Optimizing Lentiviral Vector Development for In Vivo Delivery of Cell and Gene Therapies

This content was originally published in Biocompare

Cell and gene therapies are transitioning from emerging innovations to increasingly standard treatments across therapeutic areas. Interest in this sector remains high, with more than 800 clinical trials across phases currently underway to investigate gene therapy products, according to the American Society of Gene & Cell Therapy.¹ As this market becomes increasingly competitive, drug developers face intense pressure to expedite clinical development and get their products to market as quickly as possible. Successfully crossing the finish line to commercialization for new gene therapies depends, to a great extent, on the timely development of viral vectors to deliver therapeutic material into cells.

Originally derived from the human immunodeficiency virus type-1 (HIV-1) lentivirus, LVVs are predominantly used as gene delivery vehicles for modifying cells ex vivo. Demand for LVVs as delivery vehicles for gene therapies has been rising significantly due to recent skyrocketing interest in in vivo CAR-T and in vivo gene editing approaches,² creating a need for robust, highly productive, and scalable manufacturing processes.

However, manufacturing LVVs can be a challenging proposition, especially for emerging drug developers that lack in-house expertise and developmental capacity. Moreover, downstream processing can be particularly challenging due to the low stability of lentiviruses and the fragility of the membrane envelope for these vehicles.

For many emerging gene therapy innovators, forming strategic partnerships with contract development and manufacturing organizations (CDMOs) can be key to advancing their LVV development programs. With their ability to quickly respond to emerging trends in LVV manufacturing, as well as their capabilities in process development and R&D, CDMOs can provide end-to-end support from development to manufacturing. Indeed, many of these firms regard LVV innovation expertise as an essential element of their own internal toolboxes as well as a key customer offering.

Challenges in lentiviral vector development

Lentivirus is an enveloped retrovirus, as such achieving and verifying LVV purity is a complex task with highly consequential risks. Process Impurities and low potency of final formulated LVV can lead to longer development timelines, compromise therapeutic efficacy, and result in CMC challenges, that may complicate regulatory approval. To assess LVV purity and ensure the integrity and efficacy of the particle, developers conduct infectivity assays. However, these assays often require multi-day execution, which is not amenable to real-time process monitoring and limits comprehensive process characterization to post-process assessment.

There are also several process-related issues that confound analytical characterization. For instance, it can be challenging to separate infectious from non-infectious particles, given that only a fraction of the produced LVV particles are infectious. Moreover, large impurities can closely resemble viral vectors, posing challenges in purification. Other bioprocessing hurdles include the lack of lentivirus-specific binding media and lack of current-Good-Manufacturing-Practice (cGMP) suitable high-capacity, highly selective affinity supports that can process at mild loading and elution conditions. These factors compound to make cell-based assays for LVVs highly variable and complicate decision-making during downstream processing.

The inherent instability of viral vectors requires establishing operational workflows that can maintain infectivity, remove impurities, and ensure viral safety throughout the process. LVVs are inherently sensitive to process environmental factors such as temperature, pH, conductivity, shear stress, and concentration of salts in buffers. Therefore, downstream unit operations, such as filtrations, must be kept as short as possible to preserve infectivity while maximizing yield.

For in vivo applications, where purity requirements are significantly more stringent than for ex vivo applications, low levels of LVV expression and frequently high ratios of non-infectious versus infectious LVV particles, as well as the presence of high-molecular weight impurities that are in the same size range as LVV particles (e.g., extracellular vesicles), all greatly exacerbate the challenges of developing efficient, high-yielding processes.

For adeno-associated vectors (AAVs) purification, separation of empty capsids from full capsids has long been a standard process step. In contrast LVV downstream processing currently lacks suitable tools for separating LVV particles that carry the gene of interest from those that are empty. Besides missing payload, these “empty” LVV particles can contain process impurities such as host cell protein (HCP) and host cell DNA (hcDNA), which may increase the risk of impurity related adverse effects in patients.

Emerging technologies offer potential solutions

CDMOs are seeking to stay ahead of the curve by investing in innovative methods and digital tools to address downstream processing challenges in LVV development. These innovations include advanced analytical methods such as lentivirus-specific infectivity assays, fluorescence-based technologies to assess phenotypic features of LVV particles, and methods to determine genome integrity within the particle, enabling a more comprehensive characterization of the LVV product.

Other innovations emerging in the field include lentivirus-specific affinity media, more reliable high-throughput assays, and new chromatography formats and sterilizing-grade filters to improve downstream purification of viral vectors. These emerging tools can be expected to improve manufacturability in the near future.

Technologies currently in use include stable producer cell lines (PCL), which improve batch consistency and reduce risk posed by plasmid DNA contamination. For in vivo LVV applications, in particular, host cell lines with edited major histocompatibility complex (MHC) genes have been shown to produce LVV with envelopes devoid of MHC molecules, as LVVs acquire a portion of host cell membrane during budding. This results in reduced immunogenicity compared to conventional LVV, making them more suitable for in vivo applications.³

CDMOs with internal process development and R&D capabilities are already collaborating with developers on incorporating some of the more promising technological innovations into their LVV programs. This includes alpha-testing several advanced LVV processing technologies in the field and continually evaluating methods and tools that can be quickly implemented at the GMP stage.

Ensuring continued innovation

As demand for cell and gene therapies continues to grow, the pharma industry will need to continue to address current challenges within a dynamic regulatory environment that is continuing to shift in the wake of technological advances and new medical insights. Innovators that forge LVV-focused strategic partnerships with CDMOs will play a pivotal role in enabling the next generation of cell and gene therapies to reach patients safely and efficiently.