Genetic payloads in focus: mRNA, siRNA, CRISPR, and more

Genetic medicines represent a promising frontier in therapeutics, utilizing DNA and RNA as payloads to target a wide range of diseases. These genetic materials are synthetically manufactured, versatile, and clinically validated. Below is an overview of key payload types used in genetic medicine, including their applications, challenges, and recent advances.

Messenger RNA (mRNA)

mRNA has gained prominence due to its ability to express healthy genes, making it a powerful therapeutic for previously untreatable conditions, such as certain cancers, infectious diseases, and rare disorders.

Advantages of mRNA as a drug payload:

• Transient expression: mRNA’s temporary activity ensures that it avoids the risks associated with permanent gene integration, unlike DNA-based therapies.
• Lower risk of immune reactions: mRNA drugs do not require viral vectors, reducing immune-related complications (1).

mRNA is used for producing therapeutic proteins, peptides, vaccine antigens, and gene-editing components (such as Cas9). Its non-viral delivery is a key benefit, and lipid nanoparticles (LNP) have emerged as the leading delivery system. LNP protect mRNA from degradation by nucleases, facilitate cellular uptake, and ensure efficient cytoplasmic release (2).

Challenges: delivery vehicles must protect the mRNA from degradation, and release the payload effectively.

Recent advances: NanoAssemblr™ technology innovative platform enables the rapid production of reproducible mRNA-encapsulated nanoparticles through microfluidic mixing. By fine-tuning particle size and achieving high encapsulation efficiency, NanoAssemblr™ systems address many challenges associated with therapeutic mRNA delivery (3).

Short interfering RNA (siRNA)

The discovery of RNA interference (RNAi) has unlocked new possibilities for gene silencing therapies. siRNA drugs work by silencing disease-causing genes and have already been clinically validated. In 2018, the FDA approved ONPATTRO, the first siRNA drug delivered via LNP, for treating hereditary amyloid transthyretin-mediated (hATTR) amyloidosis (4).

Advantages of siRNA:

siRNA offers a targeted approach to gene silencing, allowing for the treatment of diseases that were previously considered "undruggable."

Applications: siRNA drugs are being developed for a wide range of conditions, including:

• Cancer
• Rare genetic Disorders
• Immunotherapy

Challenges: siRNA, like mRNA, requires protection from nucleases and efficient delivery into the cytoplasm. Conventional delivery methods have batch-to-batch variability, low encapsulation efficiency, and scale-up difficulties.

Recent advances: NanoAssemblr™ technology has been effectively addressing these challenges, ensuring high reproducibility, better encapsulation efficiency, and rapid formulation development for siRNA-loaded nanoparticles (5).

CRISPR/Cas9 for genome editing

CRISPR has altered genetic medicine development by enabling precise editing of the genome. This technology has tremendous potential in treating genetic diseases, cancers, and infectious diseases.

CRISPR uses a Cas9 nuclease to cut specific sections of the genome, guided by one or more single guide RNAs (sgRNAs). Both components need to be efficiently delivered to the cell’s cytoplasm for successful gene editing.

Challenges: delivering both the Cas9 and sgRNA components to the cytosol remains a significant obstacle. LNP are being explored as an optimal delivery mechanism, but their performance still requires optimization.

Recent advances: research has shown that LNP provide a non-viral and scalable method for CRISPR delivery (6). However, continued work on optimizing the delivery of gene-editing components, ensuring specificity, and avoiding off-target effects is critical for widespread clinical use.

Plasmids

Plasmids are DNA-based molecules that can deliver therapeutic genes into cells. They are versatile and offer a longer-lasting therapeutic effect compared to mRNA-based treatments, making them suitable for diseases where continuous gene expression is required.

Applications:

• Gene editing: plasmids can produce guide RNA and Cas9 for CRISPR-based gene editing.
• RNAi pathway: plasmids can generate small RNA strands that enter the RNAi pathway, allowing gene silencing similar to siRNA.
• Therapeutic proteins: plasmids can encode proteins and peptides to treat conditions arising from malfunctioning proteins.

Challenges: delivery into target cells remains a challenge. Plasmids need to be protected from enzymatic degradation while efficiently entering the cell’s nucleus to initiate gene expression.

Recent advances: nanoparticle-based delivery systems like LNP have proven effective in overcoming many challenges with plasmid delivery, offering a viable alternative to viral vectors and ensuring safe and controlled delivery (7).

Proteins and peptides

Proteins and peptides have been used as therapeutic agents to treat cancer, autoimmune conditions, and infections. Unlike small molecules, therapeutic proteins interact with biological targets with high specificity, which can reduce side effects.

Challenges: protein-based therapies often face formulation difficulties due to solubility issues, aggregation, and stability concerns. Additionally, proteins must be encapsulated into delivery systems to ensure effective transport to target sites.

Recent advances: plasmid-LNP produced with NanoAssemblr™ technology have been used to express exogenous genes in primary neurons, and human iPSC-derived neuro progenitor cells and neurons in vitro. Successful gene expression has also been demonstrated in the brain and liver in animal models.

Small molecules

Small molecules remain the most common class of therapeutics, used to treat various conditions including cancer, infections, and psychiatric disorders.

Challenges: while they differ from genetic payloads, small molecules often face similar challenges in terms of delivery, such as:

• Solubility: small molecules must remain soluble to be effective.
• Targeting: ensuring the drug reaches the correct location in the body is essential for maximizing therapeutic efficacy while minimizing side effects.
• Controlled release: sustained release over time can improve patient outcomes and reduce the need for frequent dosing.

Recent advances: nanomedicine offers various delivery systems for small molecules, such as liposomes, polymeric nanoparticles, and micelles. Liposomal formulations like Doxil have already been approved and are in clinical use (8).

Key benefits of nanoparticle technology:

• Precise control over formulation conditions: NanoAssemblr™  technology allows for fine-tuned control over particle size and reproducibility, overcoming the inherent challenges of traditional formulation methods.
• Scalability: equipped with NxGen™ technology, the NanoAssemblr™ systems formulation process is inherently scalable, enabling the transition from lab-scale to clinical production effectively 
  
   

Genetic medicine payloads, from mRNA and siRNA to plasmids and proteins, hold transformative potential in treating a wide range of diseases. However, the success of these therapies hinges on efficient and safe delivery systems. Nanotechnology, particularly lipid nanoparticles, is a game-changer in this field, offering solutions to long-standing challenges in drug delivery, formulation, and scaling for clinical use. Advances such as NanoAssemblr™ technology are leading the way in ensuring these therapies are safe, effective, and scalable.