Gene Therapy Methods Explained

This story is part of a series on the current progression in Regenerative Medicine. In 1999, I defined regenerative medicine as the collection of interventions that restore tissues and organs damaged by disease, injured by trauma, or worn by time to normal function. I include a full spectrum of chemical, gene, and protein-based medicines, cell-based therapies, and biomechanical interventions that achieve that goal.

In this subseries, we focus specifically on gene therapies. We explore the current treatments and examine the advances poised to transform healthcare. Each article in this collection delves into a different aspect of gene therapy’s role within the larger narrative of Regenerative Medicine. This piece begins a miniseries on gene therapy vectors and their significance.

Gene therapy is likely something you’ve seen or read about in the news, the most recent being how we can treat an enzyme deficiency with mRNA. Behind all this progress we are reading about and will continue to read about in the coming years is a generation of work on methods of introducing genes into cells. Here, we review some of the most prominent methods for doing this. As the series progresses, we will give specific examples of success.


Introduction to Gene Therapy


Gene therapy is a medical field that shows promise in correcting genetic disorders by targeting the genes responsible for the disease. It involves introducing genetic material into cells to replace or repair faulty genes. The challenge with gene therapy is that the genes must be delivered to the entire body through injection or the bloodstream. However, they must enter the specific organ or cell needing treatment. The carrier cannot simply enter the cell; it must first reach the entire body. 


There are two potential solutions to this problem. One option is to extract the cell and gene from the body and reintroduce it, but this approach comes with added difficulties and costs. Another option is to target the gene through a specific organ in the body. The liver is often chosen for this purpose because it has fenestrations and blood vessels that allow it to produce many enzymes for the body. Therefore, gene delivery systems are critical for gene therapy and can be classified as viral or non-viral vectors.


Viral vectors are derived from viruses and can efficiently enter target cells and deliver the therapeutic gene to the nucleus. However, there is a risk of immune response and integration into the host genome, which can cause unintended effects. About 70% of clinical trials on gene therapy use viral vectors.


Non-viral vectors, on the other hand, are safer and have lower immunogenicity but are less efficient in gene delivery. Choosing the appropriate gene delivery system depends on various factors, such as the type of genetic disorder, target cells, and the desired outcome. 


Non-viral Vectors in Gene Therapy


Non-viral vectors, such as liposomes, cationic polymers, gold nanoparticles, exosomes, ferritin, and red cell membranes, offer a promising alternative to viral delivery systems for gene therapy. Additionally, they can carry large gene loads, display lower immunogenicity, and can be produced safely at scale


Liposomes are small spherical structures comprising two layers of lipids commonly used to transport nucleic acids to cells. Nucleic acids are genetic materials like DNA and RNA that can be used to alter specific cell functions. Liposomes are highly sought after because they are not toxic, easy to manufacture, capable of carrying large DNA fragments, and lack immunogenic protein components. Among the non-viral vectors, cationic liposomes (CLs) are one of the most effective. Cationic polymers like polyethyleneimine (PEI) and chitosan can condense these nucleic acids into nanoparticles, which can then be delivered to the target cells.


The liposome carrier can circulate throughout the body and avoid the immune system. Its lipid bilayer allows it to merge with the cell membrane, helping deliver the genetic cargo directly into the cell’s interior. Once inside the cell, the nucleic acids can be transported to the nucleus. This gene expression and cellular function alteration is possible due to the liposome’s ability to transport them.


Gold nanoparticles can also be linked with DNA or RNA and transferred to cells using electroporation or ultrasound. These nanoparticles take advantage of the enhanced permeability and retention (EPR) effect in tumors and inflamed tissues. They are tiny enough to pass through the leaky vasculature in these areas and accumulate at the intended target site. When they reach the target cells, electroporation or ultrasound techniques temporarily disrupt the cell membrane and aid the uptake of the nanoparticle-bound genetic materials into the cytoplasm. This enables the nucleic acids to travel to the nucleus and have their desired therapeutic effects.


Exosomes are small, lipid-based vesicles secreted by cells. They can be designed to carry therapeutic nucleic acids to target cells and potentially be gene-delivery vehicles. Exosomes have the potential to be used as a platform for gene therapy delivery due to their ability to cross biological barriers and their compatibility with the human body. However, despite their ability to cross cellular barriers and efficiently deliver cargo to desired cells, they face mechanisms and engineering separation limitations.


Ferritin, a protein that stores iron, can also be modified to carry nucleic acids and deliver them to cells. This method utilizes the natural attraction of ferritin to specific cell receptors, which can aid in delivering nucleic acids to the intended cells. The nanoparticles can travel throughout the body and selectively attach themselves to cells that have the required receptors. This receptor-mediated endocytosis allows ferritin-nucleic acid complexes to be absorbed into the target cells. Once inside the cytoplasm, the nucleic acids can move to the nucleus and alter gene expression. 


