Unlocking the Future: Exploring Viral and Non-Viral Delivery Methods for Gene Therapy Manufacturing

Gene therapy stands at the forefront of medical innovation, offering the potential to treat or even cure a wide range of genetic disorders by directly targeting their genetic roots. By introducing, removing, or modifying genetic material within a patient’s cells, gene therapy seeks to rectify defective genes responsible for disease development. Gene therapy holds promise for conditions previously considered untreatable, from rare genetic diseases to more prevalent issues like cancer and cardiovascular diseases.

However, despite its transformative potential, gene therapy manufacturing faces significant challenges and limitations, including complexities in delivery methods, potential immune reactions, and ethical considerations. As research advances, addressing these challenges is crucial to fully realize the potential of gene therapy in clinical applications. A critical factor in the success of gene therapy is the method used to deliver therapeutic genes into patients’ cells. In this blog post, we will delve into the diverse array of gene delivery techniques currently being explored, highlighting their unique advantages and challenges, and discussing how they are unlocking new possibilities for effective gene therapy.

Successful gene therapy manufacturing strategies rely heavily on effective gene delivery methods, which can be broadly categorized into viral vector-mediated and nonviral vector-mediated approaches. Viral vectors, such as adenoviruses, lentiviruses, and adeno-associated viruses (AAVs), use modified viruses to transport genetic material into cells, ensuring efficient delivery and integration of therapeutic genes. This method is particularly effective for long-term gene expression and is commonly used to treat various genetic disorders and cancers.

In contrast, nonviral vector-mediated gene delivery uses synthetic or natural compounds and physical methods, like lipid nanoparticles (LNPs), electroporation, and microinjection, to introduce genetic material into cells. Nonviral methods offer advantages such as lower immunogenicity, ease of production, and potential for repeat administration. They are versatile, capable of delivering a range of genetic materials, including plasmid DNA, mRNA, and gene-editing tools like CRISPR-Cas9.(1) Although traditionally seen as less efficient than viral vectors, recent advancements have significantly improved the efficacy and duration of gene expression for nonviral methods.

Each of these approaches has unique characteristics, advantages, and limitations that must be carefully considered when designing a gene therapy strategy.

Viral Vectors

Viral vectors are a cornerstone of gene therapy due to their efficiency in delivering genetic material into cells. These vectors, which include adeno-associated viruses (AAV), adenoviruses, lentiviruses, and retroviruses, are engineered to carry therapeutic genes by leveraging the natural ability of viruses to infect cells. AAVs, for instance, are favored for their low immunogenicity and ability to provide long-term gene expression, making them suitable for treating chronic conditions. Adenoviruses, known for their high transduction efficiency, are often used in cancer gene therapy and vaccination, despite their transient expression and higher immunogenicity. Lentiviruses and retroviruses are valuable for their capacity to integrate into the host genome, offering stable, long-term expression of therapeutic genes, which is particularly beneficial for treating genetic disorders. However, the use of viral vectors also presents challenges, such as the risk of immune responses and insertional mutagenesis. Despite these hurdles, ongoing advancements in viral vector design and delivery are enhancing their safety and efficacy, solidifying their role in the future of gene therapy manufacturing. (2)

1. Adenoviruses

Adenoviruses are commonly used viral vectors due to their ability to infect a wide range of cell types, both dividing and non-dividing. They can carry relatively large DNA fragments, making them suitable for delivering therapeutic genes. However, their use can sometimes trigger an immune response, which is a significant challenge in clinical applications.

2. Adeno-Associated Viruses (AAV)

AAVs are small viruses that can infect both dividing and non-dividing cells with minimal immune response. They are widely used in gene therapy because of their safety profile and long-term gene expression. However, they have a limited capacity for carrying genetic material, which restricts their use to smaller genes.
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3. Lentiviruses

Lentiviruses are a type of retrovirus that can integrate into the host genome, allowing for stable, long-term expression of the therapeutic gene. They can carry larger genetic payloads compared to AAVs and are effective in infecting non-dividing cells. This makes them suitable for a variety of gene therapy applications, including those targeting stem cells.
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Non-Viral Methods

Non-viral gene delivery methods utilize synthetic or natural compounds or physical forces to introduce DNA into cells, generally resulting in lower toxicity and immunogenicity compared to viral methods. These approaches can achieve cell or tissue specificity by incorporating cell-specific functionalities in the design of chemical or biological vectors, while physical methods offer spatial precision. Practical advantages of nonviral methods include ease of production and the potential for repeat administration. Although traditionally considered less efficacious with shorter gene expression duration than viral methods, recent advancements indicate that some physical methods now achieve clinically meaningful efficiency and expression duration. (4)

Physical Methods

Physical methods in gene delivery vectors involve using physical forces to introduce genetic material into cells without relying on viral or chemical carriers. Here are some common physical methods

Electroporation

Of the major nonviral delivery or transfection methods, electroporation is now widely employed in various applications, including cell and gene therapy, gene editing, protein expression, mRNA vaccines, and immunotherapy. This technique uses electrical pulses to create temporary pores in cell membranes, allowing genetic material to enter the cells. After the electrical current is removed, these pores spontaneously close, effectively trapping the genetic material inside. The process involves suspending cells or tissues in a conductive solution with the genetic material, followed by the application of a brief electrical pulse, typically lasting from microseconds to milliseconds. The electrical field disrupts the cell membrane, forming temporary pores through which charged molecules like DNA can pass into the cell.

