Effectively addressing cell-specific delivery has an opportunity to improve the therapeutic potential of in vivo gene therapies. We have developed a novel platform for engineering fusogens to target cellular molecules of choice, thereby enabling in vivo cell-specific delivery across many cell types with a fusogen-directed gene therapy vector. In vivo generation of CAR T cells, using gene therapy vectors with T cell targeting fusogens for in vivo CAR delivery, show promise in preclinical models and may provide broader access to CAR therapies.

Highly effective, yet complex to manufacture, simplifying CAR T cell generation is at the forefront of current research. Generating CAR T cells in vivo will heavily rely on vector technology, particularly high selectivity for T lymphocytes. Using surface-engineered lentiviral vectors targeted to T cell markers we have provided proof-of-concept for this strategy in humanized mouse models. As an alternative to lentiviral vectors, AAV vectors can be targeted to CD8+ T lymphocytes through insertion of CD8-specific DARPins into exposed loop regions. Ongoing preclinical studies are evaluating the different vector platforms and will identify potential hurdles to be solved toward clinical application.

Using targeted lipid nanoparticles (tLNP), we were able to transiently reprogram T cells in vivo by delivering modified mRNA encoding a CAR against fibroblast activation protein (FAP). This treatment resulted in the reduction of cardiac fibrosis and the restoration of cardiac function. The ability to produce transient, functional CAR T cells in vivo with mRNA addresses some of the biggest hurdles in cell therapy including manufacturing, scalability, and safety concerns.

Current CAR T cell manufacturing uses artificial ligands to induce activated T cells that are functionally distinct from T cells activated in their natural settings in vivo. In situ CAR T cells are generated directly in immune-reactive lymph nodes in just 3 days from T cells responding to virus-encoded MHC alloantigens presented by endogenous APC. Following in vivo retargeting with viruses encoding chimeric antigen receptors, the resulting in situ CAR T cells are capable of targeting antigen-positive cells systemically in the blood and in a solid tumor setting.

Cell-state specific gene expression is a major challenge in biomedicine and biotechnology. Our synthetic biology platforms limit the expression of any encodable genetic outputs only to cell states or tissue types of interest. We can rapidly and efficiently identify synthetic promoters that demonstrate superior specificity compared to native ones. Our synthetic gene circuits integrate the activity of multiple synthetic promoters via Boolean logical gates and generate output only when a predetermined promoter activity pattern is detected. This approach enhances gene expression specificity and efficacy. Our technologies are modular and could be adapted to target virtually any cell state.

We have developed a novel, synthetic, circular coding RNA platform (oRNA technology) which exhibits significant improvements in production, expression, and formulation compared to mRNAs. Given the successes as well as remaining challenges with CAR T cell therapies, we combined our oRNA technology with novel immunotropic LNPs to create an off-the-shelf “autologous” in situ CAR (isCAR) therapy that effectively delivers anti-CD19 CAR to immune cells and regresses tumors in a human-PBMC-engrafted tumor-bearing mouse model. oRNA-enabled isCAR therapies promise a transient, re-dosable, and scalable immune cell therapy without requiring lymphodepletion for the treatment of cancer. 

Ex vivo CAR T cell therapies have proven successful in the clinic, but their broad application is still facing significant challenges due to the elaborate and expensive engineering and manufacturing of T cells. Tidal Therapeutics has developed a new technology that allows the generation of CAR T cells directly in vivo. The technology uses mRNA, formulated in lipid nanoparticles that are specifically targeted to circulating T cells to transiently express CARs on the surface.

Logic-gated, two receptor systems amplify many of the challenges associated with CAR design. Here, we leverage concepts from the kinetic segregation model to design a tuneable CAR platform (Tmod) for optimal T cell activation and inhibition. Furthermore, by rationally integrating structural differences between activator and blocker targets, the Tmod platform is broadly applicable to a wide variety of therapeutic indications.

A platform that can selectively transduce specific immune cells in vivo can enable a range of personalized cellular and biologics immunotherapy. Towards this goal, our group has employed various principles from molecular biology and pharmaceutics to engineer different viral vector systems that can selectively transduce B and T cells in vivo with exceptional fidelity and potency. We will present both published and unpublished data.

Myeloid's novel in vivo engineering platform specifically targets and activates myeloid cells to elicit broader anti-tumor adaptive immunity. Through this approach, Myeloid demonstrates that delivery of lipid-nanoparticles (LNPs) encapsulating mRNA results in selective uptake and expression by myeloid cells in vivo, leading to potent tumor killing in multiple cold tumor models. These data demonstrate the potential for Myeloid's technology to program cells directly in vivo.

We have constructed many bispecific-RNA nanoparticles harboring ligands, aptamers, cell-binging chemical drugs, or Immune-checkpoint-targeting molecules to bind receptors of T cells or cancer cells. RNA's ability to form various 3D configurations allows the creation of bispecific RNA nanoparticles carrying various ligands to bridge cancer cells and T cells in therapy (Shu & Guo. Stoichiometry of multi-specific immune checkpoint RNA Abs for T cell activation and tumor inhibition using ultra-stable RNA nanoparticles. Mol Ther Nucleic Acids. 2021:24:426).

Selective transduction of cell populations has the potential to unlock a new generation of therapies. We recently developed a novel pseudotyping strategy in which VSVGmut, an affinity-ablated version of the VSVG, is coexpressed with a targeting protein to enable cell-type specific infection. Incorporating a peptide-MHC targeting protein enables antigen-specific infection of T cells, allowing us to pair pMHCs with cognate TCRs and to set the stage for cell-specific gene therapy.