In utero gene editing for monogenic lung disease

Abstract

Monogenic lung diseases that are caused by mutations in surfactant genes of the pulmonary epithelium are marked by perinatal lethal respiratory failure or chronic diffuse parenchymal lung disease with few therapeutic options. Using a CRISPR fluorescent reporter system, we demonstrate that precisely timed in utero intra-amniotic delivery of CRISPR-Cas9 gene editing reagents during fetal development results in targeted and specific gene editing in fetal lungs. Pulmonary epithelial cells are predominantly targeted in this approach, with alveolar type 1, alveolar type 2, and airway secretory cells exhibiting high and persistent gene editing. We then used this in utero technique to evaluate a therapeutic approach to reduce the severity of the lethal interstitial lung disease observed in a mouse model of the human SFTPCI73T mutation. Embryonic expression of SftpcI73T alleles is characterized by severe diffuse parenchymal lung damage and rapid demise of mutant mice at birth. After in utero CRISPR-Cas9–mediated inactivation of the mutant SftpcI73T gene, fetuses and postnatal mice showed improved lung morphology and increased survival. These proof-of-concept studies demonstrate that in utero gene editing is a promising approach for treatment and rescue of monogenic lung diseases that are lethal at birth.

Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ cells, Embryos, and Fetuses in Mice

“…Recently, two more in vivo approaches for genome editing have been reported. One of the approaches involves in utero gene delivery into postimplantation fetuses, an approach used by a group that has demonstrated the efficacy of an intraamniotic injection (as shown in b on day 12.5 in Figure 1; the day when copulation plug is found is defined as day 0 of pregnancy) of rAAVs carrying genome-editing-related genes in the successful rescue of fetuses with lethal mutations [17]. The other approach involves tail-vein injection of a solution containing a plasmid (that confers expression of both Cas9 and gRNA) into a pregnant female at the mid-gestational stage.…”

“…In these experiments, viral vectors, such as AAVs, lentiviruses, and adenoviruses have been involved in infecting the fetuses at days 9-15 of pregnancy. Experiments using in utero gene delivery of genome-editing components have been performed by several research groups [17,[91][92][93][94]. Ricciardi et al [93] demonstrated that intra-amniotic administration of polymeric, biodegradable nanoparticles derived from poly (lactic-co-glycolic acid) containing triplex-forming peptide nucleic acids and donor ssDNAs into mouse fetuses carrying mutations in the β-globin gene, results in the offspring being rescued from a disease similar to human β-thalassemia.…”

“…These rescued offspring showed sustained elevation of blood hemoglobin levels, reduced number of reticulocytes, disappearance of splenomegaly, and longer survival. Alapati et al [17] have recently used CRISPR technology to correct a lung-disease-causing gene (called SftpcI73T)-a mutated version of the pulmonary-associated protein C gene-that codes for a protein that helps prevent the lung from collapsing when emptied. Embryonic expression of SftpcI73T is known to cause severely diffused parenchymal lung damage leading to the early death of affected individuals.…”

“…Embryonic expression of SftpcI73T is known to cause severely diffused parenchymal lung damage leading to the early death of affected individuals. Alapati et al [17] performed intra-amniotic administration of CRISPR reagents into day 16 fetuses carrying the SftpcI73T gene. Gene editing was successful in the target cells of the lungs of 20% of the mice born.…”

Fluorescentin vivoediting reporter (FIVER): A novel multispectral reporter ofin vivogenome editing

“…Broadly speaking, they use one of two major pathways (9): non-homologous end-joining (NHEJ), often leading to small insertions or deletions (indels); and when a template is available, homology directed repair (HDR), resulting in precise correction of disease-causing mutations. Several fluorescence-based reporter systems for monitoring the outcomes of genome editing have been described (10)(11)(12)(13)(14)(15)(16)(17). However, these are predominantly in vitro reporters, relying on transiently transfected constructs or stable cell lines, or where available in vivo are limited to the detection of NHEJ events (14,15).…”