The clinical success of chimeric antigen receptor (CAR) T cell immunotherapy in treating multiple blood cancers has created a need for efficient methods of ex vivo gene delivery to primary human T cells for cell engineering. Here, we synthesize and evaluate a panel of cationic polymers for gene delivery to both cultured and primary human T cells. We show that a subset of comb- and sunflower-shaped pHEMA-g-pDMAEMA polymers can mediate transfection with efficiencies up to 50% in the Jurkat human T cell line with minimal concomitant toxicity (>90% viability). We then optimize primary human T cell transfection conditions including activation time, cell density, DNA dose, culture media, and cytokine treatment. We demonstrate transfection of both CD4 and CD8 primary human T cells with messenger RNA and plasmid DNA at efficiencies up to 25 and 18%, respectively, with similarly high viability.

Decreased uptake of polyplexes and an increased intracellular pH contribute to poor gene delivery to human T cells via cationic polymers.

Biomaterial properties that modulate T cell activation, growth, and differentiation are of significant interest in the field of cellular immunotherapy manufacturing. In this work, we present a new platform technology that allows for the modulation of various activation particle design parameters important for polyclonal T cell activation. We successfully create artificial antigen presenting cells (aAPCs) using supported lipid bilayers on various cell-templated silica microparticles with defined membrane fluidity and stimulating antibody density. We use this panel of aAPCs to probe the importance of activation particle shape, size, membrane fluidity, and stimulation antibody density on T cell outgrowth and differentiation. All aAPC formulations were able to stimulate T cell growth, and preferentially promoted CD8+ T cell growth over CD4+ T cell growth when compared to commercially available pendant antibody-conjugated particles. T cells cultured with HeLa and red blood cell templated aAPCs had a less differentiated and less exhausted phenotype than those cultured with spherical aAPCs with matched membrane coatings when cultured for 14 days. These results support continued exploration of silica supported lipid bilayers as an artificial antigen presenting cell platform.

While biologic drugs such as proteins, peptides, or nucleic acids have shown promise in the treatment of neurodegenerative diseases, the blood−brain barrier (BBB) severely limits drug delivery to the central nervous system (CNS) after systemic administration. Consequently, drug delivery challenges preclude biological drug candidates from the clinical armamentarium. In order to target drug delivery and uptake into to the CNS, we used an in vivo phage display screen to identify peptides able to target drug-uptake by the vast array of neurons of the autonomic nervous system (ANS). Using next-generation sequencing, we identified 21 candidate targeted ANS-to-CNS uptake ligands (TACL) that enriched bacteriophage accumulation and delivered protein-cargo into the CNS after intraperitoneal (IP) administration. The series of TACL peptides were synthesized and tested for their ability to deliver a model enzyme (NeutrAvidin-horseradish peroxidase fusion) to the brain and spinal cord. Three TACL-peptides facilitated significant active enzyme delivery into the CNS, with limited accumulation in off-target organs. Peptide structure and serum stability is increased when internal cysteine residues are cyclized by perfluoroarylation with decafluorobiphenyl, which increased delivery to the CNS further. TACL-peptide was demonstrated to localize in parasympathetic ganglia neurons in addition to neuronal structures in the hindbrain and spinal cord. By targeting uptake into ANS neurons, we demonstrate the potential for TACL-peptides to bypass the blood− brain barrier and deliver a model drug into the brain and spinal cord.