Cell and Gene Therapy

Special Delivery: can advanced intracellular delivery enable superior cell therapies for solid tumour treatment – and beyond?

By Stuart Lowe

Inspired by the promise of CAR-T cell therapy, new technologies to combat the hostile solid tumour microenvironment are being developed, underpinned by innovations in intracellular delivery, say Megan McCandless and Stuart Lowe.

Why is there so much excitement about cell therapy for solid tumours?

CAR-T cell therapy, involving the delivery of genetic material into T lymphocytes to cause expression of a chimeric antigen receptor (CAR), has revolutionised the treatment landscape for patients with haematological cancers. Trials have demonstrated up to 94% complete remission for indications such as acute lymphoblastic leukaemia (ALL) [1]. With around 93% of all cancers having a solid tumour physiology, however, the benefits of CAR-T cell therapy are yet to be felt by most patients.

What obstacles are there for CARs in treating solid tumours?

To be repurposed for the treatment of solid tumours, the next generation of cell therapies will need to overcome several obstacles, mainly associated with the solid tumour microenvironment, where [2]:

chemokines (chemotaxis-inducing proteins) prevent trafficking of T cells,

physical and chemical barriers (concentrated blood vessels, low pH) reduce infiltration,

cytokines (proteins that influence cell behaviour) reduce persistence of T cells in the tumour, and

sub-clonal populations of cancer cells may exist in a single tumour, reducing efficacy.

These factors in solid tumours demand the development of enhancements to the CAR expression platform, potentially requiring additional rounds of gene editing, direct delivery of proteins, or engineering of alternative immune cells. At present, the two leading transfection technologies struggle to deliver the necessary features. In particular:

Viral vectors provide highly efficient gene transfer but are less efficient for some cell types and can only deliver nucleic acids.

Electroporation can cause cell damage and could be lethal in multiple doses or for delicate cell types; it may also denature non-nucleic acid cargoes.

Can emerging technologies offer enhanced performance, and might they offer additional benefits for the cell therapy field?

Gene editing for souped-up CAR-T cells

Delivering multiple rounds of cell engineering, via CRISPR-Cas9 gene editing, could confer beneficial features such as: reduced expression of markers targeted by cytokines, increased expression of chemokine receptors, or expression of several antigen receptors to target multiple cell types.

Unlike established methods, several mechanical and chemical methods potentially offer the flexibility to deliver sequential CRISPR-Cas9 genetic payloads while maintaining cell viability and potency.

We spoke to Dr Michael Maguire, CEO of Avectas, about their chemical permeabilisation approach [3]:

“Developers of next generation CAR-T cell therapies are looking to rapidly add several levels of sophistication to their cell population, and need to do it in a deterministic and controllable manner. We developed the Solupore® technology as a reversible, chemical permeabilisation method that allows the desired cargo to enter cells via diffusion. We have seen that Solupore® engineered cells undergo very low levels of off-target gene expression perturbation and exhibit a high degree of potency soon after transfection. CRISPR-Cas9 gene editing is an extremely compelling use case for the Solupore® technology.”

Is the delivery vehicle right for the cargo?

Rather than relying on plasmid incorporation to induce the desired effect, it can be beneficial to directly deliver proteins into immune cells, which has been shown to:

eliminate the effect of off-target mutations in CRISPR-Cas9;

achieve precise antigen presentation on B cells, priming antigen-specific T cells; and

potentially boost delivery efficiency when co-delivered with nucleic acids [4].

Here, fundamental limitations of viral vectors include their inability to deliver non-genetic cargo, whilst electroporation-based approaches can damage delicate biomolecules. Several active areas of research target the delivery of a range of biomolecules, while preserving the functionality of the cargo, including:

An approach which involves direct penetration of the cell membrane to deliver target material, namely, delivery of CRISPR ribonucleoproteins adsorbed onto nanoneedles [5].

Mechanical methods such as squeezing cells through narrow constrictions within microfluidic devices (SQZ Biotech) [6], or subjecting cells to shear stresses that increase the tension on the cell membrane to achieve pore formation and delivery [7].

Exosomes which can be used to deliver proteins efficiently through direct fusion with the cell membrane, circumventing some of the issues of endosomal degradation seen with synthetic carrier systems [8].

The sheer multitude of non-nucleic acid cargoes could, in combination with an appropriate delivery approach, be transformative for solid tumour cell therapy. Intriguingly, the same techniques could be used to address a range of entirely different indications such as HIV and immunotolerance.

New CARs for different destinations

The techniques described above may also be beneficial for the expression of CARs in alternative immune cells, particularly natural killer (NK) cells and macrophages. These immune cells are responsible for killing cancer cells innately, meaning they may be more effective than CAR-T cells within the solid tumour environment.

Non-viral methods are in the forefront for the development of new CARs, though a finer level of control over the conditions that promote intracellular delivery while minimising unwanted cell perturbation would be desirable.

In this regard, miniaturisation and microfabrication are enabling bulk techniques to be refined, as in the Flow Electroporation® approach from MaxCyte, where cells are subjected to electric fields for short periods of time in microfluidic channels. The approach has been shown to help preserve both viability and delivery efficiency [9] when applied to NK cells targeting lymphomas.

Along for the ride: what advantages might advanced intracellular delivery approaches bring?

As well as enabling a new generation of cell therapy modalities, advanced intracellular delivery approaches could also be pivotal in ensuring widespread adoption in clinical practice. Under the direction of regulatory agencies, companies commercialising cell therapies will be looking to select processes that not only enable the desired cell functions but that also bring:

greater control over parameters influencing delivery, with the effect of increasing efficiency and scope for customization;

more subtle membrane permeabilisation means, reducing cell damage;

increased uniformity of treatment of the cell population, increasing yield and reproducibility;

less variability in transfection outcome, reducing downstream processing; and

improved scalability and versatility, suited to diverse manufacturing settings.

Hence, new methods of intracellular delivery will target expanding the therapeutic toolbox while also servicing increasing patient demand.

What is the final destination for non-viral delivery approaches?

The treatment of solid tumours is undoubtedly a niche that cell therapies will fill in the near future, with researchers generating ever more complex and creative approaches to outwit tumour physiology. To reflect this, it is likely that a new ecosystem of delivery platforms will best facilitate the production of these emerging therapies as they evolve. Crucially, the tools developed for cell therapy could even have benefits for non-cancer patients and in bioprocess development for other life science applications. In short, intracellular delivery will be driving the creation of bio-enabled products for many years to come.

TTP would like to thank Megan McCandless for guest authoring this blog article. Megan holds a First-Class Honours degree in Human Physiology from the University of Leeds. She is currently enrolled at Imperial College London in the group of Prof Molly Stevens FREng FRS under a DTP MRC iCASE PhD Studentship co-funded by TTP, researching high throughput and effective transfection of cells using innovative methods and materials.