Lipid Nanoparticles & RNA Interference: Driving a New Era in Gene Therapy Development

The technology behind gene therapy has revolutionised in recent years thanks to developments in RNAi (RNAi-targeting) technology and the fabrication of LNPs as delivery systems. RNA interference is a precise, effective way to silence genes, offering treatment for genetic disease, viral infections and cancers. But the issue of getting RNAi therapies onto the cells that matter in the body has stymied the proliferation of RNAi treatments. Lipid nanoparticles are one of the best solutions for RNA delivery, because they trap nucleic acids, improve cell absorption and confer long-term gene silencing.

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What is RNA Interference?

RNAi refers to the evolutionarily highly conserved, highly efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA). Cellular double-stranded RNA (dsRNA) is cleaved by nuclease into small interfering RNAs (siRNAs) of 21-23 nucleotides, which, together with some enzymes in the body, can form a RNA-induced silencing complex (RISC). siRNAs in the RISC recognize and bind to the mRNA sequences that are complementary to theirs, and then degrade the mRNAs under the action of nuclease, thus realizing the efficient and specific degradation of specific genes. The RISC will recognize and bind to its complementary mRNA sequence and then degrade the mRNA under the action of nuclease to regulate the expression of specific genes.

How Does RNA Interference Work?

The RNAi pathway is initiated when long double-stranded RNA (dsRNA) molecules are processed into smaller siRNA molecules by the enzyme Dicer. These siRNAs are then incorporated into the RNA-induced silencing complex (RISC), where one strand of the siRNA, the guide strand, is retained, while the other strand, the passenger strand, is degraded. The guide strand directs the RISC to the complementary mRNA, where it induces mRNA cleavage by the Argonaute (AGO) protein within the RISC complex. This leads to the degradation of the target mRNA, preventing its translation into protein. RNAi has proven to be an invaluable tool for selective gene silencing and holds significant therapeutic potential, particularly for diseases caused by genetic mutations or aberrant gene expression.

siRNA Delivery

Delivering small interfering RNA (siRNA) effectively to target cells is one of the most significant challenges in RNA interference (RNAi)-based therapeutics. The process of siRNA delivery involves overcoming multiple biological barriers, including degradation by nucleases, limited cellular uptake, poor endosomal escape, and inefficient release within the cytoplasm. For RNAi to be a viable therapeutic strategy, these obstacles must be addressed to achieve high delivery efficiency and selective silencing of target genes in specific tissues.

Challenges in siRNA Delivery

• Nuclease Degradation: siRNA molecules are inherently unstable in the bloodstream due to the presence of nucleases that degrade RNA molecules. This leads to a rapid clearance from the body, limiting the therapeutic window of the drug.
• Cellular Uptake: siRNAs are large, negatively charged molecules that cannot easily cross the lipid bilayer of cell membranes. The uptake of siRNA into cells is a critical step for its therapeutic action, and this process often requires specialized delivery vehicles to facilitate the crossing of the cellular membrane.
• Endosomal Escape: Once inside the cell, siRNA is often trapped within endosomes, where it is typically degraded before it can reach the cytoplasm, where RNAi machinery is active. Efficient escape from the endosome is essential for siRNA to function properly.
• Targeting Specific Tissues and Cells: Achieving selective delivery of siRNA to specific tissues or cells is another critical challenge. Non-specific distribution of siRNA can lead to off-target effects, toxicity, and reduced therapeutic efficacy.

siRNA Delivery Methods

Several delivery strategies have been developed to enhance the efficiency of siRNA delivery. These methods include the use of viral vectors, lipid-based carriers, polymer-based carriers, and nanoparticles. Among these, lipid nanoparticles (LNPs) have gained significant attention due to their ability to encapsulate RNA molecules effectively, protect them from degradation, and facilitate efficient cellular uptake.

Viral Vectors for siRNA Delivery

Viral vectors, such as lentiviruses and adeno-associated viruses (AAVs), have been traditionally used to deliver nucleic acids, including siRNAs, into cells. While highly efficient, the use of viral vectors raises concerns regarding safety, immunogenicity, and scalability. Additionally, viral vectors have limited packaging capacities, which restrict their use for large RNA molecules.

