Lung selective CRISPR delivery for treatment of genetic surfactant disease - Neonatal respiratory distress syndrome (RDS) is the most common respiratory cause of death
and morbidity in infants <1 year of age in the United States. Monogenic mutations in genes
regulating surfactant homeostasis, namely surfactant protein B (SFTPB), surfactant protein C
(SFTPC), and ATP binding cassette subfamily A member 3 (ABCA3), are causative drivers of
RDS in 25% of infants with severe refractory respiratory failure. Standard therapeutic regimens
for genetic lung disease are limited to symptomatic treatments and lung transplant, a procedure
with poor prognosis for long-term survival and high complication rates. These unsatisfactory
outcomes highlight the pressing need for more precise therapies that directly address the genetic
aberrations underlying RDS. Herein, we combine highly complementary expertise in neonatal
lung disease treatment (Dr. Alapati) and non-viral gene delivery (Dr. Sullivan) necessary to
develop a non-surgical approach to genetically correct lung progenitor cells during early postnatal
lung development, a widely accessible strategy designed to prevent disease manifestation. We
will establish this innovative and translationally-relevant approach via two aims: Aim 1. Design
non-viral nanocarriers (‘polyplexes’) that are biocompatible, stable in lung fluids, and capable of
cell-selective and efficient gene editing in neonatal AT2 cells. Aim 2. Engineer a partial-liquid
ventilation approach for CRISPR-Cas9 delivery to maximize AT2 cell access and gene editing
persistence in models of neonatal lung, and demonstrate this approach for durable, widespread,
and safe non-viral gene editing in lung epithelium. Our hypothesis is built on our published studies
demonstrating that (i) histone polyplex gene transfer hinges upon polyplex uptake via the
caveolin-1 transporter, a mechanism that enables highly efficient transfection in caveolin-1-
expressing cells and permits precise cell ‘targeting’ based upon differences in caveolin-1
availability; and (ii) airway delivery of CRISPR-Cas9 cargo into fluid filled fetal lungs results in
efficient pulmonary epithelial cell gene editing. This work will thus uncover important new
information on neonatal pulmonary epithelial gene transfer mechanisms while simultaneously
establishing new, more cell-selective gene therapy strategies relevant to a variety of pulmonary
genetic disorders. The study outcome will demonstrate a new delivery platform for effective, cell-
specific, and safe gene editing in postnatal lung epithelium, a strategy that would enable wide
usage even in basic-level NICUs, while simultaneously aligning with the timing of disease
diagnosis, and lay groundwork for future translation to fundamentally new, more effective, and
one-shot treatment modes for genetic surfactant protein diseases.
