Advancing therapies based on viral and nonviral vectors

Cell and gene therapies have evolved over the past decade to represent a substantial portion of advanced medicines approved by regulatory authorities around the world. Chimeric antigen receptor T cell (CAR T) is leading the way for cell therapies – for blood cancers now but potentially solid tumors and diseases beyond oncology in the near future. Beyond CAR T, other cell types are advancing in clinical trials. Several gene therapies have been approved for rare diseases with potential for treating more prevalent disorders. Both cell and gene therapies typically use viral vectors for gene delivery – lentivirus (LV) for CAR T and adeno-associated virus (AAV) for gene therapies. Nonviral methods are being explored as an alternative method for gene delivery. Solving the challenges of existing workflows as well as innovating for future possibilities will fuel the industry for many years to come and bring more treatment options to people in need.

Challenges and opportunities for CAR T and LV

As indications expand to potentially treat a dramatically higher number of people, it’s more important than ever to make manufacturing as efficient as possible, with closed processing and smart automation that minimizes the risks of contamination and operator-to-operator variability. Flexibility is on the wish list for anyone interested in fine-tuning processes and producing other cell therapy types – and it’s a must for contract development and manufacturing organizations (CDMOs) to enable them to run a client’s process as they specify. Cell processing equipment and developed processes need to be scalable to make sufficient high-quality product for a growing number of people. And of course, it’s also important to bring costs down, with the aim of lowering the prices and improving access around the globe.

In addition to the challenges with the cell processing workflow, it’s far from simple to manufacture the lentiviral vector (LV) used for gene delivery. There’s much room to improve the productivity for this particularly labile virus that must be treated gently throughout processing. Chromatography steps are not straightforward as there’s no established platform. Finally, a huge challenge comes at the sterile filtration step, where the similar size of the lentivirus compared to the filter pores leads to a loss of up to 50% of the nearly final product. The lentivirus production process would benefit from solutions for these challenges. A fully closed process offers promise and is one of the big improvements Margherita Neri, formerly Vector process development Director, now GMP Manufacturing Sr Director, AGC Biologics would like to see.

Challenges and opportunities for AAV gene therapies

While adeno-associated virus (AAV) production processes have improved incrementally, there’s still a long runway for refining the workflows further and bringing costs down. Until now, most in vivo gene therapies have targeted rare diseases. There’s a trend towards more prevalent indications with the potential to eventually expand to common disorders. With a growing number of approved therapies―and many more in the pipeline for a much larger population―it’s a challenge to meet the infectious titer requirements for today’s therapies let alone for future ones. Interest is high in improving the upstream titer and percentage of full capsids. Downstream, there’s a focus on achieving higher recoveries. With a wide variety of AAV serotypes being used, it’s particularly difficult to achieve platform processing. Manufacturers are looking for solutions to overcome these challenges while improving robustness to allow larger batch sizes. Further improvements in vector quality could minimize concerns over safety.

Aaron Dulgar-Tulloch, CTO Genomic Medicine, Cytiva, sees an immediate need on the tool and technology front but believes the industry will start to experience some productivity enhancements driven by biology. As an example, Margherita Neri says that when clients move to the more advanced clinical phases, they might consider stable cell lines, because the gene is defined and already shown to be functional for the specified treatment. She adds that stable cell lines are well accepted for their robustness. But, of course, a very deep characterization of the genetic stability is required, as is true for transient methods used today.

About viral vectors in general, Margherita Neri cites analytical methods as a key chemistry, manufacturing and controls (CMC) point, as analytical methods typically evolve along with the process. The final methods must be qualified and then validated in QC to have reliable data that helps in analysis of process performance qualification (PPQ) data and also demonstrates that the process is robust and reliable.

The future of cell and gene therapies

The therapeutic potential of autologous CAR T is moving beyond treating blood cancers. Autologous CAR Ts, in particular those targeting CD19, are also being evaluated for treating autoimmune conditions; the aim is to reset the immune system with long-acting CAR T cells (1). Acknowledging the fact that 90% of cancers are solid tumors, there are active clinical trials evaluating autologous CAR T in this area. Apart from the use of CAR T for tumor recognition, T-cell receptor (TCR) is also being applied clinically in order to enhance the potency of T cells, especially towards solid tumors. In 2024 we saw the first approvals of a TCR (2) and a tumor-infiltrating leukocyte (TIL) (3) therapy.

