Optimizing viral transduction in immune cell therapy manufacturing: key process design considerations

Abstract

Immune cell therapies have revolutionized the treatment of cancers, autoimmune disorders, and infectious diseases. A critical step in their manufacturing is viral transduction, which enables the delivery of therapeutic genes into immune cells. However, the complexity of this process presents significant challenges for optimization and scalability. This review provides a comprehensive analysis of viral transduction process in immune cell therapy manufacturing, highlighting key design considerations to support the development of safe, effective, and scalable production methods. Additionally, it examines current technological challenges in immune cell transduction and explores future innovations poised to advance the field.

Introduction

Immune cell therapies have transformed the treatment landscape for cancer and other diseases, offering unprecedented clinical outcomes. The global T-cell therapy market is expected to expand exponentially from USD 10.30 billion in 2025 to USD 161.21 billion by 2034, with a compound annual growth rate of 35.74% [1]. To meet the escalating demand, the field must achieve efficient, scalable, and reproducible manufacturing.

A pivotal step in immune cell therapy manufacturing is viral transduction, which delivers therapeutic genes into immune cells. However, transduction efficiency can be highly variable, depending on various factors such as: cell quality (e.g., activation state, donor variability), viral vector (e.g., titre, envelope pseudotyping), and process parameters (e.g., cell-vector interaction, incubation time, enhancers). Despite its central role in immune cell manufacturing, transduction process design remains largely empirical and lacks standardized methodologies. In practice, transduction processes are often product-specific and much of the foundational process knowledge is proprietary residing with leading academic institutions and pharmaceutical companies. The existing literature primarily emphasizes viral vector design, clinical applications, and large-scale vector manufacturing [2,3,4,5], with comparatively limited discourse on process optimization tailored for immune cell transduction in the context of therapy manufacturing. This gap presents a significant challenge for early-stage therapy developers and small-scale manufacturers, who often lack the resources and infrastructure for empirical optimization.

This review aims to address this gap by providing a systematic analysis of viral transduction process in immune cell therapy manufacturing and evidence-based strategies to enhance transduction efficiency, scalability, and reproducibility. We begin by outlining key immune cell types and viral vectors that form the foundation of transduction workflows. Next, we examine the Critical Quality Attributes (CQAs) of immune cells post-transduction, followed by a detailed discussion of the Critical Process Parameters (CPPs) that govern transduction outcomes. We then explore the prevailing challenges facing immune cell transduction today and highlight emerging technological advancements poised to transform the practice tomorrow. By elucidating the design principles for the transduction process, this work aims to accelerate the development, clinical translation, and commercialization of next-generation immune cell therapies. Given the diversity of immune cell types and manufacturing workflows, this discussion is meant to serves as a foundational reference rather than a prescribing guide for specific applications.

Viral transduction in immune cell therapy manufacturing: fundamentals

Common cells for immune cell therapy and their susceptibility to transduction

Immune cell therapies can be manufactured via in vivo (cells engineered inside the body) or ex vivo (cells modified in controlled laboratory settings) approaches. This review focuses on ex vivo manufacturing, the dominant paradigm for clinical applications, which typically involves isolation, activation, gene editing (often via viral transduction), expansion, and harvesting [6]. Below, we discuss key cell types for immune cell therapy and their transduction susceptibility.

T cells are the workhorses of adaptive immunity and excel at immune surveillance and targeted cytotoxicity. Their high proliferative capacity and upregulated receptor expression upon activation (e.g., via CD3/CD28 stimulation) make them highly amenable to viral transduction [7]. They are commonly transduced with Gamma-retroviral and Lentiviral vectors to express either Chimeric Antigen Receptors (CARs) [8], which recognize surface proteins independently of MHC molecules, or T-cell receptors (TCRs) [9], which recognize intracellular antigens presented by MHC molecules. T cells generally require a complex cytokine cocktail (e.g., IL-2, IL-7, or IL-15) to support their expansion, survival and function post-transduction [10].

Natural Killer (NK) cells are part of the innate immune system and play a critical role in defending against tumors and virally infected cells. They recognize and eliminate abnormal cells through a variety of activating and inhibitory receptors [11]. While Retroviral vectors and Lentiviral vectors are used to enhance their cytotoxic activity and persistence [12], NK cells usually present low baseline transduction efficiency due to their innate immune properties and susceptibility to viral restriction mechanism [13]. This may necessitate higher viral titres or tropism-engineered vectors [14]. NK cells often require the addition of co-factors, such as IL-15, to enhance cell survival and cytotoxicity after transduction [13].

Dendritic cells (DCs) and macrophages are emerging therapeutic targets due to their roles in antigen presentation and immune modulation [15,16,17]. Their transduction is typically challenging due to their lower proliferative capacity and specialized functions. While Lentiviral vectors can transduce these cells, the transduction efficiency is largely dictated by the receptor expression profiles of the target cell population [15, 18, 19]. Non-integrating vectors such as Adenoviral vectors are often favoured for transient gene expression, particularly in DC-based vaccines designed to prime adaptive immune responses [20]. In the case of macrophages, which can have pro-inflammatory or anti-inflammatory roles depending on their polarization, gene modification strategies must be carefully tailored to minimize unintended immune activation and ensure therapeutic efficacy [21].

Common viral vector systems for immune cell transduction

Viral vector system is a critical determinant of success in immune cell transduction, with each platform offering unique trade-offs between efficiency, safety, and clinical applicability. Here, we evaluate the four most clinically advanced viral vector systems for immune cell engineering.

Lentiviruses (LVs) have emerged as a leading platform for immune cell therapy. They are able to achieve stable genomic integration in both dividing and non-dividing cells, which is particularly valuable for long-term persistence of therapeutic cells, as demonstrated in Food and Drug Administration (FDA)-approved CAR-T cell therapies [22]. While early concerns about insertional mutagenesis persist, modern self-inactivating (SIN) designs and improved integration site profiling have significantly mitigated these risks [23]. Their broad tropism, enabled by pseudotyping with vesicular stomatitis virus-G (VSV-G) envelope proteins, allows efficient transduction of diverse immune cell types [24].

