The field of cell and gene therapy (CGT) has made remarkable strides in recent years, offering new hope to patients across an increasingly wide range of conditions. However, the field’s growth has also brought many of its limitations to light, several of which stem from the use of viral vectors in therapeutic manufacturing.

Modified viruses have long served as valuable tools in molecular biology, providing researchers with a reliable way to deliver genetic material (DNA or RNA) to target cells. Naturally, viruses have become foundational elements in many CGT development pipelines. But, accumulating evidence suggests the use of viral vectors can come at a cost.1–3

Inherent safety risks and complexities associated with these vectors drive up manufacturing costs while also increasing the potential for serious adverse events among patients. The FDA underscored this point in January 2024 by issuing new warning and guidance documents related to the oncogenic potential of common viruses used for cell therapy manufacturing.4,5

While such risks may be tolerable when treating advanced stage cancers, emerging applications for CGTs extend well beyond terminal conditions.6,7 It is increasingly necessary for developers to explore alternative, non-viral approaches that can enhance the safety and efficacy of these groundbreaking therapies.

Role for electroporation

Electroporation is well positioned to fill this need. Under optimized conditions, cells exposed to electrical pulses may become temporarily porous, enabling therapeutic components to enter the cell, and negating the need for a delivery vehicle (e.g., a virus). When used with clinical Good Manufacturing Processes, a clinically validated electroporation platform can be a viable way to circumvent the risks and complications associated with viral vectors, ultimately enabling simpler, safer, and more cost-effective therapeutic development.

CGTs refer to a wide spectrum of treatments that leverage cellular and genetic materials to either prevent or treat diseases, with examples ranging from mRNA vaccines to engineered tissues. The diversity of therapeutics in this field has grown in recent years, thanks to tools from synthetic biology that enable researchers to engineer novel proteins, edit genomes with precision, and transform ordinary cells into potent therapeutic agents.

James Brady, PhD
James Brady, PhD, Senior vice president, technical applications and customer support, MaxCyte


T cells fortified with chimeric antigen receptors (CARs), for example, have emerged as powerful anti- cancer agents. T cells are naturally potent regulators of adaptive immunity, functioning as sentinels that recognize pathological antigens and may trigger either inflammatory or cytotoxic outcomes when activated. Such a response can be weaponized against malignant cells by modifying T cells with DNA or RNA coding for a synthetic CAR protein, one designed to both recognize tumor-associated antigens and subsequently activate a T cell-mediated immune response.8

The success of CAR-T cell therapy in acute lymphoblastic leukemia (ALL), large B cell lymphoma (LBCL), and other such cancers has inspired hope that this approach may be useful beyond cancer.6,7 In fact, CAR-T therapy is already showing promise for treating autoimmune diseases.9

Although these therapies may be useful for a wide range of conditions, the use of CAR-T and other cell therapies is complicated by their considerable manufacturing costs and inherent safety risks.

Challenges of engineering cell therapies

Cell therapy manufacturing typically begins with the collection and isolation of immune cells, stem cells or other types of primary cells ex vivo. From there, cells can be transformed into therapeutic agents by exposing them to modified viruses that are capable of introducing custom DNA or RNA cargo into human cells. This genetic payload will contain genes and regulatory elements that enable the expression of the therapeutic construct, be it CAR or gene editing components. Altered cells are then infused into the patient for therapeutic effect.

There is a significant safety concern with viral vectors stemming from their origins as human pathogens In particular, vectors derived from retroviruses, lentiviruses, and adeno-associated viruses require complex engineering and manufacturing strategies to minimize chances that replication competent viruses might be generated during vector production. These strategies often involve dividing viral components among multiple plasmids, which must be co-transfected into producer cells, complicating manufacturing and reducing production efficiency.

Moreover, most regulatory agencies require that vector manufacturing lots intended for use in humans must be tested for replication competent viruses, which adds an additional complication to the manufacturing workflow.10 Additionally, viral vectors come with inherent safety risks related to both immunogenicity and insertional mutagenesis.

