Summary. Hemoglobinopathies such as ß-thalassemia and sickle cell disease are genetic disorders caused
by mutations in the HBB gene that codes for the ß-globin component of hemoglobin. Currently, the only gene
therapy available for these prevalent hereditary diseases is based on transplantation of genetically corrected
hematopoietic stem cells (HSPCs) from fully matched donors. However, the efficacy of this approach is
limited by multiple factors. Gene editing is a promising alternative approach for curing hemoglobinopathies.
Using this approach, synthetic mRNA-based drugs encoding nucleases that target the HBB gene can be
utilized to permanently correct the patient’s DNA. Combining nanoparticle-based drug delivery with zinc-
finger nucleases (ZFNs) has the potential to facilitate targeted gene-editing in HSPCs. However, the reliance
on in vitro screening of nanoparticles impedes the discovery of safe and efficient in vivo delivery vehicles.
Furthermore, current ZFN-mRNA based drugs targeting the HBB gene in HSPCs exhibit immunogenicity and
are expressed in off-target cells. The PIs have recently been shown that DNA barcoded nanoparticles can
‘evolve’ nanoparticles to target endothelial cells more efficiently than hepatocytes directly in vivo. The team
has also demonstrated that it is possible to (i) design low immune stimulating mRNA via nucleotide
modification and HPLC purification, and that (ii) mRNAs can be designed to completely preclude translation
in hepatocytes using rationally designed ‘on’ and ‘off’ switches. Based on these supporting data, it is posited
that nanoparticles can be evolved to specifically target HSPCs while avoiding hepatocytes, and that ZFN-
mRNA based drugs can be rationally optimized to generate safe gene editing therapeutics targeting
HSPCs. Thus, the team proposes to create an mRNA-based drug that safely and specifically edits HSPCs in
non-human primes in two phases. The development (UG3) phase will address 2 aims: (1) to iteratively evolve
nanoparticles that target HSPCs and avoid hepatocytes in vivo, and (2) to reduce mRNA immunogenicity
and improve cell type specific delivery to HSPCs. The demonstration (UH3) phase will address the aim (3) to
analyze functional gene editing in non-human primates (Rhesus macaques). These will be achieved using a
cutting edge multidisciplinary approaches recently developed. Specifically, the team will combine a DNA
barcoded nanoparticle technology to screen 4,500 nanoparticles in vivo, synthesize mRNA-based drugs with
low immunogenicity and cell type-specific expression, and utilize customized bioinformatics pipeline that
facilitates ‘big data’ experiments with a statistical power new to nanomedicine. By creating an mRNA-based
drug that safely edits HSPCs, the project is poised to advance gene editing as a viable therapeutic
approach for curing genetic blood disorders and pave the way for clinical trials.