Lastly, red cell membranes can encapsulate and deliver nucleic acids to cells. Red cells have a unique composition that allows them to evade the immune system, making them an attractive option for drug delivery. Nucleic acids can be encapsulated within red cell membranes for targeted delivery of therapeutics to specific cells, reducing off-target effects and improving treatment outcomes. These carriers protect and deliver genetic material to target cells, taking advantage of the natural biodistribution and longevity of red blood cells.


Non-viral vectors have the potential to offer several benefits in gene therapy. However, they face challenges in achieving efficient gene uptake and sustained expression in vivo. These challenges are due to limited cellular uptake, rapid immune system clearance, and nucleosome degradation. Nonetheless, the continued progress in this area provides an excellent potential for gene therapy. Lipid nanoparticles (LNPs) and cationic polymeric-based vehicles show the most promise as non-viral gene vectors and red blood cells are gaining traction in academic and clinical research.


Viral Vectors in Gene Therapy


Viral vectors are essential tools in gene therapy, and various types have been tested in clinical trials. Adenoviruses (AdV), Adeno-associated viruses (AAV), Retroviruses, Lentiviruses (LV), and Bacteriophages are the most commonly used viral vectors due to their ability to deliver therapeutic genes to target cells with high transfection efficiency.


Adenoviruses are a group of viruses that can cause various illnesses, including the common cold, respiratory, gastrointestinal, and eye infections. In gene therapy, AdV vectors are modified to remove their ability to cause disease and multiply. These modified AdV vectors then deliver therapeutic genes into target cells. AdV vectors are often used in gene therapy because they can effectively infect both dividing and non-dividing cells. Additionally, the immunogenicity of second-and third-generation adenovirus vectors has been significantly reduced, making them safer for use.


Adenovirus vectors use their natural affinity and cell entry mechanisms to bind to and infect the desired target cells. Once inside the cell, the viral DNA that carries the therapeutic gene is transported to the nucleus, where it can be expressed and produce its intended effects. The ability of adenoviruses to transduce a wide range of cell types, including non-dividing ones, makes them a versatile and effective gene delivery platform.


Adeno-associated viruses (AAVs) are generally considered relatively safe viral vectors for gene therapy applications compared to other viral vectors. AAV vectors can infect both dividing and non-dividing cells. They rely on their natural ability to bind to and infect the target cells, using their typical tropism and cell entry mechanisms. Once inside the cell, the viral DNA carrying the therapeutic gene is transported to the nucleus, where it can be expressed and produce the desired effects. AAVs can only replicate in humans in the presence of a helper virus, usually an adenovirus. However, rare AAV integrations can lead to hepatocellular carcinoma in mice. Nevertheless, the vector design can be modified to reduce this risk.


Herpes Simplex Virus (HSV) vectors are being researched as a possible gene delivery method. These vectors can be engineered to either not replicate or replicate with less harmful effects. They can carry large transgenes, making them suitable for treating neurological disorders, cancer, and research. HSV vectors have several advantages, including their ability to infect various cells, hold large amounts of genetic material, and have a low risk of causing mutations. However, challenges associated with their use include immune response, precise engineering for safety, and design and production complexity.


Retroviruses are RNA viruses that can integrate their genetic material into the DNA of the host cell. These viruses are used in gene therapy to insert therapeutic genes into the host cell’s genome. The most commonly used retrovirus vectors are based on murine Moloney leukemia virus (MLV) and human immunodeficiency virus type 1 (HIV-1), which is a type of retrovirus known as a lentivirus. 


Lentiviruses are a particular kind of retrovirus that can infect both dividing and non-dividing cells. Much research has been conducted to understand the molecular biology of the lentiviral life cycle. As a lentivirus, HIV has the unique ability to integrate its genetic material into the host cell’s genome, even in non-dividing cells. This allows lentiviral vectors derived from HIV to efficiently deliver therapeutic genes and achieve long-term, stable expression in a wide range of target cell types


Bacteriophages are viruses that infect bacteria. They are used in gene therapy to deliver therapeutic genes to bacteria. Compared to most other viruses, bacteriophages have a larger cargo capacity. They can enter cells through receptors using eukaryotic cell-targeting signals to produce therapeutic proteins, and this process is applied in gene therapy for various purposes.


Alphavirus vectors are potent tools that express transgenes and produce vaccines in vitro and in vivo. Based on alphaviruses, these vectors infect human, animal, and insect cells. Semliki Forest virus (SFV) vectors are the most popular among alphavirus vectors. These vectors offer high transgene expression and can accommodate large transgenes, making them a possible choice for genetic engineering and vaccine development researchers.


The Challenges of Viral Vectors


Viral vectors can also pose significant challenges. One of the main concerns is the potential for immunogenicity. As a result, the immune system might identify the viral vector as a foreign invader and attack it, which could reduce its effectiveness or cause severe or fatal consequences. Additionally, the production process for viral vectors is complex and expensive, which limits their availability and accessibility for patients. 


The field of gene therapy vectors is intricate and constantly evolving, with discoveries and approaches emerging regularly. Despite the challenges and intricacies involved, gene therapy holds the promise of a new era where genetic disorders can be effectively treated or even cured.

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© William A. Haseltine, PhD. All Rights Reserved.