Other ex vivo therapies using emerging cell types are advancing in clinical trials. For example, natural killer (NK) cells (i.e., CAR NK), macrophage cells (CAR M), and other T-cell therapies (such as δγT cells) are being investigated for both autologous (one-to-one) and allogeneic (one-to-many) use, the latter aiming to bring down the manufacturing cost by scaling up the production.

Another emerging trend is to move cell therapies from ex vivo genetic modification to in vivo. In vivo CAR provides great opportunities to simplify manufacturing (i.e., no more cell processing), reduce manufacturing cost, and enhance treatment convenience. The key challenge for in vivo cell therapy resides in specific targeting and gene delivery. Lentiviral vectors are being evaluated for CAR delivery and demands fit-for-purpose biological and manufacturing tools to ensure safety, purity, quantity, and potency of such lentiviral vectors.

Nonviral methods―particularly nucleic acids (including mRNAs and gene editing tools) delivered by lipid nanoparticles (LNPs)―are being evaluated for cell and gene therapies. LNPs, similar to those utilized in mRNA vaccines, provide the safety and flexibility to be applied either in vivo or ex vivo, and they can readily scale to large production volumes. In December 2023 the first CRISPR/Cas9 edited gene therapy was approved by the United States Food and Drug Administration (4). Other gene editing technologies in clinical trials include base editing, prime editing, and epigenetic editing (5).

With more drug modalities available for genomic medicine, we’re also seeing a trend of modality convergence (i.e., the combination of two or more modalities to enable a new application or create synergistic effect). The utility of combining LNPs towards cell therapies as mentioned previously is one example. Another example is the combinatorial use of CAR T therapy with an mRNA personalized cancer vaccine; the former to wipe out the cancerous cells and the latter to create a cancer-specific immunity within the recipient (6).

We’re at the crossroads of the cell and gene therapy era. Despite the hurdles—in part due to the manufacturing complexity—cell and gene therapies would benefit from a toolbox approach whereby different options are carefully evaluated and mapped into a flexible, scalable, and productive workflow to maximize the therapeutic promises of such transformative therapies.

How Cytiva can help advance cell and gene therapies

Many of the currently approved cell and gene therapies include Cytiva technologies. These are backed by decades of industry experience and the reach of a large global organization, including dedicated teams well-versed in working alongside customers to help them develop their processes and move candidates through clinical trials to commercialization.

We understand that the status quo isn’t good enough when people are waiting for new, highly effective treatments. There’s a need for more efficient, secured, and standardized processing for cell therapies, and companies are pursuing many cell types beyond CAR T. To meet these goals while preserving the flexibility to modify protocols based on individual needs, we collaborated with a key cell therapy manufacturer to develop the next system for cell therapy development and manufacturing. In addition to supporting autologous therapies, the system is well-suited to small-volume allogeneic therapies from a primary cell population.

Many cell and gene therapies in development continue to use viral vectors for gene delivery. We have refined the processes for lentiviral vector and adeno-associated viral vector to streamline the workflows, enhance yield, and maintain quality. Baseline separation of empty and full capsids is achievable for multiple AAV serotypes using a standard protocol; all that’s required for each serotype is to modify the magnesium chloride concentration or step length. To save time when developing chromatography steps in general, digital mechanistic modeling is an attractive alternative to traditional design of experiments. Looking towards simplifying AAV production and enabling scales for more prevalent conditions, a full suite of cell lines is available, including a producer cell line that requires only addition of a chemical induction agent to start production.

Understanding that nonviral methods of gene delivery are also of interest, we have expanded our portfolio and services to cover end-to-end workflows for mRNA and lipid nanoparticles (LNP). We offer an expansive library of lipids and LNP compositions designed for specific therapeutic applications, as well as an LNP manufacturing platform that enables small-scale screening for research all the way to GMP production in support of clinical trials. These LNP tools are available through off-the-shelf kits and instruments or through our service offering. Working together, we can help customers find optimal delivery solutions depending on their tissue of interest and payload, and then we can collaborate with them to progress those all the way to good manufacturing practices (GMP) environments. Formulation systems extend all the way through their preclinical and clinical development programs.

Says Dulgar-Tulloch, “We want to work with customers to help them accelerate the development, not just hand over reagents and wish them the best of luck…We can pull viral or nonviral technology together with the cell therapy and nonviral gene editing, and we can start to provide true end-to-end solutions for not just portions of their workflow, but for everything that's required for them to bring that therapeutic to market.” He adds, “We're staying at the forefront of the technologies, and it’s a great time to collaborate with us so that we can work together on not just the current generation but future generations of technologies.”