Gamma-retroviruses (γRVs) are the backbone of early CAR-T therapies, offering robust and stable transgene integration. However, unlike LVs, they require target cells to be actively proliferating for successful transduction, which has historically restricted their use to ex vivo activated T cells. While they carry higher risks of insertional mutagenesis, recent advances in vector engineering including SIN configurations and insulator elements have significantly improved their safety profile [25]. γRVs exhibit poor tropism for NK cells due to receptor incompatibility in commonly used envelope proteins (e.g., amphotropic, ecotropic) [25], and face greater antiviral defences from innate immune populations (particularly NK cells) [26].

Adenoviruses (AVs) are non-integrating viruses that provide transient transgene, presenting unique opportunities for applications requiring controlled, short-term immune modulation. High transduction efficiency across immune cell types and rapid production capabilities make AVs attractive for vaccine and cytokine delivery applications [27]. However, significant challenges remain regarding their pronounced immunogenicity and limited payload capacity (~ 8 kb), which currently preclude their use in most engineered cell therapy products [28].

Adeno-Associated Viruses (AAVs) are another non-integrating viral platform that have gained attention for their favourable safety profile and ability to transduce non-dividing cells, making them particularly suitable for delicate immune cell targets such as DCs [29, 30]. While their small payload capacity (~ 4.7 kb) has traditionally limited their use in cell therapy, innovative approaches combining AAV with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems or transposons have expanded their potential [31]. Their low immunogenicity enables repeated administration, an advantage for certain immunomodulatory strategies.

CQAs of immune cells post transduction and their control strategies

In accordance with ICH Q8 guidelines, CQAs represent essential measurable characteristics that define the safety, efficacy, and quality of cell therapy products [32]. For virally transduced immune cells, rigorous monitoring and control of these attributes throughout the manufacturing process are paramount to ensure regulatory compliance and clinical success. The following section examines six key CQAs that require careful evaluation, along with approaches for their measurement and optimization.

• Transduction efficiency

Transduction efficiency, measured as the percentage of cells successfully expressing the transgene, serves as the primary indicator of transduction success and directly correlates with therapeutic efficacy. A high transduction efficiency is usually desired to achieve adequate transgene expression and sufficient numbers of therapeutic cells. In clinical CAR-T cell manufacturing, transduction efficiencies typically range between 30–70% [33,34,35]. Low efficiencies may indicate transduction failure and compromise therapeutic potency, while excessively high rates could indicate process instability. Current analytical approaches for assessing transduction efficiency include flow cytometry for surface marker detection, quantitative PCR for Vector Copy Number (VCN) analysis, and functional assays measuring cytokine secretion upon antigen stimulation. To achieve optimal transduction efficiency, manufacturers employ several key strategies including pre-activating cells to upregulate viral receptor expression [36], using viral vectors with cell-specific pseudotype [24], spinoculation to enhance cell-vector contact [34], careful titration of multiplicity of infection (MOI) to balance efficiency and safety [33], and the use of effective transduction enhancers [37].

• Cell viability and function

Post-transduction cell viability represents a critical indicator of product quality and therapeutic potential. Poor viability may lead to manufacturing failures or ineffective therapy. Concurrent preservation of cellular function ensures the modified cells retain their cytotoxic capacity. Viability assessment commonly employs trypan blue exclusion methods or more sensitive Annexin V/7-AAD staining analysed by flow cytometry [37]. Functional evaluation often incorporates IFN-γ ELISpot assays (measure the secretion of various cytokines), cytotoxicity assays (measure target cell lysis in co-culture assays to assess cell killing capacity), and real-time cytotoxicity measurements using platforms like xCELLigence [38, 39]. Process optimization strategies focus on minimizing cell stress through reduced transduction duration, culture supplementation (e.g., IL2, IL-7 and IL-15), and careful MOI titration to prevent toxicity from excessive viral load. These approaches collectively promote cell fitness while achieving the desired genetic modification.

• VCN

VCN, the average number of viral integrations per cell genome, requires precise control to balance therapeutic transgene expression against potential genotoxic risks. Clinical programs generally maintain VCN below 5 copies per cell for optimal safety and efficacy [40, 41]. Accurate VCN quantification employs droplet digital PCR (ddPCR) as the gold standard due to its superior precision [42]. Control strategies emphasize MOI optimization to minimize multiple integration events, with lower MOI ranges typically reducing the incidence of high VCN cells [33, 43]. Vector engineering approaches, particularly SIN designs with deleted viral enhancer elements, further mitigate the risk of insertional mutagenesis [44]. These combined measures ensure sufficient transgene expression while maintaining genomic integrity of the final therapeutic product.

• Product safety

The absence of contaminants, particularly replication-competent viruses (RCVs) and residual vector particles, constitutes a fundamental safety requirement for clinical products. Current regulatory guidelines mandate rigorous testing to demonstrate adequate clearance of these impurities, which could otherwise lead to off-target transduction or adverse immune reactions [45]. Standard detection methods include extended co-culture assays for RCVs and p24 ELISA for lentiviral particle quantification [46]. Contamination control during immune cell therapy manufacturing includes optimizing transduction timing and MOI to limit excess virus exposure, and incorporating extensive wash steps to remove the residual virus before final product formulation.

