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Manufacture Advanced Medicine, Trends & Forecasts

Know Your Vectors: a 101 on Lentiviral Therapies

According to the latest available information as of July 2022, there were 2024 gene and gene-modified-cell therapies in development worldwide. And data allows us to measure the expected growth of this specific sector; indeed, by 2025 the FDA expects to approve 10–20 cell and gene therapy products per year. By 2035, this specific sector is forecast to be worth $17.3 billion, growing at a CAGR of 18 percent between 2022-2035.

Laboratory-made viruses – viral vectors – retain the natural capability of wild-type viruses of delivering genetic material to the cells that they infect and are therefore an ideal vehicle to transport genes to sick cells.

Vector selector
 

Drug developers choose from a vast array of potential vectors by considering several key aspects, including the disease being treated, the characteristics and location of cells that need to be targeted, the amount of genetic material needing to be delivered. Viral vector-mediated gene delivery can be carried out in two ways: ex vivo or in vivo. If the cells causing the disease can be collected from the patient, corrected in a dish, and then reinfused back into the patient, there is no need to give the vectors specific signals for which cells to infect. Put another way, when the cells requiring modification are the only ones that come into contact with the vectors, no specific viral tropism is required, meaning that the vectors can carry broad-tropism envelope proteins, such as the vesicular stomatitis virus envelope protein G (VSG-G). 

However, if isolating the disease-causing cells is not feasible, genetic modification of the cells must be performed in vivo. In this method, viral vectors are delivered either intravenously or locally to a specific organ. Needless to say, in such cases, off-target effects must be avoided to prevent health risks and adverse events.

Lentiviral vectors are one of the most widely used forms of viral vector. They can be delivered both ex vivo and in vivo, and can transduce both dividing and non-dividing cells, which lets them permanently correct target cell genomes and allows for long-term, stable, transgene expression. Moreover, lentiviruses are characterized by their low levels of pathogenicity.

Lentiviral vectors are enveloped viruses, which means that they bud from the producer cell membrane and are released into the cell culture medium. Their outer layer can be “pseudotyped” – meaning that the original surface proteins can be replaced with those of other viruses to direct tropism and reach a high specificity for the target cells. This feature constitutes an opportunity to personalize the vectors, and, consequently, to render gene delivery even more efficient. 

The most commonly used lentiviral vector producer cell line is HEK293. These packaging cells must be transfected with several plasmids to receive their instructions for manufacturing the lentiviruses. Plasmid transfection is the very first step of the production process. The greater the number of plasmids in which the information is distributed, the lower the recombination probability of the different sequences, which increases the biosafety of the process. The envelope protein needs to be encoded in one of the plasmids in transfection.

Know your target
 

Perhaps the most vivid example of ex vivo lentivirus-mediated therapies is CAR T therapy, in which T lymphocytes are extracted from cancer patients and genetically manipulated so that, once infused back, they specifically recognize and destroy tumor cells. The CAR-T market size was valued at $6.1 billion last year and is predicted to exceed US$21 billion by 2030. T lymphocytes can be infected fairly effectively with VSV-G pseudotyped lentiviral vectors.

Other target cells, such as human hematopoietic stem cells or NK cells, express few or no VSV-G receptors and are very “reluctant” to be infected. Consequently, higher ratios of infectious agents are required for therapies in which these cell types are the target, which impacts the batch manufacture size necessary to meet corresponding demands. Pseudotyping with alternative envelope proteins highly increases the efficiency of infection – and several pseudotyping options exist. Unfortunately, replacement of the envelope proteins is challenging; it’s not merely about diving into the literature and selecting the most efficient pseudotype – it also involves a great deal of laboratory work and fine-tuning plasmid proportions in 2D before then transferring production to 3D bioreactors.

Inside in vivo
 

When it comes to in vivo therapy, the approach to production changes considerably. As described above, in an in vivo therapy, lentivectors must be released either intravenously or locally to a specific organ. At VIVEbiotech, we manufacture lentiviruses for five different partners who administer them in vivo. In one of the five cases, the viruses are administered ophthalmically. In another case the therapy is directed to the liver, so will be administered in the patients’ portal vein. In other cases, the viruses will be administered systemically, and therefore must carry very specific instructions so that they only infect the desired cells and thus avoid off-target transduction that could trigger adverse effects. This can only be achieved by pseudotyping.

In addition to the challenges of pseudotyping, in vivo therapies require much larger virus quantities to transduce the required number of target cells – and that means the production process must be fine-tuned to ensure optimal performance. In short, we must go beyond simply increasing the number of viruses produced upstream, but also concentrate them as much as possible during the downstream process – crucially, without losing viral functionality.

Last but not least, to augment the efficacy of in vivo lentivirus-mediated therapies, we have seen some of our clients request the use of their own packaging cells to lend the lentivectors unique characteristics, such as the ability to be masked to further lower their risk of recognition by the immune system.

As is always the case when one is posed with competing options, we know and should be cognizant of the pros and cons. Lentivirus-mediated ex vivo and in vivo therapies each have their own intrinsic characteristics, and I hope that this piece has helped to strengthen your grasp on them.

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About the Author
Naiara Tejados, PhD

Head of Marketing and Technology Development

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