Polymer-Based Carriers for siRNA Delivery

Polymeric carriers, including cationic polymers like polyethylenimine (PEI), have also been explored for siRNA delivery. These carriers work by electrostatically interacting with the negatively charged RNA, forming complexes that are taken up by cells. However, polymer-based systems often suffer from toxicity and inefficient release of RNA in the cytoplasm.

Lipid-Based Carriers for siRNA Delivery

Lipid-based carriers, such as liposomes and lipoplexes, offer a safer alternative to viral vectors. These carriers consist of lipid bilayers that encapsulate the RNA molecules, protecting them from degradation and enabling their transport across cellular membranes. Liposomes, for example, have been used extensively for drug delivery and gene therapy applications. However, their limited ability to encapsulate RNA efficiently and their rapid clearance by the immune system have led to the development of more advanced formulations, such as lipid nanoparticles (LNPs).

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Lipid Nanoparticles for siRNA Delivery

Lipid Nanoparticles

Lipid nanoparticles are colloidal particles made up of lipids, which are lipid-like molecules that can encapsulate hydrophobic or hydrophilic compounds, such as RNA. The design of LNPs is based on the principles of lipid bilayer structures, which mimic the natural cell membrane, thus allowing them to interact favorably with cell membranes during uptake. LNPs typically consist of a mixture of several types of lipids, each playing a specific role in the particle's function. The composition of LNPs is critical in determining their stability, efficiency in delivering siRNA, and minimizing potential toxicity.

Diagram of lipid nanoparticles loaded with siRNA.

siRNA LNP Formulation

Lipid nanoparticles (LNPs) have emerged as one of the most promising and widely used delivery systems for small interfering RNA (siRNA). The unique properties of LNPs make them ideal candidates for overcoming the numerous challenges associated with siRNA delivery, particularly in the context of gene silencing and RNA interference (RNAi) therapies. Their ability to encapsulate siRNA and protect it from degradation, while ensuring efficient cellular uptake and endosomal escape, positions LNPs as a cornerstone in the development of targeted gene therapies.

• Lipid Composition: The choice of lipids is crucial for the formation of stable LNPs. Typically, a mixture of cationic, anionic, and neutral lipids is used to optimize the encapsulation of siRNA, enhance cellular uptake, and minimize toxicity. Cationic lipids interact with the negatively charged siRNA, while helper lipids such as DSPC (distearoylphosphatidylcholine) and cholesterol provide structural stability and enhance membrane fusion.
• Particle Size: LNPs must be small enough to facilitate efficient cellular uptake but large enough to encapsulate siRNA effectively. Particle size is typically in the range of 50-200 nm for optimal performance.
• Surface Charge: The surface charge of LNPs is an important factor in their interaction with cells. Slightly positive or neutral charges are often preferred to minimize aggregation and enhance biocompatibility.
• Encapsulation Efficiency: High encapsulation efficiency is essential to ensure that the maximum amount of siRNA is delivered to the target cells. Formulation methods, such as microfluidic mixing, allow for better control of the lipid-to-RNA ratio, leading to more efficient encapsulation.
• Stability and Release Kinetics: LNP formulations must be stable during storage and transport but also release siRNA efficiently once inside the target cells. Controlled release mechanisms, such as pH-sensitive lipids, can be incorporated to facilitate endosomal escape and siRNA release in the cytoplasm.

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Advantages of LNPs in siRNA Delivery

LNPs offer a highly efficient and versatile platform for siRNA delivery, addressing several challenges that have limited the potential of siRNA as a therapeutic agent.

