In utero gene editing for monogenic lung disease

Prenatal CRISPR gene editing results in efficient pulmonary epithelial cell editing and ameliorates a mouse model of a congenital lung disease. Editing out a lethal lung disease Surfactant, a lipoprotein mixture that reduces lung surface tension, is essential for normal lung function. In rare cases, infants are born with genetic surfactant deficiency, resulting in rapid death from respiratory failure. Because of the immediate perinatal fatality associated with this disease, any effective intervention would need to be applied before delivery. Alapati et al. used a mouse model of genetic surfactant deficiency to demonstrate the feasibility of in utero gene editing to delete the mutant allele. The authors showed that correction of the genetic defect before birth improved lung development and survival in the treated animals, demonstrating the feasibility of this therapeutic intervention. 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.

[1]  P. Davis,et al.  Epidemiology of Cystic Fibrosis. , 2016, Clinics in chest medicine.

[2]  J. Gustafson,et al.  Cystic Fibrosis , 2009, Journal of the Iowa Medical Society.

[3]  Gregg A. Duncan,et al.  The Mucus Barrier to Inhaled Gene Therapy. , 2016, Molecular therapy : the journal of the American Society of Gene Therapy.

[4]  Jianhui Gong,et al.  Correction of a pathogenic gene mutation in human embryos , 2018, Yearbook of Paediatric Endocrinology.

[5]  J. Joung,et al.  Defining and improving the genome-wide specificities of CRISPR–Cas9 nucleases , 2016, Nature Reviews Genetics.

[6]  Wei Tang,et al.  Correction of a genetic disease in mouse via use of CRISPR-Cas9. , 2013, Cell stem cell.

[7]  L. Nogee.  Interstitial lung disease in newborns. , 2017, Seminars in fetal & neonatal medicine.

[8]  Jianhui Gong,et al.  Correction of a pathogenic gene mutation in human embryos , 2017, Nature.

[9]  Eunji Kim,et al.  In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni , 2017, Nature Communications.

[10]  C. Rodeck,et al.  Percutaneous Ultrasound-Guided Injection of the Trachea in Fetal Sheep: A Novel Technique to Target the Fetal Airways , 2003, Fetal Diagnosis and Therapy.

[11]  M. Dolovich,et al.  Pulmonary drug delivery. Part I: physiological factors affecting therapeutic effectiveness of aerosolized medications. , 2003, British journal of clinical pharmacology.

[12]  P. Sly,et al.  Altered stability of pulmonary surfactant in SP-C-deficient mice , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[13]  A. Hamvas,et al.  A common mutation in the surfactant protein C gene associated with lung disease. , 2005, The Journal of pediatrics.

[14]  George M. Church,et al.  In vivo gene editing in dystrophic mouse muscle and muscle stem cells , 2016, Science.

[15]  D. Hayes,et al.  Pediatric lung transplantation: indications and outcomes. , 2014, Journal of thoracic disease.

[16]  G. Church,et al.  Cas9 as a versatile tool for engineering biology , 2013, Nature Methods.

[17]  M. Lu,et al.  HDAC3-Dependent Epigenetic Pathway Controls Lung Alveolar Epithelial Cell Remodeling and Spreading via miR-17-92 and TGF-β Signaling Regulation. , 2016, Developmental cell.

[18]  P. Alam.  ‘G’ , 2021, Composites Engineering: An A–Z Guide.

[19]  P. Zoltick,et al.  The developmental stage determines the distribution and duration of gene expression after early intra-amniotic gene transfer using lentiviral vectors , 2010, Gene Therapy.

[20]  P. Zoltick,et al.  In utero lung gene transfer using adeno-associated viral and lentiviral vectors in mice. , 2014, Human gene therapy methods.

[21]  Yang Yang,et al.  A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice , 2016, Nature Biotechnology.

[22]  J. Kent,et al.  Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR , 2016, Genome Biology.

[23]  Daesik Kim,et al.  Genome editing reveals a role for OCT4 in human embryogenesis , 2017, Nature.

[24]  Qunyuan Zhang,et al.  Outcomes of Lung Transplantation for Infants and Children with Genetic Disorders of Surfactant Metabolism , 2017, The Journal of pediatrics.

[25]  R. Crystal,et al.  Alpha 1-antitrypsin deficiency, emphysema, and liver disease. Genetic basis and strategies for therapy. , 1990, The Journal of clinical investigation.

[26]  David A. Scott,et al.  Genome engineering using the CRISPR-Cas9 system , 2013, Nature Protocols.

