Advances in manufacturing in vivo lentiviral vectors: Challenges and solutions

Lentiviral vectors (LVVs) have proven to be extremely versatile genetic delivery vehicles, gaining space as a gene transfer technology tool used both for cell and gene therapies. They are utilised for an increasing number of commercial advanced therapy technologies, from chimeric antigen receptor (CAR) T-cell therapies to ex vivo gene therapies and, earlier last year, the first approved T-cell receptor therapy. They are also increasingly popular for a wide range of in vivo applications, particularly for rare and immunological diseases. The recent authorisation of at least three in vivo CAR-T clinical trials utilising LVVs further highlights growing interest in the field.

GMP manufacturing of LVVs is complex due to the intrinsic characteristics of these vectors and their sensitivity to environmental factors, requiring a very high level of technical expertise in virology and bioprocessing for their production.

Despite these challenges, the natural mechanisms of gene transfer with LVVs make it an attractive option, due to their efficiency and well-understood behaviour compared with newly developed tools. LVVs can integrate a larger payload than other kinds of vectors, up to 9 kb. They can transduce both dividing and nondividing cells, and different envelopes are available. They have been commercially available for years, which increases the robustness of this gene transfer technology and their acceptance by regulators.

These benefits underscore the growing importance of making LVV manufacturing commercially feasible for in vivo therapies, given their potential to lead the coming wave.

Meeting in vivo LVV manufacturing requirements

In vivo gene therapies are of course used differently than ex vivo, as genetic material is applied directly to the patient. There are several implications from a vector manufacturing perspective. One is potential location: for in vivo applications, the produced LVV can be directly administered by the clinical centre caring for a patient. Generally, the overall timeline to produce the therapeutic product is shorter for in vivo applications, and the amount of LVV required is often larger, with a few exceptions.

Because of their direct use in patients, LVVs for in vivo applications require a wider analytical characterisation panel than ones used for ex vivo. Also, plasmid quality can vary starting from early clinical trials, with stricter requirements for in vivo than ex vivo applications. This will depend on the final application for which the in vivo therapy is being used.

Manufacturing LVVs has its own peculiarities and associated complexity when compared with other vector types, especially because of the intrinsic characteristics of the vectors – for example, LVVs are very sensitive to shear forces and physicochemical parameters, among other factors. Therefore, LVV production is particularly complex, and it requires highly skilled and specialised personnel.

The main challenge for in vivo LVV programmes is optimising the production process to deliver vectors that are cost-effective and functional. Because of the large amounts of vector needed per patient, the ability to deliver cost-effective LVVs will have a direct impact on the access to these therapies once marketed, especially considering the reimbursement landscape. In addition, the sensitivity of LVVs makes it especially important to optimise all the manufacturing steps, such as transfection, harvesting, clarification, purification, concentration, buffer exchange, and sterile filtration, as all of them can have an impact on the final functional titer.

Lowering costs, improving purity

This is where LVV manufacturing expertise is critical, as lowering the cost of manufacturing requires several improvements, starting with an increase in productivity by selecting high-producer clone cell lines. In addition, it may be necessary to optimise the construct or transfection ratio, as well as every single step of the upstream process, typically with a specialised manufacturing platform. The focus must be on getting the highest titers possible.

The total amount of vector being produced can be indirectly measured by p24 capsid protein, but more relevant is whether the functional vector is able to transduce cells and integrate. Therefore, it is critical to be able to adapt to LVVs’ intrinsic characteristics to obtain the greatest functional titer, better measured as the ratio between physical particles and functional viruses (p24/VCN).

Conversely, another way of increasing cost-effectiveness is diminishing the amount of lentiviral vectors to be administered in vivo per patient. This can be done through key tools like new LVV pseudotypes and modified cell lines that are able to decorate the vector. The use of such adapted pseudotypes or decorated vectors can also have an impact on the targeting of the vectors, potentially increasing their functionality and diminishing undesired side effects when directly administered to the patient.

Another challenge is reaching the purity levels that are required for in vivo LVV use, which is important from a regulatory point of view, but also for the patient, as it can potentially have a direct effect on the functionality of the vector. Very well-developed downstream processing is vital for purity, allowing not only a better recovery, but a decrease in contaminants. Meeting regulatory requirements is as relevant as getting a high productivity as it has a direct impact on the functionality of the vector itself.

Manufacturers with the right expertise can take all these aspects into consideration and address them with a single, versatile GMP manufacturing platform that meets EMA and FDA criteria for producing both in vivo and ex vivo LVVs, with multiple generations of both integrating and non-integrating, using different pseudotypes.

An in vivo future

Even though the in vivo use of LVVs is already a reality with a very promising future, there is still room for improvement in terms of manufacturing productivity and efficiency, so the cost-effectiveness of these therapies is further optimised.

In the upstream phase, efforts include different approaches, such as the use of more efficient producer cell lines, as well as the avoidance of retrotransduction of the vector by the packaging cell line. Downstream, continued work is being done to optimise every single step to enhance recovery, particularly the chromatography phase and sterile filtration.

Moreover, important efforts are being made to characterise the vector more deeply, including not only the release testing panel, but more sophisticated analytical tools that allow better understanding of what is happening all through the production process. This will allow further process optimisation according to analytical results. To prepare for this, the continued reinforcement of internal analytical development capabilities is compulsory in partnership with specialised CROs with innovative and sophisticated analytical tools. Recent advances have the potential to make the difference when implemented, including microfluidic immunoassay platforms, technology for biophysical characterisation at the level of individual LVV particles, and capabilities in specific potency assay development.

The next five to 10 years look very promising in the LVV gene therapy space, with widespread expectations that the number of approvals for therapies using LVVs will dramatically increase. These therapies are expected to be approved for increasingly prevalent diseases and for earlier lines of treatments. Following the trend of increased clinical use of LVVs in vivo in recent years, growth is expected to continue as we soon begin to see the first commercial approvals.