• Expansion capacity

Robust proliferative capacity ensures the generation of sufficient cell numbers for therapeutic dosing while serving as an indicator of cellular health. Poor expansion could result in an insufficient final cell dose, and expansion defects may indicate cellular stress or transduction-related toxicity [46]. Expansion potential is commonly monitored through population doubling calculations or more precise CFSE dilution assays that track division history at single-cell resolution [46]. Culture optimization strategies focus on cytokine cocktail formulation, with combinations such as IL-15 particularly effective for NK cell expansion [47]. Process modifications to reduce mechanical stress and avoid excessive exposure to virus, along with careful control of critical culture parameters like pH and dissolved oxygen, are also useful to support optimal cell growth post-transduction.

• Cell phenotype

Key immune cell subpopulations and effector function after transduction should be maintained. The maintenance of favourable T cell subpopulations, including balanced CD4/CD8 ratios and stem cell-like memory T (T_SCM) subsets, has demonstrated correlation with improved clinical outcomes in multiple CAR-T trials [46]. Phenotypic characterization typically employs flow cytometry assessing markers such as CD45RA and CCR7 for memory T cell subsets, along with PD-1 and LAG-3 for exhaustion states. Culture condition optimization plays a pivotal role in phenotype preservation, with strategies including Wnt pathway modulation to maintain T_SCM populations [48] and controlled activation to prevent terminal differentiation. Cytokine modulation using IL-7 and IL-15 further promotes the development of memory-like characteristics associated with prolonged in vivo persistence [49, 50]. These approaches collectively yield cell products with enhanced therapeutic potential and durability.

CPPs of immune cell transduction

Developing a robust and scalable viral transduction protocol requires careful optimization and balance of key process parameters that directly influence CQAs. This section outlines the fundamental CPPs of the process and their profound impact on transduction outcomes.

Cell source

The starting cellular material for immune cell therapies typically exhibits considerable heterogeneity, comprising diverse functional subsets and occasional non-target populations. This variability significantly impacts transduction efficiency and subsequent therapeutic performance.

Generally, less differentiated cell populations demonstrate superior genetic modification potential and expansion capacity. For example, CD8+ T cells derived from naïve T cells (T_N) (CD62L^hiCD45RO^lo) exhibited approximately 20% higher CAR expression compared to those derived from central memory T cells (T_CM) (CD62L^hiCD45RO^lo) and effector memory T cells (T_EM) (CD62L^loCD45RO^hi) following retroviral transduction [51]. This principle extends to cell sources, with umbilical cord blood-derived T cells—enriched for T_N cells (CD62L⁺CD45RO⁻)—showing 25% greater transduction efficiency than peripheral blood counterparts [33].

Non-target cells contamination presents a major challenge for immune cell transduction. Myeloid cells suppressed T cell expansion and reduced transduction efficiency during CAR-T cell production [52], while selective depletion of CD14+ monocytes from leukapheresis products resulted in a 64% increase in CAR expression of T cells after retroviral transduction [53].

These findings underscore the importance of including a pre-enrichment step prior to transduction to select favourable subsets with enhanced transduction potential such as naïve or memory stem cells, and remove inhibitory cell populations. Implementation of these approaches can potentially enhance the transduction efficiency, expansion and function of the cells post-transduction.

Activation status

Cell activation status critically determines viral vector permissiveness through multiple mechanistic pathways. While LVs do not strictly require cell division, activation markedly improves their transduction efficiency. T cells transduced with LVs 2 days post-activation showed approximately a twofold increase in transgene expression compared to non-activated cells [54]. Moreover, transduction efficiency was correlated with the degree of cell activation. T cells activated via stronger activators CD3/CD28/CD81 demonstrated nearly 30% higher transduction efficiency compared to those activated with CD3/CD28 or CD3 alone [55]. The enhanced transduction efficiency with activated cells correlates with both upregulated low-density lipoprotein receptors (LDL-Rs) (essential for VSV-G pseudotyped vector entry) [56, 57], as well reduced activity of the restriction factor SAMHD1 in cycling cells [58]. SAMHD1 hydrolyses deoxynucleoside triphosphates (dNTPs) into deoxynucleosides, thereby depleting intracellular dNTP levels below the threshold necessary for efficient viral reverse transcription [59]. γRVs show absolute dependence on cell cycling for integration. Non-activated T and NK cells exhibit negligible transduction (< 1%), while pre-activated populations achieve 65.9% and 23.0% efficiency respectively [54].

These observations suggest immune cell activation modulation as an effective strategy to enhance transduction efficiency. Manufacturers typically initiate the activation process 24–72 h pre-transduction to achieve optimal cell cycling states. Careful selection of activation reagents, such as anti-CD3/CD28 beads with or without cytokine supplementation, represents another critical consideration. A critical caution involves avoiding excessive activation, which risks inducing terminal differentiation or exhaustion that impair cell expansion and persistence [60, 61]. Optimal protocols therefore must balance sufficient activation for vector integration while preserving stem cell-like qualities that are essential for clinical efficacy.

Cell preconditioning

Cell preconditioning prior to transduction through cytokine supplementation and serum optimization represents a powerful approach to improve immune cell quality, functionality, and viral susceptibility.

Cytokine preconditioning has demonstrated particular promise in enhancing transduction outcomes. A 48-h treatment with 10 ng/ml IL-12 during TCR activation boosted the transduction efficiency of multiple TCR/GFP Retroviruses (RVs) by an average of 20% in mouse T cells and around 10% in human CD8+ T cells. This improvement correlated with IL-12-induced upregulation of Bcl-3 mRNA, which inhibited terminal differentiation and promoted memory cell formation in T cells [62, 63]. Likewise, IL-21 preconditioning for 48 h prior to Lenti-HER2 CAR transduction improved transduction efficiency in human T cells by 10–15%, likely due to its ability to maintain less differentiated T cell subsets such as T_N and T_CM that exhibit greater viral permissiveness [64, 65]. While the direct effects of IL-7 and IL-15 on transduction efficiency require further investigation, these cytokines are well-established for their ability to expand memory T cell populations (CD62L⁺CCR7⁺CD45RA⁺CD45RO⁺IL-7Rα⁺CD95⁺, D45RO⁺CCR7⁺, CD45R⁺CCR7⁺, CD45RO^–CD45RA⁺CCR7⁺CD27⁺CD95⁺) that demonstrate superior in vivo persistence and antitumor activity [66,67,68].

