Homology-independent targeted insertion (HITI) enables guided CAR knock-in and efficient clinical scale CAR-T cell manufacturing.

BACKGROUND: Chimeric Antigen Receptor (CAR) T cells are now standard of care (SOC) for some patients with B cell and plasma cell malignancies and could disrupt the therapeutic landscape of solid tumors. However, access to CAR-T cells is not adequate to meet clinical needs, in part due to high cost and long lead times for manufacturing clinical grade virus. Non-viral site directed CAR integration can be accomplished using CRISPR/Cas9 and double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) via homology-directed repair (HDR), however yields with this approach have been limiting for clinical application (dsDNA) or access to large yields sufficient to meet the manufacturing demands outside early phase clinical trials is limited (ssDNA). METHODS: We applied homology-independent targeted insertion (HITI) or HDR using CRISPR/Cas9 and nanoplasmid DNA to insert an anti-GD2 CAR into the T cell receptor alpha constant (TRAC) locus and compared both targeted insertion strategies in our system. Next, we optimized post-HITI CRISPR EnrichMENT (CEMENT) to seamlessly integrate it into a 14-day process and compared our knock-in with viral transduced anti-GD2 CAR-T cells. Finally, we explored the off-target genomic toxicity of our genomic engineering approach. RESULTS: Here, we show that site directed CAR integration utilizing nanoplasmid DNA delivered via HITI provides high cell yields and highly functional cells. CEMENT enriched CAR T cells to approximately 80% purity, resulting in therapeutically relevant dose ranges of 5.5 × 108-3.6 × 109 CAR + T cells. CRISPR knock-in CAR-T cells were functionally comparable with viral transduced anti-GD2 CAR-T cells and did not show any evidence of off-target genomic toxicity. CONCLUSIONS: Our work provides a novel platform to perform guided CAR insertion into primary human T-cells using nanoplasmid DNA and holds the potential to increase access to CAR-T cell therapies.

Electronic control of H+ current in a bioprotonic device with carbon nanotube porins.

Hybrid biotic abiotic devices can be used to interface electronics with biological systems for novel therapies or to increase device functionality beyond silicon. Many strategies exist to merge the electronic and biological worlds, one dominated by electrons and holes as charge carriers, the other by ions. In the biological world, lipid bilayers and ion channels are essential to compartmentalize the cell machinery and regulate ionic fluxes across the cell membrane. Here, we demonstrate a bioelectronic device in which a lipid bilayer supported on H+-conducting Pd/PdHx contacts contains carbon nanotubes porin (CNTP) channels. This bioelectronic device uses CNTPs to control of H+ flow across the lipid bilayer with a voltage applied to the Pd/PdHx contacts. Potential applications of these devices include local pH sensing and control.

Silicon Nanoribbon pH Sensors Protected by a Barrier Membrane with Carbon Nanotube Porins.

Limited biocompatibility and fouling propensity can restrict real-world applications of a large variety of biosensors. Biological systems are adept at protecting and separating vital components of biological machinery with semipermeable membranes that often contain defined pores and gates to restrict transmembrane transport only to specific species. Here we use a similar approach for creating fouling-resistant pH sensors. We integrate silicon nanoribbon transistor sensors with an antifouling lipid bilayer coating that contains proton-permeable carbon nanotube porin (CNTP) channels and demonstrate robust pH detection in a variety of complex biological fluids.

Carbon Nanotube Porins in Amphiphilic Block Copolymers as Fully Synthetic Mimics of Biological Membranes.

Biological membranes provide a fascinating example of a separation system that is multifunctional, tunable, precise, and efficient. Biomimetic membranes, which mimic the architecture of cellular membranes, have the potential to deliver significant improvements in specificity and permeability. Here, a fully synthetic biomimetic membrane is reported that incorporates ultra-efficient 1.5 nm diameter carbon nanotube porin (CNTPs) channels in a block-copolymer matrix. It is demonstrated that CNTPs maintain high proton and water permeability in these membranes. CNTPs can also mimic the behavior of biological gap junctions by forming bridges between vesicular compartments that allow transport of small molecules.

Impact of PEG additives and pore rim functionalization on water transport through sub-1 nm carbon nanotube porins.

Carbon nanotubes represent one of the most interesting examples of a nanofluidic channel that combines extremely small diameters with atomically smooth walls and well-defined chemical functionalities at the pore entrance. In the past, sub-1 nm diameter carbon nanotube porins (CNTPs) embedded in a lipid membrane matrix demonstrated extremely high water permeabilities and strong ion selectivities. In this work, we explore additional factors that can influence transport in these channels. Specifically, we use stopped-flow transport measurements to focus on the effect of chemical modifications of the CNT rims and chaotropic polyethyleneglycol (PEG) additives on CNTP water permeability and Arrhenius activation energy barriers for water transport. We show that PEG, especially in its more chaotropic coiled configuration, enhances the water transport and reduces the associated activation energy. Removal of the static charges on the CNTP rim by converting -COOH groups to neutral methylamide groups also reduces the activation energy barriers and enhances water transport rates.

Response to Comment on "Enhanced water permeability and tunable ion selectivity in subnanometer carbon nanotube porins".

