Viral Vectors for Gene Therapy in a Nutshell: AAVs, Lentivirus, Adenovirus and Retrovirus
Summary
Gene therapy offers great potential in the treatment and management of a variety of diseases. Viral vectors are currently the most common delivery vehicle for gene therapy owing to their fantastic capabilities of transferring many copies of therapeutic genes to host cells. Within this family of vectors, Adeno-associated vectors (AAV) and lentivral vectors (LV) are the most promising, due to their ability to transduce dividing and non-dividing cells, as well as their low immunogenic disposition. For efficient, reproducible and cost-efficient means of viral vector production, Sartorius Polyplus offers industry-leading products: PEIpro®, FectoVIR®-AAV and FectoVIR®-LV
Introduction to gene therapy
Many diseases can be traced to a faulty version of vital genes. To cure these patients, scientists are searching for ways to target and replace the defective genes within affected cells. The most popular approach is Gene Therapy, which aims at introducing nucleic acid materials such as DNA or mRNA to treat the origin of genetic disorders.
To introduce genetic materials into cells, they must be packaged within a vector. The reason for this is that nucleic acids, as well as cell membranes, have a negative charge and so naturally repel each other. Vectors act as vehicles which transport the genetic cargo into cells, and once inside can release it and enable the production of therapeutic proteins. Vectors come in a variety of forms, from peptides to lipids. For gene therapy, the most typical vectors are viruses, owing to their inherent adaptability and efficiency of gene delivery.
Viral Vectors: Adenoviral, Retroviral Lentiviral and AAVs
The fundamental goal of any virus is to produce viral progeny. To do this, viruses introduce their genetic material into host cells in order to replicate. Recombinant viruses, which carry foreign genetic material, serve as powerful tools for gene therapy. This process of transferring foreign genetic material into a cell via a viral vector is known as transduction. The range of viral vectors is extensive and includes the delivery of vectors developed for both short-term and permanent long-term gene expression. Typically, there are four main types of viral vectors utilized for gene therapy applications, these include:
Adenoviral Vectors:
They carry their genetic material in the form of DNA. These vectors are advantageous in that they can deliver many genome copies per host cell resulting in a higher gene expression. The genetic expression from adenoviruses is transient: the DNA cargo is free within the host cell’s nucleus (episomal) where it can be transcribed but will not be replicated upon cell division. This means that further administration will be required if additional gene expression is required. Adenoviral vectors can transduce both dividing and non-dividing cells, but can also induce a strong immune response, limiting their use in vivo. The reason for this immune response has been linked with several factors, including the recognition of virus capsid, virus proteins and virus genes as dangerous by the host cell. This immunity can result in rapid clearance of the virus in vivo and can also potentiate dangerous levels of inflammation.
Retroviral Vectors:
They carry their genetic material in the form of RNA. When a retroviral vector introduces its genetic cargo into a host cell it typically is accompanied by enzymes whose function is to create a DNA copy of the viral RNA via reverse transcription. Then, the DNA copy will be integrated into the hosts genome by the enzyme integrase, resulting in long-term gene expression. However, viral DNA integration can solely happen when cells are dividing, thus limiting in vivo application. Furthermore, retroviral vectors are prone to random integration into host chromosomes, which may result in potential mutagenesis.
Lentiviral Vectors (LV):
These are a subtype of retroviruses and function likewise by delivering RNA molecules in association with reverse transcriptase enzymes, with the additional capability to integrate their genome into non-dividing cells. These vectors have a limited genetic cargo capacity and include enhancer-promoter sequences that require careful management to minimize insertional mutagenesis
Adeno-associated Vectors (AAV):
They carry their genetic cargo in the form of DNA and possess the capabilities to transduce both dividing and non-dividing cells. Their DNA cargo does not integrate into the genome of the host cells, but rather persist within the nucleus as episomal DNA. AAVs have a much smaller genetic capacity compared to that of standard adenoviral vectors, however, transduced cells are minimally immunogenic and their safety profiles have been praised for in vivo use.