Serum composition similarly exerts profound effects on transduction efficiency. Recent studies demonstrate advantages of advanced formulations over traditional options. Physiologix™ Xeno-Free serum replacement achieved superior LV transduction efficiency across multiple MOIs compared to human serum [69, 70]. Human platelet lysate provided comparable transduction rates to human AB serum while modestly enhancing transgene expression levels [71]. Interestingly, serum starvation of resting T cells before transduction improved efficiency through upregulation of LDL-R [72].

These findings highlight several critical considerations for cell preconditioning to enhance transduction. Cytokines should be selected to align with both transduction goals and desired cell phenotypes, with IL-12 and IL-21 showing promise for enhancing T cell transduction efficiency, and IL-7 and IL-15 for promoting memory T cell phenotype. Serum replacement strategies should be evaluated for their ability to improve transduction while maintaining cell fitness. Importantly, the timing and duration of cell preconditioning require careful optimization, as evidenced by the 48-h pretreatment periods showing efficacy in multiple studies. Overall, the supplement type, concentration, preconditioning duration, and timing relative to transduction must all be tailored to the specific immune cell type and viral vector system. When properly optimized, preconditioning can simultaneously enhance transduction efficiency while preserving or even improving the therapeutic characteristics of the immune cell product.

Viral vector

The selection of  viral vector designs—particularly through envelope pseudotyping and promoter—plays a pivotal role in determining transduction efficiency and specificity, as well as long-term therapeutic outcomes of the cells.

• Viral envelope

The envelope pseudotype of viral vectors governs viral tropism, transduction efficiency, and vector stability [73]. Different envelope proteins interact with distinct cellular receptors, creating opportunities to target specific immune cell subsets. For example, H/F-pseudotyped LVs demonstrated superior transduction of resting CD3+ T cells (25–40% efficiency) compared to conventional VSV-G-pseudotyped LVs. This advantage stems from the H/F envelope’s use of Signalling Lymphocyte Activation Molecule as an entry receptor, which is constitutively expressed on resting T cells. In contrast, VSV-G relies on LDL-Rs that are primarily upregulated upon T cell activation [57, 74]. Similarly, baboon endogenous retrovirus (BaEV)-pseudotyped vectors, which utilize ASCT-1 and ASCT-2 amino acid transporters, achieved 30–60% transduction efficiency in IL-7-primed T cells, far exceeding the 2–12% efficiency of VSV-G vectors [75]. Recent innovations include koala retrovirus (KoRV)-pseudotyped LVs, which outperform BaEV, RD114-TR, and Gibbon Ape Leukaemia Virus envelopes in transducing peripheral blood mononuclear cell (PBMC)-derived T and NK cells by five to sixfold [76].

Beyond natural tropism, envelope engineering can further refine specificity [77, 78]. CD62L-targeted LVs preferentially transduced less differentiated human T lymphocytes, yielding products with enhanced persistence and therapeutic potential [79]. Another approach, exemplified by LentiSTIM and RetroSTIM technologies, incorporates anti-CD3 antibodies into the viral envelope, enabling simultaneous T cell activation and transduction. This strategy not only improves efficiency but also streamlines manufacturing by reducing reagent requirements [80].

• Promoter

Promoter selection is a critical determinant of transgene expression dynamics, influencing expression levels, specificity, therapeutic efficacy, persistence, and the risk of cell exhaustion [81].

Strong constitutive promoters, such as human elongation factor 1α (EF1α), cytomegalovirus (CMV) and MND, drive high transgene expression and enhance cytotoxic function. Notably, CAR-T cells engineered with EF1α exhibited superior anti-tumor activity compared to those using CMV promoters, as evidenced by heightened cytokine release and target cell killing [82]. However, excessive transgene expression driven by strong promoters can induce tonic signalling and premature T-cell exhaustion, thereby accelerating differentiation and impairing long-term persistence [83,84,85]. Maintaining optimal CAR surface density is essential to ensure effective target antigen recognition while mitigating antigen-independent tonic signalling or off-tumor, on-target cytotoxicity due to unintended target antigen recognition on normal cells [86]. Comparative studies of EF1α and MND promoters revealed that MND-driven CAR expression resulted in improved transduction efficiency while reducing CAR surface density, which correlated with lower cytokine release in patients [86].

Moderate-strength promoters, such as phosphoglycerate kinase (PGK) and murine stem cell virus (MSCV), offer a balance between potency and durability. MSCV, for example, sustained stable GFP and TCR expression in peripheral blood and tumor-infiltrating lymphocytes longer than CMV promoters [87]. For applications requiring physiological regulation, the Wiskott–Aldrich Syndrome (WAS) promoter replicates natural TCR dynamics, reducing tonic signalling while promoting central memory T cell phenotypes. WAS-driven CAR-T cells exhibited lower exhaustion markers (PD-1⁺, LAG-3⁺) and more controlled cytokine secretion (TNF-α, IFN-γ) post-activation, correlating with improved persistence in vivo [88].

The choice of promotors must align with therapeutic objectives. In acute oncology applications, potent cytotoxicity driven by strong promoters may be advantageous, whereas therapies targeting chronic conditions may benefit from the sustained persistence conferred by moderate or regulated expression systems.

Successful vector design requires a holistic approach that considers both the viral envelope and promoter elements. The envelope determines initial transduction efficiency and cellular tropism, while the promoter shapes long-term transgene behaviour and cellular fitness. Envelope selection should maximize target cell accessibility and transduction efficiency, whereas promoter choice must carefully balance therapeutic potency against the risks of T-cell exhaustion and dysfunction. Table 1 lists the retroviral backbone and promoters used in FDA-approved CAR-T cell products.