[27]  Abdullahi Umar Ibrahim,et al.  Genome Engineering Using the CRISPR Cas9 System , 2019 .

[28]  Susan E Wert,et al.  Alveolar surfactant homeostasis and the pathogenesis of pulmonary disease. , 2010, Annual review of medicine.

[29]  D. Meyerholz,et al.  Widespread airway distribution and short-term phenotypic correction of cystic fibrosis pigs following aerosol delivery of piggyBac/adenovirus , 2018, Nucleic acids research.

[30]  S. Jezzard,et al.  Factors influencing adenovirus-mediated airway transduction in fetal mice. , 2005, Molecular therapy : the journal of the American Society of Gene Therapy.

[31]  P. Nilsson,et al.  Clinical course and prognosis of never-smokers with severe alpha-1-antitrypsin deficiency (PiZZ) , 2008, Thorax.

[32]  Renzhi Han,et al.  In Vivo Genome Editing Restores Dystrophin Expression and Cardiac Function in Dystrophic Mice , 2017, Circulation research.

[33]  J. A. Maguire,et al.  A Nonaggregating Surfactant Protein C Mutant Is Misdirected to Early Endosomes and Disrupts Phospholipid Recycling , 2011, Traffic.

[34]  W. Funkhouser,et al.  A non-BRICHOS SFTPC mutant (SP-CI73T) linked to interstitial lung disease promotes a late block in macroautophagy disrupting cellular proteostasis and mitophagy. , 2015, American journal of physiology. Lung cellular and molecular physiology.

[35]  J. Doudna,et al.  The new frontier of genome engineering with CRISPR-Cas9 , 2014, Science.

[36]  A. Hamvas,et al.  Genetic Disorders of Surfactant Proteins , 2007, Neonatology.

[37]  A. Flake,et al.  Induction of Immune Tolerance to Foreign Protein via Adeno-Associated Viral Vector Gene Transfer in Mid-Gestation Fetal Sheep , 2017, PloS one.

[38]  Li Li,et al.  In utero CRISPR-mediated therapeutic editing of metabolic genes , 2018, Nature Medicine.

[39]  Daniel G. Anderson,et al.  Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing , 2017, Nature Biotechnology.

[40]  J. C. Belmonte,et al.  CRISPR-Cas9 mediated one-step disabling of pancreatogenesis in pigs , 2017, Scientific Reports.

[41]  L. Luo,et al.  A global double‐fluorescent Cre reporter mouse , 2007, Genesis.

[42]  Vivian Nguyen,et al.  Expression of mutant Sftpc in murine alveolar epithelia drives spontaneous lung fibrosis , 2018, The Journal of clinical investigation.

[43]  K. Müller,et al.  Interstitial lung disease in a baby with a de novo mutation in the SFTPC gene , 2004, European Respiratory Journal.

[44]  Zhiping Weng,et al.  In Vivo Genome Editing Partially Restores Alpha1-Antitrypsin in a Murine Model of AAT Deficiency. , 2018, Human gene therapy.

[45]  Dongsheng Duan,et al.  In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy , 2016, Science.

[46]  E. Olson,et al.  Prevention of muscular dystrophy in mice by CRISPR/Cas9–mediated editing of germline DNA , 2014, Science.

[47]  K. High,et al.  Persistent expression of hF.IX After tolerance induction by in utero or neonatal administration of AAV-1-F.IX in hemophilia B mice. , 2007, Molecular therapy : the journal of the American Society of Gene Therapy.

[48]  S. Mittal,et al.  Adenoviral vector immunity: its implications and circumvention strategies. , 2011, Current gene therapy.

[49]  E. Morrisey,et al.  Distinct Mesenchymal Lineages and Niches Promote Epithelial Self-Renewal and Myofibrogenesis in the Lung , 2017, Cell.

[50]  A. Bradley,et al.  Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements , 2018, Nature Biotechnology.

[51]  P. Sinn,et al.  Progress and Prospects: prospects of repeated pulmonary administration of viral vectors , 2009, Gene Therapy.

[52]  V. Baekelandt,et al.  Immunological ignorance allows long-term gene expression after perinatal recombinant adeno-associated virus-mediated gene transfer to murine airways. , 2014, Human gene therapy.

[53]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.

[54]  Hanmin Lee,et al.  A randomized trial of fetal endoscopic tracheal occlusion for severe fetal congenital diaphragmatic hernia. , 2003, The New England journal of medicine.

[55]  N. Arnberg.  Adenovirus receptors: implications for targeting of viral vectors. , 2012, Trends in pharmacological sciences.