R01GM144749

Programmable microvesicles for intracellular macromolecule delivery - project summary

Technologies to deliver macromolecules across the plasma membrane and bypass endosome degradation are not only instrumental for elucidating gene function but also hold enormous potential for therapeutics. Proteins, nucleic acids, and ribonucleoproteins (RNP) have become indispensable tools for biomedical research, however, their applications in human therapeutics are largely limited to modulating targets reside in the extracellular space.

Only a few percent of exogenous macromolecules can get through the cellular barriers and make it into the intracellular space. Extracellular vesicles (EVs) are increasingly being explored as potential vehicles for intracellular therapeutics delivery since they transport bioactive molecules natively between cells. Cell derived EVs are heterogeneous in size and composition and, consequently, exhibit low specific activity for delivering cargo of interest.

To address these problems, we developed an innovative macromolecule delivery system based on engineered extracellular vesicles called GECTOSOMES (G protein ectosomes), designed to co-encapsulate vesicular stomatitis virus G protein (VSV-G) with bioactive macromolecules via split GFP complementation. The reversible tethering of cargo to VSV-G provides efficient cargo loading and endosomal escape simultaneously. GECTOSOMES demonstrated efficient delivery of catalytic enzymes, interference RNA, and Cas9 RNPs to the cytosol and nucleus and successful modifications of cellular phenotypes.

We aim to develop a versatile and broadly applicable platform technology that allows rapid production of highly specific GECTOSOMES capable of modulating intracellular targets in vitro and in vivo. The objective of this application is to demonstrate the feasibility of our approach by improving the homogeneity of GECTOSOMES through CRISPR engineering of the producer cells and by creating GECTOSOMES that deliver engineered nanobodies or ubiquitin E3 ligase CRBN intracellularly to alter protein aggregation or degradation. We will also examine host immune responses to GECTOSOMES and elucidate the efficacy window of GECTOSOME delivery in vivo, which will help refine application areas.

The feasibility of proposed studies is supported by our published results showing that active loading of GECTOSOMES reduces passive incorporation of cellular proteins while CRISPR engineering of producer cells improves EV homogeneity. Three specific aims are:

SA1: Develop new producer cell lines via CRISPR-mediated cell engineering to improve the homogeneity and specificity of GECTOSOMES.
SA2: Develop GECTOSOMES to deliver antibodies or agents designed for promoting targeted protein degradation in cells.
SA3: Determine adaptive immune responses to GECTOSOMES and general toxicity profiles of GECTOSOMES.

The proposed studies will overcome current limitations in delivering biologics to the intracellular space. The improved delivery platform will also provide more accessible research tools for the wider scientific community in their endeavors to elucidate gene function or develop new therapeutic strategies for treatment of human diseases.

The Regents Of The University Of Colorado

<P>THE NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES (NIGMS) SUPPORTS BASIC RESEARCH THAT INCREASES OUR UNDERSTANDING OF BIOLOGICAL PROCESSES AND LAYS THE FOUNDATION FOR ADVANCES IN DISEASE DIAGNOSIS, TREATMENT, AND PREVENTION. NIGMS ALSO SUPPORTS RESEARCH IN SPECIFIC CLINICAL AREAS THAT AFFECT MULTIPLE ORGAN SYSTEMS: ANESTHESIOLOGY AND PERI-OPERATIVE PAIN; CLINICAL PHARMACOLOGY COMMON TO MULTIPLE DRUGS AND TREATMENTS; AND INJURY, CRITICAL ILLNESS, SEPSIS, AND WOUND HEALING. NIGMS-FUNDED SCIENTISTS INVESTIGATE HOW LIVING SYSTEMS WORK AT A RANGE OF LEVELSFROM MOLECULES AND CELLS TO TISSUES AND ORGANSIN RESEARCH ORGANISMS, HUMANS, AND POPULATIONS. ADDITIONALLY, TO ENSURE THE VITALITY AND CONTINUED PRODUCTIVITY OF THE RESEARCH ENTERPRISE, NIGMS PROVIDES LEADERSHIP IN SUPPORTING THE TRAINING OF THE NEXT GENERATION OF SCIENTISTS, ENHANCING THE DIVERSITY OF THE SCIENTIFIC WORKFORCE, AND DEVELOPING RESEARCH CAPACITY THROUGHOUT THE COUNTRY.</P>

93.859 - Biomedical Research and Research Training

National Institute of General Medical Sciences (NIGMS) [HHS - NIH]

Boulder, Colorado 803095005 United States

Single Zip Code

Focused Technology Research and Development (R01 Clinical Trial Not Allowed) (PAR-25-203)

Amendment Since initial award the End Date has been extended from 11/30/25 to 04/30/30 and the total obligations have increased 840% from $342,660 to $3,220,426.