Highly Efficient Repair of the ΔF508 Mutation in Airway Stem Cells of Cystic Fibrosis Patients with Functional Rescue of the Differentiated Epithelia

Ciaran Lee

doi:10.1101/561183

Highly Efficient Repair of the ΔF508 Mutation in Airway Stem Cells of Cystic Fibrosis Patients with Functional Rescue of the Differentiated Epithelia

Ciaran Lee

School of Biochemistry and Cell Biology

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Abstract

Cystic fibrosis (CF) is a monogenic autosomal recessive disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl- channel. CF results in multiorgan dysfunction and ultimately mortality from respiratory sequelae. Although pharmacologic approaches have demonstrated efficacy in reducing symptoms and respiratory decline, a curative treatment modality remains elusive. Gene therapy, a promising curative strategy, has been limited due to poor correction efficiencies both in vitro and in vivo. Here, we use Cas9 and adeno-associated virus 6 (AAV6) to correct the ΔF508 mutation (found in ~70% of CF alleles and ~90% of CF patients in North America) in upper airway basal stem cells (UABCs) obtained from CF and non-CF patients undergoing functional endoscopic sinus surgery (FESS). In UABCs from homozygous (ΔF508/ΔF508) and compound heterozygous (ΔF508/Other) CF patients, we achieved 28 ± 5 % and 42 ± 15% correction, respectively. In homozygous human bronchial epithelial cells (HBECs), we achieved 41 ± 4 % correction. Upon differentiation in air-liquid interface (ALI), cultures of corrected CF cells displayed partial restoration of CFTRinh-172 sensitive Cl- currents relative to non-CF controls: 31 ± 5 % in UABCs and 51 ± 3 % in HBECs (both from subjects homozygous for ΔF508 CFTR). Finally, gene edited cells embedded successfully and retained expression of cytokeratin 5 (KRT5), a basal cell marker, on a FDA-approved porcine small intestinal submucosal (pSIS) membrane previously shown to improve re-mucosalization after FESS. In summary, we present an efficient, feeder-free, selection-free and clinically compatible approach to generate cell-based therapies for CF from autologous airway stem cells. This approach represents a first step towards developing patient-specific autologous airway stem cell transplant as a curative treatment for CF.

Original language	Undefined/Unknown
DOIs	https://doi.org/10.1101/561183
Publication status	Published - 26 Feb 2019

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@misc{87a631ff1c9f424498451b18cab99223,

title = "Highly Efficient Repair of the ΔF508 Mutation in Airway Stem Cells of Cystic Fibrosis Patients with Functional Rescue of the Differentiated Epithelia",

abstract = "Cystic fibrosis (CF) is a monogenic autosomal recessive disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl- channel. CF results in multiorgan dysfunction and ultimately mortality from respiratory sequelae. Although pharmacologic approaches have demonstrated efficacy in reducing symptoms and respiratory decline, a curative treatment modality remains elusive. Gene therapy, a promising curative strategy, has been limited due to poor correction efficiencies both in vitro and in vivo. Here, we use Cas9 and adeno-associated virus 6 (AAV6) to correct the ΔF508 mutation (found in \textasciitilde{}70\% of CF alleles and \textasciitilde{}90\% of CF patients in North America) in upper airway basal stem cells (UABCs) obtained from CF and non-CF patients undergoing functional endoscopic sinus surgery (FESS). In UABCs from homozygous (ΔF508/ΔF508) and compound heterozygous (ΔF508/Other) CF patients, we achieved 28 ± 5 \% and 42 ± 15\% correction, respectively. In homozygous human bronchial epithelial cells (HBECs), we achieved 41 ± 4 \% correction. Upon differentiation in air-liquid interface (ALI), cultures of corrected CF cells displayed partial restoration of CFTRinh-172 sensitive Cl- currents relative to non-CF controls: 31 ± 5 \% in UABCs and 51 ± 3 \% in HBECs (both from subjects homozygous for ΔF508 CFTR). Finally, gene edited cells embedded successfully and retained expression of cytokeratin 5 (KRT5), a basal cell marker, on a FDA-approved porcine small intestinal submucosal (pSIS) membrane previously shown to improve re-mucosalization after FESS. In summary, we present an efficient, feeder-free, selection-free and clinically compatible approach to generate cell-based therapies for CF from autologous airway stem cells. This approach represents a first step towards developing patient-specific autologous airway stem cell transplant as a curative treatment for CF.",

