shRNA in Neurodegenerative Diseases-The mystery Gene Silencers

Introduction of shRNA in Neurodegenerative Diseases

Short hairpin RNA (shRNA) is a form of gene silencing technology based on RNA interference (RNAi). Short hairpin RNA can be synthesized from a DNA vector and processed into small interfering RNAs (siRNAs) in the cell. Short hairpin RNA interacts with the RNA-induced silencing complex (RISC) and guides it to the degradation of target mRNAs. In the treatment of neurodegenerative diseases, shRNA can play a role as a therapeutic agent. These diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD), are often caused by the accumulation of misfolded proteins and neuronal death. Targeting mRNAs of the genes that are responsible for the pathogenesis of the diseases, the shRNA can inhibit the production of toxic proteins and eventually alleviate the disease symptoms. For example, shRNA against BACE1 reduces the formation of Aβ plaques in the mouse model and has an effect on improving the cognitive function. For PD, shRNA against α-synuclein reduces the accumulation of α-synuclein, which is the cause of the disease. In HD, shRNA targets the mutant huntingtin gene and inhibits the death of neurons and restores motor function in the animal model.

What are Neurodegenerative Diseases

Neurodegenerative diseases are disorders associated with progressive neuronal loss, together with the deposition of proteins with altered physicochemical properties in the brain and in peripheral organs. Molecular classification of neurodegenerative disease is based on proteins. This classification of neurodegenerative disorders is emphasizing the importance of protein-processing systems in the pathogenesis of these disorders. The most frequent proteins involved in the pathogenesis are amyloid-β, prion protein, tau, α-synuclein, TAR-DNA-binding protein 43 kDa, and fused-in sarcoma protein. Additional proteins are involved mostly in hereditary disorders, such as proteins encoded by the genes responsible for trinucleotide repeat disorders, neuroserpin, ferritin and familial cerebral amyloidoses. The clinical presentations are defined by the specific involvement of functional systems and do not necessarily reflect the molecular pathologic background.

shRNA Mechanism and Design in Neurodegenerative Diseases

• Mechanism

ShRNA consists of 19-20 base pair RNA sequence containing a short hairpin loop of 4–11 nucleic acid. These RNA are synthesized within the cell after transfection of shRNA-plasmid containing RNA sequence. When these plasmids are incorporated within the cellular DNA they start transcribing shRNA by RNA polymerase II and III. Individual pre-shRNA after transcription is transported to the cytoplasm. In the cytoplasm these RNA form Dicer complexes and produce double stranded RNA which are consequently degraded by the cellular enzymes. The main advantage of shRNA in therapeutic applications is that these macromolecules have a long standing effect on cellular function than siRNAs. They are highly specific in their approach in gene silencing and not like miRNAs which have a wider effect on several genes at a time.

Fig.1 Graphic scheme of RNA interference^1,4.

• Design Principles

A proper design of shRNA is of great importance to achieve high efficiency and specificity. The choice of the target sequence is the first step to be taken into account. The target sequence should be 19-21 nt in length and be located in the 3' UTR of the target mRNA, and shRNAs can target other regions too. The target sequence should be unique to the gene of interest and does not share any similarity with other genes. In addition, the structure of shRNA is of importance for its efficiency. The stem length should be at least 19 bp to be processed by Dicer. And the length of loop should be no less than 4 nt to avoid tension on the stem region and decrease Dicer's access. Moreover, shRNA should have a 2nt 3' overhang which is necessary for Dicer recognition and processing. As for the choice of promoter, high level of shRNA expression across various cell types is required, and the most frequently used promoters are RNA polymerase III promoters, such as U6 and H1. RNA polymerase II promoters can also be chosen for the purpose of shRNA expression, which has some advantages for certain applications.