Table 1 Retroviral backbone and promoters used in FDA-approved CAR-T cell products

Transduction conditions

Precise control of transduction conditions and careful balancing of multiple variables determine both the transduction efficiency and the quality of the transduced cells. The interplay between viral particle availability, cellular exposure conditions, and enhancer systems requires systematic optimization to achieve robust transduction outcome.

• MOI

MOI, defined as the ratio of the number of viral particles to target cells, serves as a primary determinant of transduction outcomes. While increased MOI generally enhances transduction efficiency [95], this relationship follows a nonlinear pattern with diminishing returns at higher levels. Increasing MOI from 1 to 10 boosted lentiviral GFP transduction with cord blood-derived CD8+ T cells from 30 to 80%, albeit with a marginal reduction in viability [33]. The consequences of MOI escalation extend beyond simple efficiency metrics. In anti-CD19 CAR-T cell production, progressive MOI increases from 1 to 20 led to corresponding rises in VCN (0.63 to 1.30 copies/cell) while exhibiting an inverse correlation with cell viability [96].

These findings underscore the need to identify an optimal MOI range to achieve sufficient genetic modification without compromising cellular fitness—a balance particularly crucial in clinical manufacturing where both product potency and patient safety are paramount.

• Virus titre

The concentration of viral particles in the transduction medium influences gene delivery by affecting particle-cell interaction dynamics. Concentrated VSV-G pseudotyped LV preparation (5 ml volume) achieved five-fold greater transduction of PBMCs than diluted preparations (50 ml volume) containing identical total particle numbers [54]. This concentration-dependent enhancement likely reflects improved collision frequency between viral particles and target cells. On the other hand, excessive viral titres introduce challenges including particle aggregation, reduced viability, and inefficient vector utilization [97].

Past studies emphasize the importance of titre optimization alongside MOI adjustment, particularly in large-scale production where viral vector costs represent a significant portion of manufacturing expenses. The development of standardized titre measurement methods and their correlation with functional transduction units remain an active area of investigation to improve process consistency.

• Transduction enhancers

Transduction enhancers have become indispensable tools in immune cell manufacturing, offering targeted solutions to overcome biological barriers at different stages of viral entry and integration. These compounds are broadly categorized based on their mechanism of action, addressing either pre-entry or post-entry bottlenecks in the transduction process.

The initial contact between viral particles and target cells faces electrostatic repulsion due to negatively charged membrane surfaces. Pre-entry enhancers including polybrene [98], protamine sulfate [99], dasatinib [100], and dextran [101, 102] represent classical solutions to this challenge, functioning as cationic bridges that neutralize these charges. While effective, these traditional reagents are increasingly being supplemented or replaced by more sophisticated alternatives in clinical manufacturing. RetroNectin employs a biomimetic approach through its dual-domain structure. Its heparin-binding domains capture viral particles while cell-binding domains engage surface integrins, creating a molecular bridge that enhances interaction efficiency. Clinical data demonstrate its superiority over polybrene, achieving 63% CAR expression versus 35% while reducing cytotoxicity from 49 to 5% [34].

Vectofusin-1 and LentiBOOST represent alternative strategies targeting membrane dynamics. Vectofusin-1's amphipathic peptides transiently destabilize lipid bilayers, facilitating viral entry across diverse vector systems. Its application elevated lentiviral transduction from 9 to 61% while maintaining viability [103]. Similarly, LentiBOOST’s poloxamer-mediated enhancement of membrane fluidity improved transduction of cord blood derived T cell without viability trade-offs even at low MOIs [33].

Following viral entry, additional hurdles emerge during cytoplasmic trafficking and genomic integration. This has been addressed by post-entry enhancers which modulate intracellular processes that affect viral integration, reverse transcription, or transgene expression. For example, rapamycin improved LV transduction in CD8+ T cells by 90% while maintaining cellular function [104]. A more recently identified GSK compound targeting the MDN1 pathway achieved three-fold vector dose reduction while preserving phenotype and cytotoxicity—a critical advantage for scalable manufacturing [105]. These intracellular modulators open new possibilities for enhancing efficiency without increasing viral load.

While transduction enhancers offer clear benefits, their implementation requires careful parameterization. The choice between pre-entry and post-entry enhancers should be guided by the specific rate-limiting step in each system. Concentration thresholds must be established to avoid membrane disruption or interference with viral uncoating. Temporal considerations are equally important, with some enhancers requiring pre-incubation while others are co-delivered with vectors.

• Duration

The duration of viral exposure presents a fundamental trade-off between efficiency and cellular stress. Conventional 18–24 h transduction windows are being reevaluated in light of emerging rapid manufacturing paradigms. Recent studies demonstrate that shortened 4-h protocols can maintain therapeutic levels of gene transfer while significantly improving viability—a critical advantage for products requiring immediate infusion [106, 107]

This temporal compression aligns with the growing recognition that prolonged vector exposure may trigger stress responses independent of MOI effects. The optimal window appears system-specific, influenced by vector type, enhancer use, and target cell characteristics.

• Temperature

Temperature exerts multifaceted effects on transduction biology, influencing both viral particle stability and cellular physiology. In one comparison study, 2-h transduction of CD3+ T cells with Lenti-CD19 and CD123 CAR vectors at different temperatures (4 °C, 25 °C, 32 °C, and 37 °C) led to different outcomes. Transduction at 4 °C resulted in the highest transduction efficiency, which was correlated with the possibly improved viral stability at this lower temperature. In comparison, transduction at 37 °C promoted the fastest proliferation of CAR-T cells, indicating that higher temperatures may facilitate cellular division and expansion. Cells transduced at 32 °C comprised the highest proportion of T_N cells (CD62L⁺CD45RO⁻) and demonstrated enhanced cytotoxic activity and improved cytokine secretion, suggesting a more functional phenotype. As a result, transduction at 32°C provided the best balance between maximizing transduction efficiency, preserving T cell function, and promoting an ideal cellular phenotype for CAR-T cell therapy [108].

