In the context of in vivo genetic medicines, targeting approaches on a particle surface have limitations and may result in payload delivery to millions of off-target cells, resulting in safety and tolerability issues. Immune cell-selective synthetic promoters have the potential to control gene expression within desired cell types and thus enable the high level of precision anticipated for next-generation in vivo genetic medicines.

We present two key enabling technologies with transposon-based gene transfer relying on mRNA and DNA-based vectors, and AAV-based vector systems. These techniques are compatible with highly scalable, automated point-of-care manufacturing as well as in vivo generation of CAR T cells. Being platform technologies, they are widely applicable (e.g., for expression cassettes that comprise a CAR transgene and/or additional genetic cargo to enhance potency or safety of a cell product).

Most recent cell and gene therapy approaches require extraction of patient cells, expansion, and genomic editing ex vivo, which is expensive and time-consuming. In this talk, I will describe selective in vivo targeting of mRNA therapeutics and interventions to specific cells and cell subtypes such as T cells and hematopoietic stem cells (HSCs) via antibody-modified lipid nanoparticles. I will also discuss the potential applications we explored with this platform technology such as gene editing.

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.

Most gene delivery vectors are based on proteins from a limited subset of a huge number of naturally occurring viruses. We have implemented large-scale sequence search algorithms to identify candidate virus proteins in deep sequencing databases. We are building gene delivery vectors from these naturally occurring viruses and testing them in vitro and in vivo for specificity and efficiency of gene delivery in various cell types.

Cellular targeting and intracellular delivery of oligonucleotide therapeutics remain critical barriers to the clinical application of RNA interference. To overcome these barriers, novel peptidic MGSs were identified to deliver cargo to discrete cell types. These MGSs trigger rapid endocytosis and achieve significant levels inside the cells of interest. Importantly, off-target effects are minimal, and oligonucleotide therapeutics remain functional once inside the targeted cell or tissue type.

Cystine-knot peptides (CKPs) have attracted a lot of attention as a promising class of pharmacological ligands. A few CKPs have been shown to penetrate cells. However, the cell-penetrating properties and cellular-uptake mechanisms of many CKPs remain unclear. Our study reports a cell-penetrating CKP that could be used to deliver different cargoes into cytosol, and hence enable future exploration of its utility in drug discovery and delivery.

Currently approved gene therapies rely on viral vectors harboring a broad cell tropism. The ultimate goal for gene therapy vectors is to achieve manipulation of only therapy-relevant cells. Using surface engineered lentiviral vectors targeted to T cell markers we have provided proof-of-concept for in vivo generation of CAR T cells in humanized mouse models. In addition, AAV vectors can be efficiently targeted to lymphocytes, tumor cells, or neuronal cells through insertion of target-specific DARPins into exposed loop regions. Ongoing preclinical studies are evaluating the different vector platforms and will identify potential hurdles to be solved towards clinical application.

Cell-specific transduction remains one of the next frontiers for virally delivered gene therapy. Our lab developed a “receptor-blinded” version of VSVG, enabling co-display of a new LV pseudotype ligand to drive specific lentiviral tropism. Initial experiments have shown modularity of this platform for achieving potent transduction of on-target cells via a range of co-expressed host proteins, across a range of affinities and at frequencies as low as 1-in-100,000.

In vivo delivered CAR-T cell therapy has the potential to combine the transformative clinical benefit of auto-CAR with the ease of administration of a traditional biologic. kelonia’s in vivo gene placement system (iGPSTM) comprises a lentiviral particle with modified envelope proteins that enables highly specific cell targeting and efficient gene transfer. By eliminating the need for ex vivo manufacturing and toxic lymphodepleting chemotherapy, we believe our iGPS technology will remove barriers that currently prevent patients from accessing transformative genetic medicines.

Direct in vivo engineering of various immune cells not only can greatly reduce costs and broaden access to cellular therapy. In this talk, we will share our published and unpublished data on engineering CAR T by transducing circulating PBMCs in situ that can effectively eradicate aggressive tumors, as well as engineering CAR B cells that can secrete immunoglobulins of interest.

Adeno-associated viruses (AAV) are commonly used delivery vehicles for gene therapies. We have evolved AAV variants targeting human and mouse T cells. In this talk, I will describe how these AAVs can be used in vivo to specifically target T cells for gene editing and more. Additionally, I highlight the use of targeted AAVs as gene therapies to improve T cell therapeutics in immunocompetent tumor models