Electroporation is utilized in various contexts, including in vitro transfection of different cell types, in vivo gene delivery to tissues such as the brain, muscle, skin, and tumors, DNA vaccination,(5) and the delivery of gene-editing tools like CRISPR-Cas9.(6) This method offers several advantages, including its ability to transfect a wide range of cell types and its applicability to both in vitro and in vivo settings. The effectiveness of electroporation is influenced by multiple factors, such as the strength and duration of the electrical pulses and the composition of the surrounding solution. Carefully adjusting these parameters is essential for optimizing gene transfer efficiency while minimizing cell damage.

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The Gene Gun, also known as particle bombardment or microprojectile gene transfer, was first developed in 1987 for gene transfer into plants and has since been adapted for mammalian cells both in vitro and in vivo. This technique propels DNA-coated gold particles into cells to achieve intracellular DNA transfer, using methods such as high-voltage electronic discharge, spark discharge, or helium pressure discharge. Key parameters for efficient gene transfer include gas pressure, particle size, and dose frequency. Typically, gold, tungsten, or silver particles with a diameter of 1 μm are used. The Gene Gun allows for precise DNA dosage delivery, making it particularly useful for gene therapy research, especially in ovarian cancer. However, gene expression via this method is transient, and significant cell damage can occur at the discharge site’s center. In vivo applications have mainly targeted the liver, skin, muscle, and other surgically accessible organs. (7,8)

Chemical-based vectors

Chemical-based vectors in gene delivery are nonviral methods that utilize synthetic or natural chemical compounds to encapsulate and transport genetic material into cells. These vectors are designed to protect the genetic material from degradation and facilitate its entry into target cells. Key types of chemical-based vectors include:

1. Lipid nanoparticles (LNPs)

Lipid nanoparticles (LNPs) have become a pivotal tool in gene therapy, providing a versatile and efficient method for delivering genetic material to target cells. LNPs can encapsulate various types of genetic material, including small interfering RNA (siRNA), messenger RNA (mRNA), self-amplifying RNA (saRNA), plasmid DNA, and gene editing tools like CRISPR-Cas9, (9) protecting these cargos from degradation by nucleases in the bloodstream and enhancing their stability and circulation time. They facilitate cellular uptake through endocytosis and enable the release of genetic material into the cytoplasm by using ionizable lipids that become protonated in the acidic endosome. As a non-viral vector, LNPs reduce the risk of immunogenicity (9) and can accommodate both nucleic acids and proteins, making them suitable for diverse therapeutic applications. They can be modified with targeting ligands for specific cell or tissue delivery and their production can be easily scaled up due to established protocols and available small- and large-scale production instruments. LNPs have been used to deliver siRNA for treating rare genetic disorders like transthyretin-mediated amyloidosis (Onpattro), (10), mRNA vaccines for infectious diseases such as COVID-19, and are being researched for cancer treatment and neurodegenerative diseases. Lipid-based nanocarriers can overcome both intracellular and extracellular barriers, enabling efficient delivery of genetic materials. These non-viral vectors are particularly appealing due to their versatility, biocompatibility, ease of synthesis, and cost-effectiveness. However, ensuring efficient delivery to specific extrahepatic target cells or tissues and avoiding off-target effects are ongoing challenges. (11)

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2. Polymer-Based Vectors

Polymer-based vectors use synthetic polymers to encapsulate and deliver genetic material to target cells. These vectors can be tailored to improve stability, reduce toxicity, and enhance cell-specific targeting. While they offer a versatile and scalable option for gene delivery, optimizing their efficiency and minimizing potential side effects require further research. (12)

3. Liposomes

Liposomes are spherical vesicles that can encapsulate nucleic acids and deliver them into cells. They merge with cell membranes to release their genetic payload, which can then be expressed by the host cell. Liposomal delivery is less likely to trigger an immune response compared to viral vectors, but it may be less efficient in delivering genes to target cells. Numerous types of liposomes have been developed over the years, with those composed of cationic lipids proving to be the most effective for gene delivery. (13)

 

The choice of gene delivery techniques is a critical factor in gene therapy manufacturing that significantly influences the success and efficacy of the treatment. The table below summarizes key considerations for selecting gene delivery methods for therapeutic applications.

Emerging technologies & Pioneering Methods in Gene Therapy Manufacturing

With numerous gene therapy approvals in recent years, gene delivery technologies are continuously advancing. Scientists are exploring exosomes, which are naturally occurring vesicles that can be engineered to carry genetic material to target cells with high specificity and minimal immunogenicity. (15) Proteolipid Vehicles (PLVs) are a novel type of nanoparticle delivery system that combines a viral fusion protein with a lipid core. PLVs have shown improved distribution throughout the body, decreased toxicity, and high cargo delivery. (16) These technologies are paving the way for more effective and targeted gene therapies, offering hope for treating a wide range of genetic and acquired diseases.

Conclusion

Selecting the appropriate gene delivery technique is a crucial decision in the development of gene therapies. It requires a thorough understanding of the specific needs of the therapy, the characteristics of the target cells, and the advantages and limitations of each delivery method. By carefully selecting and optimizing the gene delivery strategy, researchers can enhance the efficacy and safety of gene therapies, bringing us closer to effective treatments for a wide range of genetic diseases.

Resources:

1. Production of AAV at the 1,000L scale in the DynaDrive Bioreactor using the CTS AAV-MAX production system
2. Small-scale purification of AAV produced with the AAV-MAX system
3. Achieving efficiency with CTS TrueCut Cas9 Protein

 

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References:

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16. Safe and effective in vivo delivery of DNA and RNA using proteolipid vehicles
    Brown, Douglas W. et al. Cell, Volume 187, Issue 19, 5357 – 5375.e2