The interdependence of different transduction conditions necessitates holistic optimization. Shortened transduction durations may require compensatory adjustments in temperature or enhancer concentration. Similarly, temperature-mediated phenotype modulation must be balanced against efficiency targets. Advanced modelling approaches and design-of-experiment methodologies can be invaluable in navigating these complex interactions during process development.

Transduction platforms

Four primary platforms are utilized today for immune cell transduction, with each system offering distinct features impacting efficiency, scalability, and process control.

• Static incubation in conventional cultureware

Static incubation in standard culture plates, flasks, or bags remains the most common and accessible transduction approach today. In this platform, gravitational settling of cells creates a concentration gradient where viral particles must diffuse through the medium to reach their targets. This physical separation becomes particularly problematic in deep culture vessels and with high medium height, where viral particles in upper layers may never encounter settled cells due to Brownian motion limitations. This resulting inefficiency manifests in both suboptimal transduction efficiency and poor viral particle utilization.

While static systems offer flexibility and require minimal infrastructure, their inherent limitations in facilitating virus-cell interactions have driven the development of optimization strategies. One such approach involves reducing medium height to minimize diffusion distances, thereby enhancing transduction efficiency. However, despite these modifications, static methods typically exhibit lower efficiency and reproducibility compared to more advanced platforms, which offer enhanced control over transduction parameters and will be discussed in the following section.

• Spinoculation

Spinoculation applies centrifugal force to colocalize cells and viral particles during transduction, improving transduction efficiency compared to static incubation. This method can be implemented using conventional laboratory centrifuges or automated systems such as the Sepax C-Pro. A 1-h spin using Sepax C-Pro achieved transduction efficiency comparable to a 2-h spin with conventional bag centrifugation and significantly higher than static incubation (Sepax C-Pro: 83.4%; bag centrifugation: 72.8%; static incubation: 35.7%). Importantly, this enhancement in transduction efficiency did not compromise cell viability or expansion potential, making spinoculation particularly valuable for patient-derived cells [109].

Conventional centrifuge based spinoculation is hampered by manual operation and scalability challenges, underscoring the need for purpose-built solutions compatible with Good Manufacturing Practice (GMP) environments. In comparison, Sepax C-Pro reduces contamination risks through closed processing while enabling seamless integration with automated workflows. These advantages enhance process standardization and reproducibility, supporting its accelerated adoption in clinical manufacturing.

Despite its proven efficacy, the exact working mechanisms underlying spinoculation remain poorly understood. Emerging evidence suggests beyond facilitating viral sedimentation, spinoculation may induce biophysical changes such as cytoskeletal reorganization, which enhances viral entry and uptake [110]. Further elucidation of these mechanisms could inform the refinement of transduction protocols to improve efficiency and reproducibility in clinical manufacturing.

• Engineered cultureware

Innovative culture systems leverage high surface-area-to-volume ratio design principles to enhance cell-viral interactions, addressing the limitations of conventional static incubation methods [111,112,113]. Microfluidics chips based on this approach enabled a six-fold reduction in viral load while simultaneously improving transduction kinetics [114]. Similarly, a transverse-flow microchamber achieved peak efficiency within 90 min—2.4 times faster than overnight static culture—while requiring only half of the viral dose [115].

Despite their promising performance in research settings, these engineered systems face significant challenges in scaling up to therapeutic cell quantities, making them a key focus of ongoing biomedical engineering efforts [116]. Among currently available solutions, the MFX-T module represents one of the few commercially accessible options, though broader adoption in clinical manufacturing will require further validation and regulatory alignment.

• Automated and integrated manufacturing systems

Integrated manufacturing platforms, such as CliniMACS Prodigy and Cocoon, have transformed clinical manufacturing by facilitating decentralized, end-to-end workflows. Transduction efficiency in these systems varies based on donor cell characteristics and process parameters, with lentiviral CAR-T production typically achieving 60–65% efficiency in healthy donor cells [117]. Protocol customization with γ-retroviral spinoculation achieved 72–83% BCMA-CAR expression [118]. However, patient-derived samples often exhibit lower transduction rates (~ 30%), underscoring the need for further process optimization [119].

These automated systems provide three key advantages for viral transduction: (1) precise temporal control over viral exposure, (2) reduced manual handling through automated fluidics, and (3) compliance with GMP closed-system requirements. Although these platforms are not inherently designed to enhance transduction efficiency, their ability to regulate critical parameters, such as temperature and viral vector exposure, supports the development of robust and scalable clinical manufacturing processes.

Overall, static incubation remains the preferred approach for early-stage research due to its accessibility and minimal infrastructure requirements, though it often yields suboptimal transduction efficiencies. Spinoculation offers a significant improvement in efficiency but requires specialized centrifugation equipment. Emerging engineered cultureware has shown promise in enhancing vector utilization and transduction kinetics; however, its feasibility for large scale clinical manufacturing remains to be fully validated. Finally, while automated and integrated systems do not inherently enhance transduction efficiency, they facilitate streamlined, end-to-end manufacturing with regulatory compliance, reducing process variability and manual handling. Continued advancements in product-specific transduction platform design will be crucial for bridging the gap between laboratory-scale optimization and scalable clinical production.

