The demand for gene therapies based on viral vectors has increased to unprecedented levels, thanks to their potential to help treat previously incurable diseases. The two most outstanding vectors? Lentiviral Vectors (LVs) and Adeno-Associated Viral Vectors (AAVs): Due to the increased research and positive clinical results they are seeing in a wide range of applications, including cancer, heart disease, and hematologic and genetic disorders. The more drug developers look to expand this range of therapeutic areas, the greater the demand for commercial-scale development. Therefore, it is important to understand not only how these two vectors can be applied to drug development, but also the capabilities required for scale-up that allow us to bring these innovative therapies to patients.
LV vectors are derived from the single-stranded RNA retrovirus HIV-1 and have been widely used due to their ability to infect non-dividing cells, efficiently integrate into the host genome, carry large transgenic payloads, and allow for long-term transgenes. expression. They are predominantly used as delivery vehicles to introduce genetic modifications in cell therapies, such as CAR-T and HSC gene therapies. Importantly, recent regulatory approvals and clinical successes with LV vectors are sparking even more interest among drug developers.
Let’s look at the benefits of LV vectors in more detail:
- Volume. LV vectors can carry a large volume of transgenes, up to 8 kilobytes, into host cell DNA, helping to address more indications.
- Gene delivery. The viral genome is passed on to daughter cells during division, leading to stable, long-term expression of foreign genes.
- Applicability. Unlike other types of retroviruses, lentiviruses can infect cells whether they are dividing or not, allowing them to transduce and genetically modify non-replicating cells.
- Immunogenic profile. Recent lentiviral vector designs have few negative side effects; an advantage they share with AAV vectors.
However, LV vectors also present two important safety risks.
The first is the risk of accidental exposure because HIV can self-replicate during manufacturing thanks to the high rate of mutation and recombination of the lentivirus.Although research shows the risk to be low, it remains a significant safety concern for engineers and lab workers during development. Before using a lentiviral vector system, a risk assessment must be completed and documented. Lentiviral vectors can generally be handled safely using BSL-2 or BSL-2 enhanced controls, depending on the risk assessment.
The second risk is the possibility of oncogenes being produced in cells through insertional mutagenesis. For this reason, lentiviral vectors are predominantly used for cell therapy applications with ex-vivo genetic modification of cells. Only limited use is seen for direct in vivo therapies.
Unlike their LV cousins, AAV vectors are single-stranded DNA parvoviruses that can replicate only in the presence of helper viruses, such as adenovirus, herpesvirus, human papillomavirus, and vaccinia virus. Following several landmark approvals, AAV vectors are now being used for in vitro, ex vivo, and in vivo research. AAV therapies predominantly target rare genetic disorders for which the patient population tends to be very limited. Because the market is so small, drug developers feel great pressure to be first to market with their therapies.
The biological elements of AAV vectors make them a very attractive candidate for gene therapy for several reasons:
- Safety. AAVs do not cause any known human disease and therefore have very low pathogenicity and require less equipment to handle.
- Immune response. AAVs have a low immunogenic profile, complementing their low pathogenicity during gene delivery and reinforcing their biosafety.
- infectivity. Thanks to their ability to deliver genetic material to dividing and non-dividing cells, AAVs can be applied in different indications, an advantage they share with LV vectors.
As with LV vectors, AAV vectors have several drawbacks that affect their applications and efficiency.
First, AAV vectors are limited by their restricted ability to insert transgenic DNA; due to their relatively small transgene size, they cannot deliver genes larger than 4.8 kilobytes. Second, the generation of neutralizing antibodies against AAVs in nonhuman primates (NHPs) and humans may attenuate the curative effects of AAV-mediated gene therapies and limit the size of patient populations suitable for these therapies. Third, there are several different serotypes and capsids for AAV, all of which have different production and purification requirements and vary greatly with respect to function and efficacy. Fourth, AAV pharmaceuticals have varying degrees of empty and partially filled capsids, and this has implications for safety and efficacy. In general, the highest possible percentage of AAV particles with the complete transgene DNA is desired, and this varies significantly depending on the method of production, the AAV serotype, and the transgene itself. The latter two factors present significant manufacturing challenges for AAV therapies.
Overall, the industry’s collective ability to successfully scale LVV and AAV vectors faces two challenges:
i) Currently, the manufacture of each viral vector requires different processes, so companies cannot apply a single approach for all their upstream and downstream processes. Therefore, manufacturing requires extensive scientific and market expertise to make the informed decisions necessary to develop a sound plan.
ii) Given the limited industry experience with supplying viral vectors on a commercial scale, companies should work closely with regulatory agencies. This can be especially challenging during the transition from preclinical to commercial, where complexities arise that can cause potential delays that result in increased costs.
As demand continues to increase, pharmaceutical companies must understand how to meet these challenges in order to continue delivering their life-saving medicines.
Head of Business Development for Viral Vector, Cell and Gene Technologies (CGT) at Lonza
It works closely with the innovation, operations, engineering, strategic marketing, and business teams to enable prioritization, strategic development, and commercialization of viral vector production services for CGT. Suparna’s background is in neuroscience and she earned her Ph.D. in neuropharmacology from the University of Toronto. She has over 15 years of extensive pharmaceutical and CDMO experience driving innovation, drug discovery, product and service development for the CNS, oncology, and cell and gene therapy.