In Vivo CRISPR CAR T Cells Eradicate Tumors in Mice

CAR T Therapies: Promising Yet Challenging In theory, T cells, which serve as the primary attackers in our adaptive immune system, ought to effectively destroy cancer cells. However, in many instances, these T cells either do not identify tumor cells as threats or become inhibited by the surrounding

CAR T Therapies: Promising Yet Challenging

In theory, T cells, which serve as the primary attackers in our adaptive immune system, ought to effectively destroy cancer cells. However, in many instances, these T cells either do not identify tumor cells as threats or become inhibited by the surrounding tumor environment.

CAR T cell therapy addresses these shortcomings by modifying T cells genetically to produce a synthetic receptor known as a chimeric antigen receptor (CAR). This receptor directs the T cells to target and eliminate cancer cells that display a specific protein, such as CD19 found on leukemia cells. Currently, seven FDA-approved treatments of this nature exist, offering long-lasting remissions for patients with blood cancers who have exhausted all other treatment avenues.

The conventional procedure involves extracting T cells from the patient and transporting them to a dedicated facility. There, a gene encoding the CAR is introduced using retroviral or lentiviral vectors. The cells are then cultured and multiplied before being reinfused into the patient. This entire operation spans 3 to 5 weeks, incurs costs in the hundreds of thousands of dollars, and yields inconsistent outcomes. Frequently, the cells fail to proliferate adequately. Beyond financial barriers, patients often succumb while awaiting treatment.

Delivering CRISPR Directly In Vivo

Researchers from the University of California San Francisco have published a groundbreaking study in Nature, detailing a method to generate CAR T cells directly within the body, bypassing the traditional ex vivo process entirely. Building on a 2017 discovery, they utilized a precise CRISPR-based DNA-editing system paired with a repair template to insert the CAR sequence at a designated genomic location in T cells: the T cell receptor alpha constant (TRAC) locus. Placing the CAR gene at this site provides key benefits, including enhanced control over its expression, which helps prevent T cell fatigue. Previously, this technique had only been applied outside the body.

To accomplish in vivo engineering, the team employed dual delivery mechanisms. Initially, they used enveloped delivery vehicles (EDVs), which are virus-like particles constructed from viral proteins. These EDVs transported the CRISPR components responsible for precise targeting and DNA cleavage. Separately, an adeno-associated virus (AAV) vector carried the CAR gene, complete with homology arms matching the sequences flanking the CRISPR cut site. The cell's inherent DNA repair processes then incorporated the CAR gene accurately into the TRAC locus during break repair.

Following successful in vitro validation, the researchers implanted immunodeficient mice with a blend of human immune cells, encompassing T cells, B cells, and monocytes (collectively termed peripheral blood mononuclear cells or PBMCs). Subsequently, the mice were administered an intravenous dose of the EDV-AAV combination encoding the CD19-targeted CAR.

After two weeks, analysis of the mice spleens revealed the presence of TRAC-CAR T cells. Notably, these animals exhibited a significant reduction in CD19-positive B cells, confirming the CAR T cells' ability to eliminate their intended targets. Through iterative refinements to their delivery system, the researchers attained high transfection efficiency without triggering any widespread inflammatory responses.

Further examination of the cells' characteristics indicated not only their survival but also their active proliferation, functionality, and retention of a memory-like state, positioning them to respond effectively to subsequent antigen encounters. This study marks the pioneering achievement of targeted integration of a substantial DNA segment into primary human T cells within a live organism.

Taking on Aggressive Cancers in Models

The researchers proceeded to evaluate their approach against aggressive leukemia in mouse models. Three days post-tumor inoculation, human PBMCs were introduced, followed by the experimental therapy the next day. This protocol was replicated using PBMCs from four distinct donors to verify consistency. Remarkably, 18 out of 20 mice across all donors experienced complete tumor clearance.

To benchmark their innovation, the team compared it to an existing lentiviral system designed for in vivo CAR T production, which is undergoing Phase I clinical trials. Both in vitro and in vivo versions were assessed. The TRAC-CAR T in vivo method significantly surpassed competitors, delivering complete responses in all six tested mice. Moreover, these TRAC-CAR T cells expanded far more rapidly and exhibited superior, more consistent CAR expression compared to the lentiviral alternative. The researchers attribute this edge to the precision of site-specific integration at a regulated locus versus the haphazard insertion typical of lentiviral methods.

Justin Eyquem, PhD, associate professor of medicine at UCSF and senior author of the study, remarked, "What stood out most was that the cells generated in vivo appeared superior to those produced in laboratory settings. We believe that extracting cells from the body for lab culturing diminishes their stem-like qualities and growth potential, an issue avoided in this in vivo strategy."

Undeterred, the team tested their therapy on multiple myeloma, a distinct cancer requiring a different CAR target antigen. Once again, every one of the eight mice achieved full tumor eradication.

The ultimate challenge involved sarcoma, a solid tumor notoriously resistant to CAR T therapies due to challenges like limited T cell penetration, suppressive microenvironments, and variable antigen expression. Results varied by donor: with one donor, five of six mice saw complete responses; with the second, three of eight did. This underscores the persistent issue of donor-to-donor variability.

Eyquem elaborated, "Should we successfully adapt this for human use, it could slash expenses, remove delays, and empower local hospitals—not solely elite cancer institutions—to deliver these vital treatments. This would genuinely broaden access to CAR T cell therapy."