• Enhanced Stability: The lipid core of LNPs provides excellent protection against enzymatic degradation, ensuring that the siRNA remains intact as it travels through the bloodstream.
• Efficient Cellular Uptake: LNPs are able to cross cellular membranes more efficiently than free RNA molecules due to their lipid composition, which mimics the cell membrane. Their size and charge can also be optimized to enhance cellular uptake via endocytosis.
• Endosomal Escape: One of the biggest challenges in RNA delivery is ensuring that the RNA cargo escapes from the endosome and enters the cytoplasm. LNPs have been engineered to disrupt the endosomal membrane, facilitating the release of siRNA into the cytoplasm, where it can interact with the RNA-induced silencing complex (RISC).
• Biodegradability: The lipids used in LNP formulations are typically biodegradable and do not accumulate in tissues, reducing the risk of long-term toxicity.
• Scalability: The production of LNPs is scalable, making them an attractive option for industrial applications. Techniques such as microfluidic mixing and ethanol injection have been developed to produce LNPs with high consistency and quality.

Applications of LNP siRNA

Lipid nanoparticles (LNPs) carrying small interfering RNA (siRNA) have emerged as a transformative tool in RNA-based therapeutics, offering targeted gene silencing for treating various diseases. These formulations enable precise modulation of gene expression in genetic disorders, cancers, and viral infections, with several clinical applications already underway.

Genetic Disorders

LNP-siRNA technology has shown great potential in treating genetic disorders by targeting mutant genes responsible for disease development.

• Transthyretin (TTR) Amyloidosis: TTR amyloidosis is a fatal genetic condition caused by the buildup of abnormal TTR proteins in organs. LNP-siRNA targeting the TTR gene can reduce TTR protein levels, offering a promising therapy. ALN-TTR02, developed by Alnylam Pharmaceuticals, has demonstrated a 50-80% reduction in serum TTR levels in clinical trials, showing significant improvements in patient outcomes.
• Cystic Fibrosis: In cystic fibrosis (CF), mutations in the CFTR gene cause thick mucus buildup in the lungs and digestive system. LNP-siRNA formulations targeting the CFTR mutation could restore normal gene function and provide a novel therapeutic strategy. Although still in early clinical stages, this approach holds potential for CF treatment.

Cancer Therapy

LNP-siRNA formulations are being explored for cancer therapy by silencing oncogenes that drive tumor growth.

• Oncogene Silencing: Targeting specific oncogenes such as KRAS, MYC, or BCL-2 with LNP-siRNA formulations could inhibit tumor growth and enhance treatment efficacy. For instance, silencing KRAS in cancers like pancreatic or lung cancer could potentially halt tumor proliferation.
• Tumor-Specific Targeting: LNPs can be engineered to deliver siRNA specifically to cancer cells by incorporating tumor-specific ligands. This targeted delivery reduces off-target effects, making LNP-siRNA an effective tool in cancer therapy.

Viral Infections

LNP-siRNA is also effective in treating viral infections by silencing key viral genes, preventing replication and viral spread.

• Hepatitis B: LNP-siRNA formulations targeting the hepatitis B virus (HBV) genome can reduce viral loads and may provide a cure for chronic HBV infection. Early-phase clinical trials have shown encouraging results in reducing HBV replication and improving liver function.
• HIV: LNP-siRNA targeting HIV genes such as Tat and Rev may provide long-term viral suppression and could potentially offer a cure for HIV. Although still in research stages, this approach is gaining attention as a novel way to treat HIV.

Cardiovascular Diseases

LNP-siRNA is being explored in the treatment of cardiovascular diseases, especially those linked to lipid metabolism.

• PCSK9 Inhibition: LNP-siRNA targeting PCSK9 can lower LDL-C levels, reducing the risk of cardiovascular events. ALN-PCS, an LNP-siRNA formulation targeting PCSK9, has shown a 40% reduction in LDL-C in clinical trials, offering a potential alternative to traditional statins.

Neurological Disorders

LNP-siRNA technology is being investigated for treating neurodegenerative diseases and spinal muscular atrophy (SMA). By silencing inhibitory genes, LNP-siRNA could promote the expression of necessary proteins like SMN in SMA, potentially improving motor function in affected individuals.

Regenerative Medicine

LNP-siRNA formulations are also being explored in regenerative medicine to modulate gene expression in stem cells or promote tissue regeneration. Targeting specific genes in tissue fibrosis or stem cell differentiation holds potential for advancing tissue repair therapies.