The progress of gene therapy, largely powered by adeno-associated viruses (AAVs), has transformed treatment possibilities for previously untreatable rare disorders. With several FDA approvals and many more patient success stories, AAV-based therapies represent a breakthrough in medicine. While AVVs continue to play a key role in advanced therapies, researchers are actively exploring new vectors to expand the reach of these transformative treatments.
This article examines two rapidly growing modalities poised to expand the cell and gene therapy landscape: lentiviral (LV) vectors and plasmid DNA (pDNA). Though serving different clinical applications, both hold significant promise for developing treatments beyond AAVs’ current capabilities.
For manufacturers in this space, strategic positioning is critical. The explosive growth from mRNA vaccine platforms and continuous innovations in CAR T cell therapy have created both opportunities and challenges. Understanding these emerging vector technologies could determine whether your operations thrive at full capacity or struggle to adapt to changing market demands.
To gain some firsthand insights, we consulted Chaira Pacini, a Cytiva field application specialist (FAS) specializing in downstream operations, and Peiqing Zhang, Scientific Director of Advanced Therapies at Cytiva. Their combined knowledge and hand-on experience provides valuable guidance for those navigating the evolving landscape of gene delivery systems.
Why lentiviral vectors and plasmid DNA?
AAVs have established themselves as a leading vector in gene therapies due to their favorable characteristics—ability to enter multiple cell types, long-lasting expression of gene-of-interest, low immunogenicity, and non-pathogenicity. Across the 12 most common AAV serotypes, a wide range of tissues can be targeted. However, AAVs are limited by their ability to carry genetic payloads above 5 kb, which has prompted research to explore alternative delivery systems.
LVs offer a compelling advantage: they can carry nearly double the genetic payload of AAVs, vastly expanding the range of treatable diseases with a single viral vector. “No serotype selection is needed to transduce a wide range of cell types”, Chiara explains. LVs are integrating vectors that provide long-term transgene expression. This characteristic, combined with an improved safety profile compared to other retroviruses, has positioned LVs as the preferred vector for ex vivo gene transfer applications, particularly in CAR T cell therapy.
Meanwhile, pDNA has experienced its own surge in demand. Though it has long served as a key raw material in mAbs production, pDNA now plays a crucial role in transient expression applications—serving as templates for mRNA vaccines and enabling the transfection process for viral vector production. The COVID-19 pandemic demonstrated the remarkable potential of mRNA vaccines for illness prevention, which catalyzed widespread interest from many researchers and biotech companies alike. The result was a worldwide push to build and scale sovereign mRNA manufacturing capabilities, with pDNA production being a critical foundation of this process. The industry now faces a pressing challenge: producing high purity, GMP-grade pDNA with the flexibility and speed required to meet rapidly growing demand across multiple therapeutic platforms.
What that future of LV and pDNA may hold
CAR T therapy has made remarkable strides in recent decades, with substantial untapped potential still awaiting discovery. LV vectors play a fundamental role in this therapeutic approach by facilitating gene transfer in CAR T cells. “Behind each CAR T program, there is very likely an LV program,” emphasizes Peiqing. With well over a thousand ongoing CAR T clinical trials worldwide, demand for LV production is expected to continue its upward trajectory.
The future of CAR T could be in vivo, Peiqing adds. “So far, there are well over 100 clinical programs using LV vectors for in vivo applications,” suggesting a potential for faster and more accessible treatments. From a manufacturing perspective, “in vivo treatments come with more scrutiny on the quality and purity of the viral vector product”. Optimizing the media, cell line, bioprocess, chromatography, and filtration technologies will be essential to achieve the quantity and purity levels necessary for direct administration of in vivo CAR T therapies to patients.
We’ve already established that the surging interest in mRNA vaccines has motivated many countries to ramp up pDNA manufacturing, but what about the other applications of pDNA? Beyond serving as a template for mRNA, pDNA is also an essential across the viral vector landscape. “As long as there’s viral vectors, there’ll be demand for pDNA,” Peiqing explains, highlighting its critical role in producing various viral vectors, including LV and AAV.
Emerging applications aim to use pDNA as the active drug substance, such as in DNA-based vaccines or protein replacement therapy. These novel applications introduce different—typically more stringent—purity requirements and manufacturing considerations compared to traditional uses.
This convergence of advancing CAR T technologies, mRNA platforms, and innovative therapeutic approaches creates significant opportunities for contract development and manufacturing organizations (CDMOs). “In the advanced therapy field, we see a lot of molecules originating from academia, translational centers, clinical centers, hospitals, and so on—these are not necessarily the places known for large-scale manufacturing,” notes Peiqing. “Startups are also likely to rely on CDMOs to support their scale up and meet viral vector manufacturing demand.” This positions experienced manufacturing partners as critical enablers for translating promising research into clinical-stage therapies.