Nanoparticle based delivery of shRNA in Neurodegenerative Diseases

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

The dimensions of nanoparticle and its intercalate are among the main focuses of present research. Resaerchers tested PLGA nanoparticle loaded with shAnnexin A2. The shRNA plasmid was 11.7 kb and became 165 nm after encapsulation. Primary endothelial cells are hard to transfect, but this final shRNA-PLGA nanoconjugate showed high transfection efficiency and effective cellular uptake. Annexin A2 downregulation confirmed the treatment efficacy of this nanoconjugate in diabetic retinopathy, retinopathy for prematurity, cancer, and macular degeneration. Another ocular disease, e.g., corneal neovascularization, was planned to be treated by this nanoconjugate. In this study, PLGA NP–encapsulated pSEC shRNA VEGF-A plasmids decreased VEGF-A mRNA in the neovascularized corneas. The objective of this study was to oppose angiogenesis by gene therapy with an FDA-approved, biodegradable, nonviral vehicle (PLGA NPs) and deliver the shRNA plasmid directly to the site of pathology via intrastromal injection. Five days after the intrastromal injection, the shRNA-PLGA nanoconjugate demonstrated an evident advantage over naked shRNA plasmid by reducing the corneal VEGF-A protein expression. This work provided nontoxic, highly efficacious, and long-lasting regression of corneal neovascularization. Conjugating with PLGA in shRNA, they restrained neural apoptosis in the OGD model of cerebral ischemia.

• Polymeric nanoparticles

Polyethylenimines (PEIs) are the most commonly used polymer for the delivery of nucleic acids. PEIs are polycationic polymers with high amine content. There are two types of PEIs, linear and branched. There are also PEIs with different molecular weights. Because of the polycationic structure of PEI, it has strong electrostatic interaction with nucleic acids. Furthermore, it can cross the cell membrane through endocytosis and has weak-base buffering property to protect nucleic acids from endolysosomal degradation. The proton sponge effect leads to endosome's burst and nucleic acids are released to cytoplasm. After intranasal administration, PEI nanoparticles can deliver shRNAs to different brain cells efficiently. However, low biocompatibility and non-biodegradability of PEI limit its clinical application. Cytotoxicity of PEI generally increases with the increase of molecular weight and positive charge density. But low molecular weight reduces transfection efficiency. To address the problem, some modifications are performed to PEI. Conjugation of PEI (12 kDa) with negatively charged deoxycholic acid can significantly reduce its cytotoxicity while keeping high transfection efficiency. The iNGR cell-penetrating peptide is suggested to modify the surface of PEI (800 Da) for its targeted delivery.

• Lipid-based nanoparticles

Liposomes are nanoscale vesicles formed by a single or multiple phospholipid bilayers enclosing an aqueous cavity that allows nucleic acid encapsulation. It is one of the widely used carrier systems for shRNA delivery and already available on the market for mRNA vaccine. Solid lipid nanoparticles with a solid hydrophobic core and phospholipid monolayer can also transfer nucleic acids and have an electrostatic binding to shRNA at the outer surface. Lipid-based nanoparticles have a very stable structure to protect nucleic acids from degradation, however, the half-life of the particle is limited by the rapid clearance in the liver and spleen. For extending the circulation time, PEGylation is the golden standard. Moreover, they can be modified with several types of targeting molecules such as T7 peptide and iRGD (tumor-penetrating peptide) for higher uptake into the brain and tumor.

• Peptide-based nanoparticles

Nanoparticles loaded with peptides are usually prepared by covalent binding of peptides with RNA therapeutics or with other nanoparticles to make a functional component. However, recent developments have been made using peptides as the backbone of nanoparticles and form complexes with nucleic acids by non-covalent interactions. Researchers designed a series of peptide-based nanoparticles that contained RVG as the neuron targeting peptide and RVG as the intracellular trafficking peptide. After intravenous administration of the optimized nanoparticles, high accumulation of shRNA was observed at the injury site of the brain.

shRNA Applications in Neurodegenerative Diseases

• Alzheimer's Disease(AD)

AD is a progressive neurodegenerative disorder that manifests with the aggregation of amyloid-beta (Aβ) plaques and neurofibrillary tangles. A major enzyme that contributes to Aβ production is beta-secretase 1 (BACE1). Several studies have demonstrated the feasibility of using shRNA targeting BACE1 to inhibit Aβ production. For example, a study delivered shRNA targeting BACE1 via a lentiviral vector in a transgenic mouse model of AD. It was found that BACE1 suppression substantially attenuated amyloid plaque formation and neuropahtological and behavioral alterations. Memory and learning behaviors improved in SAMP8 mice when BACE1 was targeted through shRNA treatment. Besides BACE1, other targets such as APP (amyloid precursor protein) and presenilin1 (PS1) have also been tested. The use of shRNA against APP resulted in enhanced synaptic performance alongside better mitochondrial operation within human neuroblastoma cell lines. shRNA targeting PS1 decreased the level of Aβ42 in human neuroblastoma cells.

