculoviruses may also be advantageous over human-derived gene therapy vectors, as they are non-pathogenic in humans and do not replicate in human cells but can transduce many vertebrate cell types with high efficiency. The baculovirus Autographa californica nuclear polyhedrosis virus was adapted to express shRNAs targeting reporter and endogenous genes in many cell lines.

Nanoparticle-based delivery of shRNA

• Chitosan

The emerging concept of Theranostic nanocarriers enables diagnosis and therapy to be performed in a single step. The diagnosis and therapeutic agents are conjugated in a single nanocarrier. A novel theranostic nanocarrier was prepared from polyethyleneimine (PEI)-modified Fe₃O₄-SiO2 (Fe₃O₄-SiO₂-PEI), containing high loaded VEGF shRNA or Notch-1 shRNA for VEGF or Notch-1 silencing genes and magnetic resonance (MR) imaging properties. The resent publication reported co-encapsulation of DOX and two fluorescent materials (fluorescein isothiocyanate (FITC) and carbon quantum dots (C-dots)) within chitosan nanoparticles for dual imaging and targeted therapy (FA–CS–FITC(DOX/C-dots)/VEGF shRNA nanocomplexes). However, biocompatibility, chemotherapeutics/gene codelivery and dual imaging property of chitosan hybrid nanoparticles made it a suitable candidate of nano-probes for cellular fluorescence imaging. Chitosan gold nanoparticle was synthesized to reduce the toxicity and reported in a publication. shRNA was conjugated with the conjugate and was well-internalized in human lung adenocarcinoma cells (A549 cells). The conjugate showed good gene silencing efficiency of voltage-gated K⁺ channels (Kv1.3) involved in cancer cell proliferation by the help of the shRNA against the same.

• Dendrimer

A type of 3D, highly branched macromolecule, known as dendrimers has one specific feature, i.e. amine groups at the end which can complex with the shRNA by electrostatic interaction. A new carrier α-cyclodextrin (G3, DS 2) was employed for conjugation with pDNA expressing shRNA for pGL3 luciferase gene (shGL3). In a comparison study, it was found that the shGL3 was bound with dendrimer (G3) as well as with α-CDE (G3, DS 2) to examine the complex formation between shGL3 and G3 or α-CDE (G3, DS 2). It was reported that α-CDE (G3, DS 2) is more efficient in inhibiting the degradation of shGL3 without showing any off-target effect.

• Gold nanoparticle

As an shRNA delivery vehicle, gold nanoparticle–DNA oligonucleotide conjugate (AuNP–DNA oligo) was used as a universal carrier. The in vitro synthesized p53 shRNA was successfully delivered into HEK293 and HeLa human cell lines using an AuNP–DNA oligo. The gene silencing achieved an 80–90% knockdown of p53 gene expression. The same delivery vehicle was used to deliver Mcl-1 shRNA and also suppressed the expression of the MCL-1 gene. The knockdown efficiency of shRNA-AuNP–DNA oligo was comparable to a liposome-based shRNA delivery method and provided an alternative method of delivery system for shRNA that could be used for any other gene of interest. Gold nanoparticle was also used in treatment of human periodontal ligament stem cells (hPDLSC). When it was treated with LRP5 shRNA and β-catenin siRNA, the results showed that the Wnt/β-catenin signaling pathway plays a crucial role in promoting hPDLSC proliferation by gold nanoparticle.

• Lipid nanoparticle

Due to their straightforward synthesis and biocompatible nature lipid-based nanoparticles represent the best available delivery system for safe therapeutic use. Solid lipid nanoparticle (SLN) stands out as a potential delivery system for shRNA macromolecules. A study was carried out on a new approach to target the chronic infection of Hepatitis C virus (HCV). In this study shRNA74 targeting the stem loop II of the 5' UTR in SLNs was developed to block the internal ribosome entry site (IRES) of human hepatocellular carcinoma cell line HepG2 expressing this viral protein. Solid lipid nanoparticle was also employed for the delivery of shRNA-encoding plasmid against 5-α reductase (p5α-Red). In vitro study showed considerable decrease in the specific protein level of human prostate cancer cell line (DU-145 cell line). Another study was carried out on in vivo efficacy of lipid nanoparticle (LNP) conjugated with sshRNAs (short synthetic shRNAs) targeting the HCV IRES. sshRNA SG220 was formulated into the LNP and injected into a mouse which was expressing HCV IRES-luciferase. In vivo imaging showed substantial amount of uptake of this formulation and dose dependent inhibition of luciferase expression.

• Poly(D,l-lactide-co-glycolide) (PLGA)

A major area of concern today is the size of the nanoparticle and its intercalate. To overcome this, a study was conducted on PLGA nanoparticle loaded with shAnnexin A2. The size of the shRNA plasmid was 11.7 kb and after encapsulation, the diameter became 165 nm. Endothelial cells were difficult to transfect; however, with this final shRNA-PLGA nanoconjugate, it showed a high transfection efficiency and cellular uptake. Annexin A2 downregulation confirmed the treatment efficiency of this nanoconjugate in the disease such as diabetic retinopathy, retinopathy for prematurity, cancer, and macular degeneration. Another ocular disease, i.e., corneal neovascularization was intended to be treated with similar nanoconjugates. In this work, PLGA NP-encapsulated pSEC shRNA VEGF-A plasmids downregulated VEGF-A mRNA in the neovascularized corneas. This work was intended to combat angiogenesis by gene therapy with an FDA-approved, biodegradable vehicle that is nonviral (PLGA NPs) and bring the shRNA plasmid directly to the site of pathology via intrastromal injection. Five days after the intrastromal injection, the shRNA-PLGA nanoconjugate showed a clear advantage over the naked shRNA plasmid in downregulating the corneal VEGF-A protein expression. When PLGA was conjugated with JNK3-shRNA, it inhibited neural apoptosis in the cerebral ischemia, oxygen and glucose deprivation model.

