Spleen SORT LNP Generated in situ CAR T Cells Extend Survival in a Mouse Model of Lymphoreplete B Cell Lymphoma.

Chimeric Antigen Receptor (CAR) T cell immunotherapy is revolutionizing treatment for patients suffering from B-cell lymphoma (BL). However, the current method of CAR T cell production is complicated and expensive, requiring collection of patient blood to enrich the T cell population, ex vivo engineering/activation, and quality assessment before the patient can receive the treatment. Herein we leverage Spleen Selective ORgan Targeted (SORT) Lipid Nanoparticles (LNPs) to produce CAR T cells in situ and bypass the extensive and laborious process currently used. Optimized Spleen SORT LNPs containing 10 % 18 : 1 PA transfected CD3+, CD8+, and CD4+ T cells in wild-type mice. Spleen SORT LNPs delivered Cre recombinase mRNA and CAR encoding mRNA to T cells in reporter mice and in a lymphoreplete B cell lymphoma model (respectively) after intravenous injection without the need for active targeting ligands. Moreover, in situ CAR T cells increased the overall survival of mice with a less aggressive form of B cell lymphoma. In addition, in situ transfected CAR T cells reduced tumor metastasis to the liver by increasing tumor infiltrating lymphocytes. Overall, these results offer a promising alternative method for CAR T cell production with pre-clinical potential to treat hematological malignancies.

Optimization of phospholipid chemistry for improved lipid nanoparticle (LNP) delivery of messenger RNA (mRNA).

Lipid nanoparticles (LNPs) have been established as an essential platform for nucleic acid delivery. Efforts have led to the development of vaccines that protect against SARS-CoV-2 infection using LNPs to deliver messenger RNA (mRNA) coding for the viral spike protein. Out of the four essential components that comprise LNPs, phospholipids represent an underappreciated opportunity for fundamental and translational study. We investigated this avenue by systematically modulating the identity of the phospholipid in LNPs with the goal of identifying specific moieties that directly enhance or hinder delivery efficacy. Results indicate that phospholipid chemistry can enhance mRNA delivery by increasing membrane fusion and enhancing endosomal escape. Phospholipids containing phosphoethanolamine (PE) head groups likely increase endosomal escape due to their fusogenic properties. Additionally, it was found that zwitterionic phospholipids mainly aided liver delivery, whereas negatively charged phospholipids changed the tropism of the LNPs from liver to spleen. These results demonstrate that the choice of phospholipid plays a role intracellularly by enhancing endosomal escape, while also driving organ tropism in vivo. These findings were then applied to Selective Organ Targeting (SORT) LNPs to manipulate and control spleen-specific delivery. Overall, selection of the phospholipid in LNPs provides an important handle to design and optimize LNPs for improved mRNA delivery and more effective therapeutics.

Testing Amyloid Cross-Toxicity in the Vertebrate Brain.

While amyloid proteins such as amyloid ß (Aß),α-synuclein, tau, and lysozyme are known to be prion-like; emerging data have revealed that they are also able to seed the misfolding of prion-like proteins differing in sequence. In the present study, we have developed a tool designed to test neurohistochemical outcomes associated with the entry of an amyloid protein into heterotypic neurons, i.e., neurons that do not express the invading amyloid and, instead, endogenously express amyloids differing in sequence. The stereotaxic introduction of Aß into the rodent tegmental area of the mid-brain revealed that the foreign amyloid had infiltrated into nigral neurons. Furthermore, Aß was found colocalized with α-synuclein, an amyloid endogenous to the substantia nigra and differing in sequence relative to Aß. Disruption of α-synuclein status in the substantia nigra is associated with Parkinson's disease onset and progress. In addition to the study findings, a significant inroad to future neurodegenerative research was made via the stereotaxic introduction of the foreign amyloid. This technique limits the presence of confounding neurometabolic variables that may be prevalent in transgenic animal models of cross-toxicity and, thereby, better addresses the role of individual neuronal factors in cross-toxicity. Finally, the data from this work may help reconcile the high frequency of clinical comorbidity seen in neurodegenerative diseases.