• Parkinson's Disease (PD)

The hallmark of PD is the accumulation of the alpha-synuclein (α-syn) protein and the formation of Lewy bodies and the loss of dopaminergic neurons. As a potential treatment option, shRNA targeting the α-syn protein was investigated for its potential in preventing protein aggregation and delaying the progression of the disease. One study found that anionic liposomes with a rabies virus glycoprotein-derived peptide delivered shRNA targeting α-syn to neuronal cells from newborn C57BL/6J mice reduced α-syn levels. A study using viral vectors to deliver shRNA targeting α-syn to the substantia nigra in a mouse model of PD demonstrated reduced α-syn expression and ameliorated the behavioral deficits. Other targets such as ROCK-II have also been explored. ShRNA against ROCK-II was demonstrated to enhance axonal growth in neural stem cells.

• Huntington's Disease

HD is caused by the expansion of a CAG trinucleotide repeat in the huntingtin (HTT) gene, resulting in the production of a mutant HTT protein that is prone to aggregation and neuronal cell death. shRNA has been used to silence the mutant HTT gene as a potential treatment. For example, one study injected naked shRNA into the cerebral cortex of a transgenic mouse model of HD. This resulted in the inhibition of HTT expression and a reduction in the size and number of neuronal intranuclear inclusions (NIIs). Other studies with HD mouse models also show that injection of AAV-HTT shRNA into the striatum reduces HTT expression, which improves motor and behavioral deficits without neurotoxicity. The safety of this strategy in NHPs has also been reported. One benefit of targeting total HTT is that, in theory, one therapeutic agent can be used for all patients with HD, regardless of their mutation.

• Amyotrophic lateral sclerosis(ALS)

The loss of motor neurons triggers ALS which typically leads to respiratory failure and death between three and five years following disease onset. Between 5% and 10% of cases have a family history of mutations (fALS), and some mutations are found in otherwise apparently sporadic cases (sALS). The three most commonly mutated ALS genes (SOD1, C9orf72, TDP43 and FUS) have been targeted for gene therapy of their associated mutations in clinical trials. Intravenous administration of AAV9–SOD1 shRNA decreased SOD1 expression, reduced disease progression and increased lifespan in a transgenic SOD1 mouse model.

Future Directions

One of the major limitations to the clinical application of shRNA therapy in neurodegenerative diseases is the delivery of the therapeutic shRNA to the CNS. The BBB prevents the vast majority of molecules from accessing the brain. One promising strategy to overcome this limitation is the use of AAV vectors that have been made safe and efficient for gene delivery. Because they allow stable, long-term transgene expression in non-dividing cells, AAV vectors can be used to deliver shRNA to the brain. For example, AAV vectors have been delivered intranasally and were shown to overcome the BBB and downregulate the 5HT-2A receptor in specific neuronal populations. An alternative method is to use extracellular vesicles (EVs) that have been loaded with shRNA. These vesicles can be targeted to the brain after intravenous injection, leading to gene silencing. For example, human RVG-EVs loaded with shRNA minicircles (MCs) reduced α-synuclein expression and protected dopaminergic neurons in a mouse model of PD.

References

1. Liu, Zehua, et al."Non-viral nanoparticles for RNA interference: Principles of design and practical guidelines." Advanced Drug Delivery Reviews 174 (2021): 576-612. https://doi.org/10.1016/j.addr.2021.05.018.
2. Rohn, Troy T., et al. "Intranasal delivery of shRNA to knockdown the 5HT-2A receptor enhances memory and alleviates anxiety." Translational Psychiatry 14.1 (2024): 154. https://doi.org/10.1038/s41398-024-02879-y.
3. Wang, Lingling,et al. "Significance of gene therapy in neurodegenerative diseases." Frontiers in Neuroscience 19 (2025): 1515255. https://doi.org/10.3389/fnins.2025.1515255.
4. Distributed under Open Access license CC BY 4.0, without modification.