Manufacturing for high yield and high purity
For organizations seeking to incorporate LV or pDNA manufacturing into their portfolio, early preparation and strategic planning are essential. Working with an experienced team for process development and optimization is crucial for building a robust manufacturing platform efficiently. According to Charia, innovators and biotech companies increasingly prioritize CDMOs that already have experience with LV or pDNA processes and a proven track record of delivering both quality and quantity.
Recent years have seen significant advancements in manufacturing processes for both LV and pDNA, focused on achieving high-yield production while maintaining stringent quality standards.
Lentiviral vectors present unique manufacturing challenges. As an enveloped virus, LV exhibits a particular sensitivity to process stressors, including pH fluctuations and varying salt concentrations. Its larger size compared to AAV creates complications for maintaining consistent recovery rates. Improving LV yields requires streamlined manufacturing processes with carefully optimized conditions. Effective strategies may include reducing the number of processing steps and automating sample handling (1). Charia notes that many of her customers have successfully made the switch from adherent cell lines to suspension cell cultures. "This approach helps increase the cell density and ultimately produce more doses using fewer operator touchpoints," she explains, highlighting how this shift enhances both productivity and process consistency.
For pDNA, the manufacturing challenges center on both scale and specificity. “Meeting the upcoming demands for GMP-grade material will involve not only optimizing yields but also isolating the desired form of pDNA”, Peiqing explains. In particular, manufacturers must separate the therapeutically preferred supercoiled pDNA from less effective open circular forms. “To accomplish this separation, we’ve developed a two-step chromatography purification scheme that, when combined with appropriate filtration steps, can achieve high-purity supercoiled pDNA.” These high-precision requirements for molecular configuration represent exactly the type of complex processing challenges driving biotechnology companies toward partnerships with experienced CDMOs capable of supporting their scale-up needs.
Workflows for plasmid DNA and lentiviral vectors
Meeting current and future manufacturing demands effectively while minimizing risk requires thoughtful workflow design that prioritizes flexibility and agility. As production standards for both LV vectors and pDNA continue to evolve—with increasingly stringent purity requirements and higher yield expectations—implementing adaptable manufacturing systems becomes essential. Equipment that can be reconfigured to accommodate process improvements or even support multiple modalities helps prevent costly downtime and resource inefficiency.
Single-use technology can play a big role in achieving this needed flexibility. The changeover time between different products can be dramatically reduced through implementation of single-use bioreactors in upstream processes and filtration and chromatography systems in downstream operations. This approach not only accelerates product transitions but also reduces cross-contamination risks, especially important in facilities handling multiple vector types.
For organizations already manufacturing AAV vectors, there may be opportunities to leverage existing equipment. Many downstream processing technologies—particularly chromatography and filtration systems—can be repurposed for LV vector production with minor modifications. This flexible equipment utilization can significantly reduce costs when expanding into new modalities.
Most importantly, working with a supplier who knows how to implement single-use technology in biologics manufacturing and has experience with supporting robust workflows for LV and pDNA will help you stay ahead of the curve and ready to meet demand for next-gen therapeutics.
Beyond equipment considerations, process knowledge and expertise are critical success factors. Partnering with suppliers who possess extensive experience implementing single-use technologies in biologics manufacturing and have demonstrated success supporting robust workflows for both LV and pDNA production can help you stay one step ahead. As competition intensifies in the advanced therapy space, the combination of flexible manufacturing infrastructure and specialized knowledge will differentiate successful manufacturers.
CDMOs: Are you ready for tomorrow’s viral vector manufacturing demands?
While AAV-based therapies will continue to play a pivotal role in the cell and gene therapy landscape, LV vectors and pDNA are rapidly expanding treatment possibilities beyond previous limitations. These emerging modalities are enabling breakthrough approaches like CAR T cell therapy to address conditions once considered untreatable. With manufacturing technologies advancing at unprecedented speed, the production of high-quality vectors at commercially viable scales is increasingly within reach.
Innovations driving this field forward will likely continue to emerge from research institutions and agile biotechnology startups. These organizations will depend heavily on experienced CDMOs with specialized expertise to develop, optimize, and scale their manufacturing processes. In this evolving marketplace, competitive advantage will belong to CDMOs that have invested in flexible manufacturing platforms capable of adapting to these emerging modalities. Those positioned with technical capability, process knowledge, and manufacturing agility stand to become essential partners in bringing the next generation of advanced therapies to patients worldwide.