• Magnetic nanoparticle

A novel magnetic targeted thermosensitive drug and gene co-delivery system (TSMCL) was prepared. Doxorubicin (DOX) and Special AT-rich binding protein (SATB1) shRNA was co-delivered for gastric cancer treatment. The result of this co-delivery system inhibited the growth of gastric cancer cells in vitro and in vivo more effectively than single delivery system. In another study, magnetic Fe₃O₄ nanoparticle [MNP (Fe₃O₄)] was used as conjugate with MDR1 shRNA expression vector. Human leukemia cell line (K562) and its Adriamycin-selected P-gp-over expressing subline (K562/A02) was selected for this study which showed reverse multidrug resistance. Other cancer is lung cancer which has over-expressed Type 1 insulin-like growth factor receptor (IGF-1R) mediates cancer cell proliferation. A commercially available nanoparticle (CombiMAG) was conjugated with plasmids expressing green fluorescent protein (GFP) and shRNA targeting IGF-1R. The entire study was performed in vivo and it showed remarkable decrease of tumor size and weight. Osteosarcoma is another cancer which was treated with bacterial magnetosomes (BMs) and Doxorubicin (DOX) and pHSP70-Plk1-shRNA (heat shock protein 70-polo-like kinase 1-short hairpin RNA) co-delivery system. BMs are membrane-bound nanocrystals with a magnetic iron oxide or iron sulfide core and isolated from magnetotactic bacteria.

• Polyethylene glycol (PEG)

Castration-resistant prostate cancer (CRPC) exhibits characteristics of malignant neoplasm recurrence accompanied by poor prognosis. A mPEG-PEI nanoparticle was used as a nonviral carrier of EZH2 shRNA in a report which is commonly overexpressed in CRPC. The result was encouraging with non-viral gene carrier for EZH2 shRNA delivery to human prostate carcinoma cell line (PC3 cells) for advanced PCa therapy. Monomethoxypolyethylene glycol-chitosan (mPEG-CS) nanoparticles were synthesized in a report conjugated with survivin shRNA for PCa treatment. Cell proliferation was inhibited and apoptosis was facilitated and this was found to be a better outcome than the treatment with individual shRNA. MUC16 promoter-driven gro-α shRNA plasmid vectors conjugated with FSH peptide-conjugated PEG nanoparticle in a report on ovarian cancer showed decreased tumor growth without toxicity in a nude mouse model of ovarian cancer. The research utilized a copolymer FSH-PEG-PEI conjugated with gro-α shRNA for ovarian cancer treatment. The nanoconjugate was used to overcome the in vivo application and showed safe antitumor efficacy. A targeted delivery platform was prepared in a report using 10CPEG with 10-bromodecanoic acid (10C) and modified PAMAM. Bcl-xL protein was knocked down by shRNA and showed increased transfection efficiency in lung carcinoma cell line (A549 cells).

Applications of shRNA in Disease Therapy

• Cancer Treatment

There are many other nano carriers including supramolecular micelles formed by the self-assembly of host PEI-CyD (PC) which is β-cyclodextrin (β-CyD) and polyethylenimine (PEI) modified with survivin shRNA and adamantine-paclitaxel (Ada-PTX) for anticancer therapy. Conjugated shRNA/PTX delivery to human ovarian cancer cell line (SKOV-3 cells) showed the significant reduction of survivin and Bcl-2 expression and induce cellular apoptotic effect in the in vitro model. Combined shRNA/PTX delivery showed more efficiency compared to individual delivery. Another H1 (folate–PEI600–cyclodextrin) nanopolymer conjugated with Androgen receptor (AR) shRNA for the treatment of hormone-independent prostate cancer (HIPC) therapy. The study showed that AR-shRNA increased the radiosensitivity of prostate cancer by inhibition of cell growth, cell cycle arrest and apoptosis in ARdependent HIPC in vitro. A new nanoparticle was used to combat malignant pleural mesothelioma (MPM) via anti-thymidylate synthase (TS) shRNA. This shRNA increased the chemotherapy and showed more clinical results.

• Neurological Disorders

Knock down of endogenous α-synuclein in the adult rat substantia nigra by adeno-associated virus-mediated shRNA targeting the endogenous rat Snca transcript. Inhibition of α-synuclein by ~35% did not cause motor dysfunction or degeneration of nigral dopaminergic neurons in control rats. However, in rotenone-treated rats, progressive motor dysfunction was attenuated contralateral to α-synuclein knock down. In parallel, rotenone-induced degeneration of nigral dopaminergic neurons, their dendrites and their striatal terminals were decreased ipsilateral to α-synuclein knock down. The knock down of α-synuclein by shRNA is neuroprotective in the rotenone model of Parkinson's disease (PD) and suggests that endogenous α-synuclein contributes to the regional specificity of dopaminergic neuron vulnerability to systemic mitochondrial inhibition. shRNA targeting the SNCA transcript should be considered as a potential neuroprotective therapeutic for PD. Rabies virus glycoprotein [RVG] peptide-exosomes were used to deliver anti-alpha-synuclein shRNA minicircles (shRNA-MCs) therapy to the alpha-synuclein preformed-fibril-induced model of parkinsonism. Therapy decreased alpha-synuclein aggregation, reduced the loss of dopaminergic neurons and improved the clinical symptoms.

References

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5. Distributed under Open Access license CC BY 4.0, without modification.