Background: CF is a progressive genetic disease leading to reduction in function or complete loss of the CFTR chloride channel that is normally located on the apical plasma membrane of specialized respiratory epithelial cells.Treatments combining CFTR modulators and a potentiator has been very successful, but a significant fraction of people with CF are not amenable to currently approved treatments.To address this unmet need, ReCode Therapeutics is advancing an mRNA-based treatment to rescue CFTR function using its proprietary lipid nanoparticle (LNP) platform to deliver optimized CFTR mRNA as an inhaled aerosol.Methods: Sequence-optimized CFTR (hemagglutinin tagged) mRNA containing modified nucleotides was used in patient-derived primary human bronchial epithelial cells (hBECs).Different CF genotypes (R553X/W1282X, W1282X/W1282X, F508del/F508del, G542X/F508del) and non-CF hBECs were grown at an air-liquid interface (ALI) and treated with aerosolized mRNA LNPs using commercially available mesh nebulizers and exposure systems.Read-outs for the nebulization experiments included transepithelial resistance (TEER) and forskolin-induced chloride conductance measured by multi-transepithelial current clamp (MTECC24), protein levels (Western blot), cytotoxicity (lactate dehydrogenase), and cell tropism (immunofluorescence). Results: We evaluated the impact of cell tropism and mucus accumulation on rescue of CFTR function in hBECs derived from people with different genotypes.Forskolin-induced Cl -currents showed significant rescue after a single dose of aerosolized SORT LNPs.Analysis of protein expression by Western blot indicated successful delivery of mRNA using optimized LNPs and expression of CFTR protein in all donor cultured cells.Data suggest that CFTR is being translated, folded, and glycosylated, whereas immunofluorescence results showed that the newly translated hemagglutinin-tagged protein trafficked to the apical membrane in pulmonary ionocytes and secretory cells.ReCode's leading formulation to treat primary ciliary dyskinesia, which was developed for delivery of DNAI1 mRNA to ciliated cells for rescue ciliary activity, promoted inefficient CFTR functional rescue.Mucus accumulation was studied by subjecting hBECs to stringent dithiothreitol washes (3 mM or 10 mM).Chloride flux results showed a modest effect of sterile mucus in transfection efficiency after LNP exposure.Further studies are being conducted to explore the connection between LNP-specific cell tropism patterns and ability to restore CFTR-dependent Cl -currents.We are also investigating the impact of mucus on LNP survivability and cell transfection in patient-derived mucus and in vivo models.Conclusions: Our results demonstrate the capability of proprietary ReCode SORT LNPs to deliver LNP-formulated CFTR mRNA as an aerosol and increase CFTR function in well-differentiated CF hBEC cultures, including cells from people with mutations unresponsive to current modulator therapies.ReCode's leading formulations to treat CF efficiently transfect pulmonary ionocytes and secretory cells in vitro.These cell-based observations support further development and may provide future treatment options for a significant fraction of people with CF who do not benefit from current CFTR modulator therapy.

Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the gene encoding alpha-galactosidase A (alpha-Gal A), with consequent accumulation of its major glycosphingolipid substrate, globotriaosylceramide (GL-3). Over 500 Fabry mutations have been reported; approximately 60% are missense. The iminosugar 1-deoxygalactonojirimycin (DGJ, migalastat hydrochloride, AT1001) is a pharmacological chaperone that selectively binds alpha-Gal A, increasing physical stability, lysosomal trafficking, and cellular activity. To identify DGJ-responsive mutant forms of alpha-Gal A, the effect of DGJ incubation on alpha-Gal A levels was assessed in cultured lymphoblasts from males with Fabry disease representing 75 different missense mutations, one insertion, and one splice-site mutation. Baseline alpha-Gal A levels ranged from 0 to 52% of normal. Increases in alpha-Gal A levels (1.5- to 28-fold) after continuous DGJ incubation for 5 days were seen for 49 different missense mutant forms with varying EC(50) values (820 nmol/L to >1 mmol/L). Amino acid substitutions in responsive forms were located throughout both structural domains of the enzyme. Half of the missense mutant forms associated with classic (early-onset) Fabry disease and a majority (90%) associated with later-onset Fabry disease were responsive. In cultured fibroblasts from males with Fabry disease, the responses to DGJ were comparable to those of lymphoblasts with the same mutation. Importantly, elevated GL-3 levels in responsive Fabry fibroblasts were reduced after DGJ incubation, indicating that increased mutant alpha-Gal A levels can reduce accumulated substrate. These data indicate that DGJ merits further evaluation as a treatment for patients with Fabry disease with various missense mutations.