Essentially, achieving optimal transduction begins with a clear definition of CQAs that govern both immediate process outcomes and long-term therapeutic performance. Achieving these CQAs requires multidimensional optimization of cell-intrinsic elements (e.g., activation state, differentiation status, and receptor expression profiles), vector characteristics (including envelope pseudotyping, promoter strength, and integration safety), process parameters (e.g., MOI, duration, temperature, and enhancement strategies), and manufacturing platforms that balance efficiency with scalability (Fig. 1). While heterogeneous starting cell materials and varying process workflows are common in immune cell therapy manufacturing, it is important to adapt the viral transduction process according to the specific cell and manufacturing requirements to achieve the best transduction outcome. The refinements of the discussed CPPs are driving the evolution of transduction protocols, shaping next-generation manufacturing paradigms that enhance therapeutic potential while maintaining the practicality of large-scale production. Continued advancements in these areas will be critical to bridging the gap between laboratory-scale optimization and industrial implementation.

Fig. 1

Critical quality attributes (CQAs) and critical process parameters (CPPs) essential for immune cell transduction in therapy manufacturing

Challenges and potential solutions

Viral transduction for clinical and commercial immune cell manufacturing presents several challenges, such as process variability, efficiency limitations, and regulatory constraints. This section critically examines these challenges and discusses emerging strategies to improve manufacturing robustness, transduction efficiency, and therapeutic efficacy.

Variability in transduction efficiency

Transduction efficiency varies significantly across different immune cell types, with T cells generally achieving higher modification rates (37–80%) [41, 120, 121] compared to NK cells (10 to 77%) [122,123,124] and DCs (< 35%) [18]. This variability can be largely attributed to intrinsic cellular factors including receptor expression, antiviral defence mechanisms, and cell cycle status.

Recent advancements have sought to improve transduction efficiency, particularly in refractory cell types such as NK cells. One promising approach involves engineering receptor-targeted viral vectors with NK-specific envelopes, such as CD48 or NKG2D ligands, to enhance vector-cell interactions [125]. Additionally, cytokine preconditioning with IL-15 has been shown to increase NK cell permissiveness by upregulating viral entry receptors while preserving cytotoxic function [126]. Another promising strategy involves co-culture systems incorporating feeder cells or artificial antigen-presenting cells, which mimic physiological signalling environments and provide essential stimuli to enhance NK cell transduction efficiency [127]. These advancements highlight the need for cell type-specific optimization strategies that integrate vector engineering, cytokine modulation, and process innovations to enhance transduction efficiency and therapeutic potential.

Inconsistent transgene expression and stability

Heterogeneous transgene expression remains a critical challenge in immune cell manufacturing, primarily resulting from random viral integration events and epigenetic silencing [128]. Even under identical MOI conditions, studies reported varied expression levels, directly impacting product potency and therapeutic consistency [129].

To mitigate positional effects and enhance expression stability, targeted genomic integration using CRISPR-guided lentiviral insertion into safe harbour loci, such as AAVS1 or CCR5, has been employed [130]. Additionally, SIN vector designs improve long-term transgene expression and reduce the risk of insertional mutagenesis [131,132,133]. Furthermore, the incorporation of endogenous promoter systems (e.g., TCR or CD8α) further improves physiological transgene regulation, reducing exhaustion marker expression compared to conventional viral promoters [134, 135]. These innovations are now being integrated into next-generation clinical vector designs to enhance transgene expression predictability, ensuring greater consistency and therapeutic efficacy in immune cell therapies.

Toxicity and cell stress

The viral transduction process can induce significant cellular stress and toxicity, particularly when using high MOI or prolonged vector exposure.

To mitigate these effects, careful optimization of viral exposure time and MOI balance is essential to ensure efficient transduction while preserving cell health. Strategies to minimize MOI requirements include the use of more potent viral vectors with enhanced transduction capabilities [33], and the adoption of transduction-enhancing protocols such as spinoculation, which improves vector-cell contact without necessitating high MOI [34]. Additionally, post-transduction recovery strategies, such as supplementing cultures with cytokines like IL-7 for T cells and IL-15 for NK cells, have been shown to support cell survival and expansion while reducing stress-related effects [47]. These approaches collectively enable high-efficiency modification while preserving immune cell viability and function.

Cell exhaustion and differentiation

Prolonged viral exposure or suboptimal culture conditions during transduction can drive T cell exhaustion and unwanted differentiation, ultimately impairing therapeutic efficacy [136, 137].

To counteract these effects, precise modulation of transduction timing and culture conditions is necessary. Transducing immune cells at an early activation stage, when they retain a more naïve state, has been shown to preserve their functional potential while reducing differentiation and exhaustion risks [137]. Moreover, SIN LVs enable tighter control over transgene expression, reducing unintended cellular activation and differentiation [138]. These refined strategies contribute to maintaining a functionally competent, long-lasting therapeutic immune cell population.

Scaling up without compromising quality

Scaling up viral transduction while maintaining consistent efficiency and product quality poses significant challenges. While efficient transduction can be achieved at small scales, ensuring uniform outcomes in large-scale manufacturing requires addressing critical factors such as maintaining homogeneous transduction efficiency, preserving immune cell phenotype and function, and minimizing batch-to-batch variability [139, 140].

To overcome these challenges, the use of closed-system bioreactors offers precise control over key culture conditions such as pH, temperature, cell density, and viral exposure, while maintaining sterility and minimizing contamination risks [121, 122]. Additionally, the development and integration of advanced real-time monitoring technologies into bioreactors enables continuous assessment of critical parameters, such as viral titre, and allows dynamic adjustments to optimize transduction conditions [141]. The incorporation of continuous perfusion systems can also support high-density cell culture, improving nutrient exchange and enhancing both transduction efficiency and post-transduction expansion [7]. Collectively, these innovations help facilitate the transition from laboratory-scale to commercial-scale immune cell manufacturing, addressing the complexities of large-scale viral transduction.

Regulatory and quality control issues in scaled transduction

The transition from research-scale to clinical manufacturing of virally transduced immune cell therapies introduces complex regulatory and quality control challenges that necessitate systematic oversight. Regulatory agencies including U.S. FDA (21 CFR 1271) and the European Medicines Agency (EMA) (ATMP Regulation 1394/2007) mandate rigorous process control from vector qualification to final product release.

