CAR T-cell therapy is a revolutionary treatment for cancer that uses genetically modified immune cells to selectively kill cancerous cells in the body. The first FDA-approved CAR T-cell therapy was released in 2017 for acute B-cell lymphoblastic leukemia, and since then, several other CAR T-cell therapies have been developed and have achieved remarkable remission rates against a range of blood cancers. However, despite this progress, current methods for producing CAR T-cells are complex and resource-intensive, while attempts to utilize these cells against solid tumors have yielded limited success.
In this article, SelectScience® speaks with Dr. Krishanu Saha, an associate professor of biomedical engineering at the University of Wisconsin-Madison, to learn how his team is using a nascent CRISPR-Cas9 gene editing method to generate CAR T-cells for the treatment of solid tumors. Saha reveals how this approach can circumvent the challenges of conventional viral-based methods, highlights the potential of CRISPR to improve CAR T-cell efficacy, and shares how support and tools from Thermo Fisher Scientific are helping advance his lab’s research toward clinical use.
A “hit and run” approach to CAR T-cell production
CAR T-cells are human immune cells that have been genetically engineered to express a chimeric antigen receptor (CAR), an artificial receptor that can recognize specific types of cancer. Currently, all approved CAR T-cell therapies rely upon the use of viral vectors to introduce, or knock-in, a CAR transgene into a patient’s T cells. These viral vectors, such as lentiviruses or γ-retroviruses, enable stable and efficient gene integration. However, they are limited by a tendency for their nucleic acid payload to broadly integrate into the host genome – in up to tens of thousands of different locations. The use of viruses also raises safety and regulatory considerations due to the potential for off-target effects. “There are concerns about insertional oncogenesis for some of these viral vectors, where one can unintentionally generate cancer in the patient after infusion of the cell product,” explains Saha. “There are also other unpredictable effects on CAR gene expression and receptor levels through viral gene delivery.”
“Advances in genome editing provide a path to do this genetic engineering in a cleaner way,” he continues, noting that the CRISPR/Cas9 method allows the insertion of the CAR transgene at precise target regions of the genome. There is still the potential for off-target insertions using CRISPR, but this can be minimized by limiting the duration of editing, and specifically the lifetime of the editor. “We deliver the Cas9 protein directly, meaning it doesn’t need to be transcribed and translated – as with some other CRISPR delivery strategies such as plasmids or mRNA,” he explains. “The protein degrades away within a matter of hours, so by using CRISPR in this ‘hit and run’ type strategy, it doesn't have the time to affect off-target areas in the genome as much.”
Learn more about how to improve gene editing fidelity and efficiency along every stage of your workflow with this free eBook, available for download here >>
Targeting solid tumors
Saha and his team recently used this approach to tailor CAR T-cells to target solid tumors1. Historically, solid tumors have posed a persistent challenge to CAR T-cell therapy, owing to factors including T cell homing, T cell fitness, and the hostile tumor microenvironment (TME). “Tumor-associated macrophages, fibroblasts, and other aspects of the TME, including it being hypoxic in some cases, can all make it challenging for T cells to function appropriately,” Saha explains. “There are also various modifications to the extracellular matrix and cell surface that occur in a solid tumor, including sugars that may help the tumors grow more efficiently by capturing various growth or immunosuppressive factors.”
Building upon prior work on hematological malignancies, the group genetically engineered T cells to specifically target a sugar molecule found on the surface of neuroblastoma solid tumor cells. They found that not only did these CRISPR-generated CAR T-cells induce solid tumor regression in mouse models, but they were also less prone to exhaustion compared to CAR T-cells produced by viral methods.
“We’re now in a very exciting and interesting phase of moving towards an Investigational New Drug (IND) application with a fully non-viral CAR T product,” Saha enthuses. “This work has taken several years and has been aided by the development of advanced CRISPR tools and instruments in the field.”
One such instrument that the team has recently started using is the Gibco™ CTS™ Xenon™, a benchtop closed electroporation system designed to provide high-performance non-viral gene editing and transfection. “What's unique about the Xenon is that it allows us to get under the hood of the details of electroporation that could be critical for the CRISPR gene editing of T cells," says Saha. The system enables users to optimize the electroporation process by tailoring parameters such as voltage and pulse width, number, and interval, and can provide efficient transfection in volumes of up to 25 mL in less than 25 minutes. "It’s on its way to being appropriate for clinical use and has been a key component in moving our protocols from a research bench to a clinical manufacturing facility,” he adds.
Saha also praises the team at Thermo Fisher Scientific, whose expertise has proven invaluable to the progression of this research. “One of the nice features of working with Thermo Fisher is that the team that has supported us through this journey is very knowledgeable and has really assisted us in moving our protocols to a more stringent set of requirements for GMP-compliant manufacturing," he says. “I think Thermo Fisher's array of products will allow us to quickly move this into the clinic."
Looking ahead, Saha sees great opportunity in improving CAR T-cell therapies to achieve longer durability in patients and overcome barriers within the body that can influence their efficacy, specifically in the tumor microenvironment. “Using CRISPR, not only can we go into any area of the genome to insert our functionality, but also add in additional genetic payloads, such as protective cytokines or additional therapeutic modalities like BiTEs and engagers,” he says. “These are all possibilities that bioengineers have been working on for decades now, and those types of functionalities have tremendous promise for programming human cell therapeutics.”
“As an engineer, I'm very excited to be able to design these types of constructs essentially on my laptop by looking at the genetic code, and then trying to execute that on a technology stack that can be quickly implemented in a clinical setting,” he continues. “Of course, this is heavily mediated by and involves partnership with clinicians and patients, but they have been very willing partners thus far, and I hope will continue to be so in the future.”
Looking for more expert advice on how to improve your gene editing workflows?
1. Mueller, Katherine P., et al. Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression. Journal for ImmunoTherapy of Cancer (2022)