Horner and Pohl argue that high water transport rates reported for carbon nanotube porins (CNTPs) originate from leakage at the nanotube-bilayer interface. Our results and new experimental evidence are consistent with transport through the nanotube pores and rule out a defect-mediated transport mechanism. Mechanistic origins of the high Arrhenius factor that we reported for narrow CNTPs at pH 8 require further investigation.

Enhanced water permeability and tunable ion selectivity in subnanometer carbon nanotube porins.

Fast water transport through carbon nanotube pores has raised the possibility to use them in the next generation of water treatment technologies. We report that water permeability in 0.8-nanometer-diameter carbon nanotube porins (CNTPs), which confine water down to a single-file chain, exceeds that of biological water transporters and of wider CNT pores by an order of magnitude. Intermolecular hydrogen-bond rearrangement, required for entry into the nanotube, dominates the energy barrier and can be manipulated to enhance water transport rates. CNTPs block anion transport, even at salinities that exceed seawater levels, and their ion selectivity can be tuned to configure them into switchable ionic diodes. These properties make CNTPs a promising material for developing membrane separation technologies.

Real-time dynamics of carbon nanotube porins in supported lipid membranes visualized by high-speed atomic force microscopy.

In-plane mobility of proteins in lipid membranes is one of the fundamental mechanisms supporting biological functionality. Here we use high-speed atomic force microscopy (HS-AFM) to show that a novel type of biomimetic channel-carbon nanotube porins (CNTPs)-is also laterally mobile in supported lipid membranes, mimicking biological protein behaviour. HS-AFM can capture real-time dynamics of CNTP motion in the supported lipid bilayer membrane, build long-term trajectories of the CNTP motion and determine the diffusion coefficients associated with this motion. Our analysis shows that diffusion coefficients of CNTPs fall into the same range as those of proteins in supported lipid membranes. CNTPs in HS-AFM experiments often exhibit 'directed' diffusion behaviour, which is common for proteins in live cell membranes.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'.

Synthesis, lipid membrane incorporation, and ion permeability testing of carbon nanotube porins.

Carbon nanotube porins (CNTPs) are 10- to 20-nm-long segments of lipid-stabilized single-walled carbon nanotubes (CNTs) that can be inserted into phospholipid membranes to form nanometer-scale-diameter pores that approximate the geometry and many key transport characteristics of biological membrane channels. We describe protocols for CNTP synthesis by ultrasound-assisted cutting of long CNTs in the presence of lipid amphiphiles, and for validation of CNTP incorporation into a lipid membrane using a proton permeability assay. In addition, we describe protocols for measuring conductance of individual CNTPs in planar lipid bilayers and plasma membranes of live cells. The protocol for the preparation and testing of the CNTPs in vesicle systems takes 3 d, and single CNTP conductance measurements take 2-5 h. The CNTPs produced by this cutting protocol remain stable and active for at least 10-12 weeks.

Structure of Carbon Nanotube Porins in Lipid Bilayers: An in Situ Small-Angle X-ray Scattering (SAXS) Study.

Carbon nanotube porins (CNTPs), small segments of carbon nanotubes capable of forming defined pores in lipid membranes, are important future components for bionanoelectronic devices as they could provide a robust analog of biological membrane channels. In order to control the incorporation of these CNT channels into lipid bilayers, it is important to understand the structure of the CNTPs before and after insertion into the lipid bilayer as well as the impact of such insertion on the bilayer structure. Here we employed a noninvasive in situ probe, small-angle X-ray scattering, to study the integration of CNT porins into dioleoylphosphatidylcholine bilayers. Our results show that CNTPs in solution are stabilized by a monolayer of lipid molecules wrapped around their outer surface. We also demonstrate that insertion of CNTPs into the lipid bilayer results in decreased bilayer thickness with the magnitude of this effect increasing with the concentration of CNTPs.

Ultrafast proton transport in sub-1-nm diameter carbon nanotube porins.

Proton transport plays an important role in many biological processes due to the ability of protons to rapidly translocate along chains of hydrogen-bonded water molecules. Molecular dynamics simulations have predicted that confinement in hydrophobic nanochannels should enhance the rate of proton transport. Here, we show that 0.8-nm-diameter carbon nanotube porins, which promote the formation of one-dimensional water wires, can support proton transport rates exceeding those of bulk water by an order of magnitude. The transport rates in these narrow nanotube pores also exceed those of biological channels and Nafion. With larger 1.5-nm-diameter nanotube porins, proton transport rates comparable to bulk water are observed. We also show that the proton conductance of these channels can be modulated by the presence of Ca(2+) ions. Our results illustrate the potential of small-diameter carbon nanotube porins as a proton conductor material and suggest that strong spatial confinement is a key factor in enabling efficient proton transport.

Bioelectronic light-gated transistors with biologically tunable performance.

Light-activated bioelectronic silicon nanowire transistor devices are made by fusing proteoliposomes containing a bacteriorhodopsin (bR) proton pump onto the nanowire surface. Under green-light illumination, bR pumps protons toward the nanowire, and the pH gradient developed by the pump changes the transistor output. Furthermore, co-assembly of small biomolecules that alter the bilayer permeability to other ions can upregulate and downregulate the response of field-effect transistor devices.