The Pros and Cons of Lentiviral and Adeno-Associated Viral Vectors

Demand for viral-vector-based gene therapies has risen to unprecedented levels, thanks to their potential to help treat previously incurable diseases. The two vectors most in the spotlight? Lentiviral (LV) vectors and adeno-associated viral (AAV) vectors – due to the increased research and positive clinical results they are seeing across 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. So it’s important to understand not only how these two vectors can be applied to drug development, but also the capabilities required for scale-up that allows us to bring these innovative therapies to patients.

LV vectors are derived from the single-stranded RNA retrovirus HIV-1, and have been used extensively because of their ability to infect non-dividing cells, efficiently integrate into the host genome, carry large transgene loads, and allow for long-term transgene expression. They are predominantly used as delivery vehicles for introducing genetic modifications into cell therapies, such as CAR-T, and HSC gene therapies. Importantly, recent regulatory approvals and clinical successes with LV vectors are spurring even more interest among drug developers.

Let’s look at the benefits of LV vectors in more detail:

• Volume. LV vectors can carry a high volume of transgenes – up to 8 kilobytes – into the DNA of host cells, which helps address more indications.
• Gene delivery. The viral genome is passed onto daughter cells during division, leading to long-term and stable expression of exogenous genes.
• Applicability. Unlike other types of retroviruses, lentiviruses can infect cells whether or not they are dividing, which allows them to transduce and genetically modify cells that do not replicate.
• Immunogenic profile. The recent lentiviral vector designs have low negative side effects; an advantage they share with AAV vectors.

However, LV vectors also present two major risks to safety. 

The first is a risk of accidental exposure because HIV can self-replicate during manufacturing thanks to the lentivirus’s high mutation and recombination rate.Though research shows that the risk is low, it remains a major safety concern for lab engineers and workers during development. Before using a lentiviral vector system, a risk assessment must be completed and documented. Typically, lentiviral vectors may be safely handled using either BSL-2 or BSL-2 enhanced controls, depending upon the risk assessment.

The second risk is the potential for oncogenes to occur in cells through insertional mutagenesis. For this reason, lentiviral vectors are predominantly used for cell therapy applications with genetic modification of cells ex-vivo. 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 the adenovirus, herpes virus, human papillomavirus, and vaccinia virus. Following several landmark approvals, AAV vectors are currently 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 highly limited. As the market is so small, drug developers feel immense pressure to be first to market to commercialize their therapies.

The biological elements of AAV vectors make them a very attractive candidate for gene therapy for several reasons:

• Safety. AAVs do not produce any known human diseases and thus 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 across different indications – an advantage they share with LV vectors. 

As with LV vectors, AAV vectors come with several drawbacks that affect their applications and efficiency.

Firstly, AAV vectors are limited by their restricted capacity for insertion of transgene DNA; because of their relatively small transgene size, they are unable to deliver genes larger than 4.8 kilobytes. Secondly, the generation of neutralizing antibodies against AAV in non-human primates (NHP) and humans may attenuate the curative effects of AAV-mediated gene therapies and limit the size of patient populations suitable for these therapies. Thirdly, there are several different serotypes and capsids for AAVs, all of which have different production and purification requirements and vary greatly with respect to function and efficacy. Fourthly, AAV drug products have varying degrees of empty and partially filled capsids, and these have implications for safety and efficacy. Generally, the highest possible percentage of AAV particles with the full transgene DNA is desired, and this varies significantly depending on the production method, AAV serotype, and the transgene itself. The latter two factors introduce significant manufacturing challenges for AAV therapies.

Overall, the industry’s collective ability to successfully scale up LVV and AAV vectors faces two challenges: 

i) Manufacturing each viral vector currently requires different processes, so companies cannot apply a one-size-fits-all approach to their upstream and downstream processes. Therefore, manufacturing requires immense scientific and market expertise to make the informed decisions necessary for developing a robust plan. 

ii) Given the industry’s limited experience with commercial-scale viral vector supply, companies need to 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 resulting in increased costs. 

As demand continues to rise, pharma companies must understand how to navigate these challenges to continue delivering their life-saving medications.