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T1 - Highly Efficient Repair of the ΔF508 Mutation in Airway Stem Cells of Cystic Fibrosis Patients with Functional Rescue of the Differentiated Epithelia

AU - Lee, Ciaran

PY - 2019/2/26

Y1 - 2019/2/26

N2 - Cystic fibrosis (CF) is a monogenic autosomal recessive disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl- channel. CF results in multiorgan dysfunction and ultimately mortality from respiratory sequelae. Although pharmacologic approaches have demonstrated efficacy in reducing symptoms and respiratory decline, a curative treatment modality remains elusive. Gene therapy, a promising curative strategy, has been limited due to poor correction efficiencies both in vitro and in vivo. Here, we use Cas9 and adeno-associated virus 6 (AAV6) to correct the ΔF508 mutation (found in ~70% of CF alleles and ~90% of CF patients in North America) in upper airway basal stem cells (UABCs) obtained from CF and non-CF patients undergoing functional endoscopic sinus surgery (FESS). In UABCs from homozygous (ΔF508/ΔF508) and compound heterozygous (ΔF508/Other) CF patients, we achieved 28 ± 5 % and 42 ± 15% correction, respectively. In homozygous human bronchial epithelial cells (HBECs), we achieved 41 ± 4 % correction. Upon differentiation in air-liquid interface (ALI), cultures of corrected CF cells displayed partial restoration of CFTRinh-172 sensitive Cl- currents relative to non-CF controls: 31 ± 5 % in UABCs and 51 ± 3 % in HBECs (both from subjects homozygous for ΔF508 CFTR). Finally, gene edited cells embedded successfully and retained expression of cytokeratin 5 (KRT5), a basal cell marker, on a FDA-approved porcine small intestinal submucosal (pSIS) membrane previously shown to improve re-mucosalization after FESS. In summary, we present an efficient, feeder-free, selection-free and clinically compatible approach to generate cell-based therapies for CF from autologous airway stem cells. This approach represents a first step towards developing patient-specific autologous airway stem cell transplant as a curative treatment for CF.

AB - Cystic fibrosis (CF) is a monogenic autosomal recessive disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl- channel. CF results in multiorgan dysfunction and ultimately mortality from respiratory sequelae. Although pharmacologic approaches have demonstrated efficacy in reducing symptoms and respiratory decline, a curative treatment modality remains elusive. Gene therapy, a promising curative strategy, has been limited due to poor correction efficiencies both in vitro and in vivo. Here, we use Cas9 and adeno-associated virus 6 (AAV6) to correct the ΔF508 mutation (found in ~70% of CF alleles and ~90% of CF patients in North America) in upper airway basal stem cells (UABCs) obtained from CF and non-CF patients undergoing functional endoscopic sinus surgery (FESS). In UABCs from homozygous (ΔF508/ΔF508) and compound heterozygous (ΔF508/Other) CF patients, we achieved 28 ± 5 % and 42 ± 15% correction, respectively. In homozygous human bronchial epithelial cells (HBECs), we achieved 41 ± 4 % correction. Upon differentiation in air-liquid interface (ALI), cultures of corrected CF cells displayed partial restoration of CFTRinh-172 sensitive Cl- currents relative to non-CF controls: 31 ± 5 % in UABCs and 51 ± 3 % in HBECs (both from subjects homozygous for ΔF508 CFTR). Finally, gene edited cells embedded successfully and retained expression of cytokeratin 5 (KRT5), a basal cell marker, on a FDA-approved porcine small intestinal submucosal (pSIS) membrane previously shown to improve re-mucosalization after FESS. In summary, we present an efficient, feeder-free, selection-free and clinically compatible approach to generate cell-based therapies for CF from autologous airway stem cells. This approach represents a first step towards developing patient-specific autologous airway stem cell transplant as a curative treatment for CF.

UR - http://dx.doi.org/10.1101/561183

U2 - 10.1101/561183

DO - 10.1101/561183

M3 - Other output

ER -

Highly Efficient Repair of the ΔF508 Mutation in Airway Stem Cells of Cystic Fibrosis Patients with Functional Rescue of the Differentiated Epithelia

Abstract

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