The majority of CGTs are produced using lentivirus (LV), murine γ-retrovirus, adenovirus (AD), and adeno-associated virus (AAV).11 Each differs from the other in many crucial ways, including their ability to transduce various cell types, genomic complexity, the size of genetic cargo they can carry, and the ultimate fate of that cargo.10 LV, for example, is designed to integrate its payload with the host’s genome where it can endure through successive rounds of cellular division. By using a virus that embeds in the host’s genome, researchers gain long term, stable expression of therapeutic genes and the ability to expand the population of engineered cells.10, 12 

viral vector
Most regulatory agencies require that vector manufacturing lots intended for use in humans must be tested for replication competent viruses, which adds an additional complication to the manufacturing workflow. Additionally, viral vectors come with inherent safety risks related to both immunogenicity and insertional mutagenesis. [ttsz/Getty Images]
However, manipulating the LV genome is more complex than other viruses and researchers have little control over where payload integration happens.13 When it occurs in or near a proto-oncogene, the vector’s powerful promoters may drive overexpression of the endogenous oncogene and, ultimately, lead to malignant transformation of the cell. 1–4

It has also recently been reported that persistence of viral proteins in transduced cells may result in immunogenic responses with the potential to cause cytokine release syndrome—a life threatening adverse reaction.14 Therefore, use of any integrating vector comes with the added risk of potential insertional mutagenesis. The risk is great enough that the FDA saw fit to release a guidance document related to cell therapy manufacturing and integrating vectors, calling on researchers to consider alternatives when possible.3,4

Non-viral alternatives

Electroporation stands out as a validated non-viral method for delivering genetic material into cells that can support the growth of CGT development. Where viral vectors transfer genetic material into a cell by binding to surface proteins, electroporation allows naked nucleic acids to permeate the cell by creating temporary perturbations in the cell membrane with electrical pulses.15 By avoiding the use of viruses, electroporation offers several distinct advantages for therapeutic development.

One such advantage is the lack of packaging limitations. With viral vectors, genes must be packaged inside the finite space of a capsid which limits researchers to approximately 4–10 kb of transgenic material, with diminishing packaging efficiency as they approach the upper limits for each virus.16,17 This is a substantial constraint for CRISPR-based therapies that may require the delivery of a Cas9 enzyme, sgRNA, regulatory elements, and potential donor templates.18

To achieve this, multiple transductions may be necessary and the overall efficiency of the workflow decreases. Electroporation has a much larger size limit, and it allows simultaneous delivery of multiple and diverse loading agents. Therefore, researchers can readily deliver complex payloads when developing advanced therapies.

Such is the case with Vertex’s Exagamglogene autotemcel (trade name Casgevy), the world’s first FDA-approved CRISPR gene therapy. To correct transfusion-dependent beta thalassemia or sickle cell disease, an enhancer controlling the BCL11A gene is CRISPR modified in patient bone marrow stem cells, ultimately driving an increase in fetal hemoglobin production and reduced anemia or red blood cell sickling.

A CRISPR ribonucleoprotein complex—consisting of Cas9 protein and a sgRNA—are delivered to the stem cells ex vivo using electroporation.19 Not only does this allow for the efficient delivery of large CRISPR components, but it eliminates concern of virus induced immunogenicity and insertional mutagenesis. None of the 31 patients treated with Casgevy in clinical trials developed malignant side effects—a stark contrast to a similar vector-based CRISPR therapeutic for sickle disease which has received a black-box warning due to the risk of hematological malignancy.20

As Casgevy demonstrates, electroporation enables researchers to deliver a wide range of payloads, from proteins to RNA, transposons, and reporters. This flexibility is key for improving the safety of cell therapies as it allows for innovation. Electroporation does not inherently eliminate the risk of insertional mutagenesis, but rather it allows researchers to use payloads and alternative strategies that avoid the issue altogether (as in the case of Casgevy) or greatly reduce the odds of oncogenic insertion.15,21

Lastly, the use of electroporation in place of viral vectors requires less stringent biosafety precautions because it eliminates concerns about producing replication competent viruses. This significantly simplifies and reduces the cost of product manufacturing.

Viral vectors have historically been preferred because they allow cell engineering on a much larger scale. However, the development of flow electroporation technologies like those offered by Maxcyte have made it possible to perform automated, aseptic electroporation across a wide range of scales (5 × 105 cells to 2 × 1010 cells and beyond).22 Accordingly, Maxcyte’s flow electroporation platform is used for the manufacturing of Casgevy and several other CGTs currently in development.

Electroporation has proven to be a robust, versatile, and safer alternative to viral vectors for CGT development. Its ability to efficiently deliver large and complex genetic payloads, combined with a reduced need for stringent biosafety measures, lower immunogenic risks, and flexibility in payload type, positions electroporation as a key technology in the future development of successful and safe cell and gene therapies.

James Brady, PhD, is senior vice president, technical applications and customer support, MaxCyte.


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