AAV-TCs: T Cell Targeting Capsids
T cells are central to adaptive immunity and are key effectors in cancer immunotherapy, autoimmune disease, and infectious disease treatment. While ex vivo genetic modification of T cells has achieved remarkable clinical success, particularly with CAR-T therapies, in vivo T cell gene delivery remains a major unmet challenge. Adeno-associated virus (AAV) vectors are widely used for in vivo gene therapy due to their favorable safety profile and durable expression; however, native AAV serotypes show poor transduction efficiency in T cells. Recent advances in AAV capsid engineering, directed evolution, and genome optimization have begun to overcome these barriers.
Biological Barriers to AAV Transduction of T Cells
Despite the broad clinical success of adeno-associated virus (AAV) vectors in multiple tissues, T cells remain one of the most refractory cell types to AAV-mediated gene delivery. This resistance arises from a combination of extracellular, intracellular, and nuclear barriers that collectively limit efficient vector entry, genome trafficking, and transgene expression.
- Limited Cell-Surface Receptor Availability. Most natural AAV serotypes rely on glycan-based receptors, such as heparan sulfate proteoglycans, galactose, or sialic acid, for cell attachment and entry. Resting T cells express low levels of these canonical AAV receptors, resulting in inefficient viral binding and internalization. This contrasts sharply with highly permissive tissues such as liver and muscle, where receptor abundance facilitates robust transduction.
- Reduced Endocytic Activity. Naïve and resting T cells exhibit intrinsically low endocytic and macropinocytic activity, further restricting AAV uptake. Even when viral attachment occurs, internalization efficiency is markedly lower than in epithelial or endothelial cells. T cell activation can partially enhance endocytosis, which explains the modest improvement in AAV transduction observed in activated T cells compared with resting populations.
- Inefficient Intracellular Trafficking and Endosomal Escape. Following internalization, AAV particles must escape the endosomal compartment to reach the cytoplasm and subsequently the nucleus. In T cells, endosomal escape is inefficient, and viral particles are frequently targeted for lysosomal degradation. In addition, strong proteasomal activity in T cells contributes to capsid degradation before productive infection can occur.
- Restrictive Nuclear Entry and Genome Processing Successful AAV transduction requires nuclear entry of the viral genome and conversion to a transcriptionally active episome. T cells present additional challenges at this stage, including:
- Limited nuclear import of AAV capsids. Reduced efficiency of second-strand synthesis for single-stranded AAV genomes. A chromatin environment that is less permissive to episomal persistence, particularly in resting T cells. These factors significantly delay or prevent transgene expression, even when vector genomes reach the nucleus.
- Cell State–Dependent Permissiveness. T cell susceptibility to AAV transduction is highly dependent on activation state. Activated T cells display increased metabolic activity, enhanced DNA repair pathways, and greater chromatin accessibility, all of which modestly improve AAV transduction efficiency. However, activation-dependent transduction is incompatible with many therapeutic goals, particularly for in vivo gene delivery to resting T cells.
Together, these biological barriers explain why native AAV serotypes exhibit poor tropism for T cells and why conventional AAV vectors have failed to achieve therapeutically relevant transduction in vivo. Overcoming these challenges requires advanced capsid engineering, often combined with genome-level optimization, to enable efficient and selective T cell gene delivery.
Natural AAV Serotypes and T Cell Transduction
Naturally occurring AAV serotypes have been extensively characterized for gene delivery to tissues such as liver, muscle, central nervous system, and retina. However, none of the native AAV serotypes exhibit strong intrinsic tropism for T cells, either in vitro or in vivo. This limitation represents a major barrier to the development of AAV-based in vivo T cell gene therapies.
| AAV Serotype | Primary Attachment Receptor | Resting T Cells | Activated T Cells (ex vivo) | In Vivo T Cell Transduction | Comments |
|---|---|---|---|---|---|
| AAV2 | Heparan sulfate proteoglycan | Very low | Very low | None detected | Widely studied; efficient in adherent cells but poorly transduces T cells |
| AAV6 | Sialic acid (α2,3) | Very low | Low–moderate | low | Best-performing natural serotype for activated T cells ex vivo |
| AAV8 | Galactose | Very low | Very low | None detected | Strong liver tropism; ineffective for T cells |
| AAV9 | Galactose / N-linked glycans | Very low | Very low | None detected | Excellent CNS and muscle tropism; poor T cell transduction |
| Other natural serotypes | Various | Very low | Very low | None detected | No native serotype shows meaningful T cell tropism |
AAV6 for T Cell Gene Delivery
AAV6 is widely regarded as the most effective natural AAV serotype for T cell transduction, particularly in activated human T cells ex vivo. Among all native AAV serotypes, AAV6 consistently demonstrates superior entry and transgene expression in T cells and is therefore commonly used as the benchmark serotype for evaluating engineered AAV capsids targeting T cells.
AAV6 is most effective in activated or proliferating T cells, where enhanced endocytosis, chromatin accessibility, and DNA repair activity facilitate AAV genome processing. Consequently, AAV6 has been extensively applied in ex vivo T cell engineering, including delivery of donor templates for CRISPR/Cas-mediated genome editing, such as targeted knock-in at the TRAC locus for CAR-T cell generation.
Despite these advantages, AAV6 has notable limitations. Transduction efficiency remains low in resting T cells, and systemic in vivo T cell targeting with AAV6 has not been convincingly demonstrated. High vector doses are often required, and transgene expression can be variable, limiting its utility for in vivo gene therapy applications.
Overall, AAV6 serves as a valuable tool for ex vivo T cell modification and as a reference standard for next-generation capsid engineering efforts. Its limitations highlight the need for engineered AAV capsids capable of efficient and selective gene delivery to resting T cells in vivo.