Viral Transfection
For viral vector production, plasmids containing the viral DNA and therapeutic genetic material are typically introduced into host cells in vitro. This equips the cells with the capabilities to produce viral particles containing the therapeutic gene. These vectors can then be harvested from the cell culture, purified, and subsequently used. This process is known as transfection and can be performed in various ways. Examples include physical methods such as electroporation and microinjection, as well as chemically mediated transfection agents, which have proven to be the most effective method.
Chemical mediated transfection involves the use of engineered polymers with a positive charge. These positive polymers spontaneously form complexes with negatively charged nucleic acids via electrostatic interaction. These complexes conceal the nucleic acids negatively charged allowing them to cross the lipid membrane and be internalized into cells through endocytosis. Examples of chemical mediated transfection reagents include peptides, lipoplexes, and synthetic polymers. Chemical mediated transfection has many advantages over other forms of transfection as it is efficient at all scales, simple, fast, and reproducible, as well as cost-efficient.
Sartorius Polyplus offers gold standard reagents for efficient production of viral vectors at all scales. These include PEIpro® , FectoVIR®-AAV and FectoVIR®-LV
PEIpro® for viral vector manufacturing
PEIpro® is the leading chemical-based transfection reagent for DNA delivery, allowing for flexible and scalable viral vector production. Extensive research studies were performed to develop and optimize this PEI polymer chemistry to achieve the highest transfection efficiency in both adherent and suspension cell systems. Thanks to a thorough process development conducted at Sartorius Polyplus, directly implementable protocols are available for large scale manufacturing of both preclinical and clinical batches of viral vectors with higher quality grades of PEIpro®, including PEIpro®-HQ and PEIpro®-GMP. PEIpro® is the gold standard r for the sustainable large-scale manufaturing of viral vectors (adenovirus, lentivirus, etc).
FectoVIR®-AAV for AAV manufacturing
AAVs are fast becoming the leading viral vector and are widely used in the development of gene-based advanced therapy medicinal products (ATMPs). The success of AAVs is owed, not only to their episomal DNA placement, but also to their broad tropism. Currently, one major challenge in the field is the manufacturing of f AAVs, in particular scaling up to obtain sufficient viral titers for the treatment of large patient groups. In this regard, FectoVIR®-AAV offers a revolutionary solution.
FectoVIR®-AAV is a novel transfection reagent, used for the industrial scale production of recombinant AAV (rAAV) vectors using suspension and adherent HEK-293 cells. FectoVIR®-AAV is an animal –free product, with guaranteed high viral titers production, improved flexibility/scalability, also available in GMP-grade quality, making industrial scale-up more efficient and easier. In comparison to competitors, the use of FectoVIR®-AAV results in a 10-fold increase in functional virus titer production. Compared to PEIpro®, FectoVIR®-AAV improves yields by at least 2-3 fold. FectoVIR®-AAV has been optimized to reduce complexation volume up to 1% of the culture media volume (standard complexation volume is 10% for others chemical transfection reagents). This major advantage reduces greatly production cost and alleviates technical constraints for the preparation and transfer of complexes into large-scale bioreactors. Unlike adeno-associated viruses (AAVs), which are primarily used for in vivo gene therapy, lentiviruses are preferred for ex vivo gene delivery into host cells. Lentiviral vectors are particularly favored for generating autologous cell therapies, such as CAR-T cells, due to their ability to permanently integrate the viral genome into host cell DNA. Typically, lentiviral vectors are produced by transfecting HEK-293 cells with viral and therapeutic DNA, a process that can be limited if the transfection reagent is not suitable for large-scale manufacturing.
To address this challenge, Sartorius Polyplus has developed FectoVIR®-LV, a transfection reagent specifically designed for lentivirus production. FectoVIR®-LV offers increased complex stability and reduced complex volume, enhancing viral titers by up to threefold compared to competitor reagents. This improvement allows for the production of more doses to treat a greater number of patients while simultaneously reducing lentivirus manufacturing costs