To ensure regulatory compliance, manufacturers must establish robust Standard Operating Procedures and validate every step of the transduction process. Quality control testing should include transduction efficiency assessments via flow cytometry or PCR-based assays, alongside evaluations of cell viability, phenotype, and functionality. The integration of Process Analytical Technologies for real-time in-line monitoring of CQAs would enable proactive process adjustments, allowing for proactive process adjustments to maintain consistency and regulatory adherence [142, 143]. These measures are essential for ensuring the safety, efficacy, and reproducibility of virally transduced immune cell therapies in both clinical and commercial manufacturing environments.

Future development

The rapid growth of the immune cell therapy market has driven an increasing demand for more efficient, scalable, and cost-effective viral transduction technologies. Below, we highlight key technology development poised to influence the future of viral transduction in immune cell manufacturing.

Development of next-generation viral vectors

Conventional viral vectors like LVs and RVs have been foundational to immune cell transduction. Future advancements will focus on engineering viral vectors with improved capabilities to transduce more challenging immune cell types, such as NK cells. Expanding vector tropism to target a range of immune cell types—including T cells, NK cells, and dendritic cells—will help address the variability in transduction efficiency across different cell populations. Additionally, advancements in viral envelope protein modifications are expected to enhance target specificity, optimize vector-cell interactions, and reduce off-target effects. These next-generation viral vectors will offer improved efficiency, precision, and safety for future immune cell transduction and manufacturing [144].

Integration of advanced gene editing technologies

Advanced gene editing technologies like CRISPR/Cas9, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs), enable precise modifications, improving both the safety and efficacy of immune cell therapies [145]. For example, CRISPR/Cas9-mediated knockout of immune checkpoint genes, such as PD-1, has been shown to enhance T cell function and prevent cell exhaustion in CAR-T therapy [146]. It has also been employed to modify cell surface receptors, enhancing tumor targeting and enabling the development of highly customized immune cells with improved therapeutic potential [147, 148].

The combination of viral transduction with these advanced gene editing tools represents new opportunities to achieve more efficient and precise modifications. This integrated approach holds the potential to significantly enhance process efficiency and facilitate the creation of next-generation immune cell therapies with improved functionality and persistence.

Process automation and artificial intelligence (AI)

Automation and AI are poised to play an increasingly critical role addressing challenges related to scalability, reproducibility, and regulatory compliance. Automated platforms can streamline viral transduction workflows, minimize manual intervention, and improve batch-to-batch consistency [149]. AI-powered systems have the potential to analyse vast datasets generated throughout the manufacturing process, allowing for real-time optimization of transduction protocols, culture conditions, and quality control measures [150, 151].

The convergence of automated workflows and AI-driven predictive models is expected to revolutionize immune cell transduction, providing scalable and standardized solutions that ensure both precision and efficiency. Such advancements will not only optimize manufacturing processes but also contribute to making immune cell therapies more accessible and commercially viable.

Future perspectives and conclusion

Despite its widespread adoption, viral transduction in immune cell therapy manufacturing faces significant challenges, particularly concerning safety risks associated with viral vectors, as well as the long lead times and high costs of GMP-grade vector production. These constraints have spurred growing interest in non-viral transfection methods as alternative gene delivery strategies [152]. Nevertheless, given its established efficacy, safety and industry-wide adoption, viral transduction remains the cornerstone of genetic modification in immune cell therapy manufacturing.

While T cell therapies have benefited from relatively efficient lentiviral and retroviral transduction, other immune effectors—particularly NK cells and myeloid populations—pose unique biological barriers to gene delivery that demand innovative solutions. As manufacturing shifts toward automated, closed-system production, the field must reconcile the competing priorities of process intensification (achieving higher efficiencies) and product quality (maintaining therapeutic cell fitness).

Ongoing innovations in viral vector engineering hold great promise for improving transduction efficiency and safety. Furthermore, the integration of transduction with advanced gene-editing tools presents exciting opportunities for precise genomic modifications, potentially improving both transduction efficiency and the therapeutic functionality of modified immune cells. Additionally, the adoption of process automation and AI is poised to transform manufacturing workflows, improving scalability, reproducibility, and real-time quality control. The coming decade will likely witness the maturation of viral transduction from an empirical process to a predictive science, driven by a deeper mechanistic understanding and the implementation of advanced analytical technologies. This evolution will be critical to expanding the accessibility of immune cell therapies across diverse clinical indications.

This review has systematically examined the CQAs and CPPs essential for developing robust, scalable immune cell transduction processes that satisfy three fundamental requirements: (1) high-efficiency gene delivery, (2) preservation of cellular fitness and function, and (3) compliance with evolving regulatory standards for therapeutic cell products.

While this work focuses on in vitro manufacturing—where immune cells are genetically modified in vitro before reinfusion, direct in vivo administration of viral vectors is emerging as an alternative strategy [153]. In vivo transduction offers the potential to bypass complex ex vivo processing, reducing costs and expanding accessibility. However, major challenges remain, including off-target effects, immune responses, and insertional mutagenesis risks. The success of in vivo transduction hinges on precise vector biodistribution and cell-type-specific targeting, areas that require further refinement [154, 155]. Looking ahead, ex vivo immune cell therapies are expected to remain the dominant approach due to their controlled and reliable nature, while in vivo therapies may play a growing role in systemic genetic and metabolic disorders, provided that safety and specificity challenges can be addressed.

Ultimately, the field must strive to develop transduction strategies that are both reliable and effective, enabling the scalable production of potent immune cell therapies. Achieving this vision will require a concerted effort in innovation, optimization, and stringent quality control—a challenge the industry is well-positioned to meet.