In Vivo Base Editing of Scn5a Rescues Type 3 Long QT Syndrome in Mice.

BACKGROUND:Pathogenic variants in SCN5A can result in long QT syndrome type 3, a life-threatening genetic disease. Adenine base editors can convert targeted A T base pairs to G C base pairs, offering a promising tool to correct pathogenic variants.METHODS:We generated a long QT syndrome type 3 mouse model by introducing the T1307M pathogenic variant into the Scn5a gene. The adenine base editor was split into 2 smaller parts and delivered into the heart by adeno-associated virus serotype 9 (AAV9-ABEmax) to correct the T1307M pathogenic variant.RESULTS:Both homozygous and heterozygous T1307M mice showed significant QT prolongation. Carbachol administration induced Torsades de Pointes or ventricular tachycardia for homozygous T1307M mice (20%) but not for heterozygous or wild-type mice. A single intraperitoneal injection of AAV9-ABEmax at postnatal day 14 resulted in up to 99.20% Scn5a transcripts corrected in T1307M mice. Scn5a mRNA correction rate >60% eliminated QT prolongation; Scn5a mRNA correction rate <60% alleviated QT prolongation. Partial Scn5a correction resulted in cardiomyocytes heterogeneity, which did not induce severe arrhythmias. We did not detect off-target DNA or RNA editing events in ABEmax-treated mouse hearts.CONCLUSIONS:These findings show that in vivo AAV9-ABEmax editing can correct the variant Scn5a allele, effectively ameliorating arrhythmia phenotypes. Our results offer a proof of concept for the treatment of hereditary arrhythmias.

Generation of a doxycycline-inducible ETV2 expression cell line using PiggyBac transposase system.

E26 transformation-specific variant 2 (ETV2), as a member of Ets family of transcription factors, plays an important role in embryonic vasculogenesis, angiogenesis and hematopoiesis. Here, based on H9 cell line and PiggyBac technology, we generated a doxycycline-inducible ETV2 (Dox-ETV2-H9) embryonic stem cell (ESC) line. The Dox-ETV2-H9 cells maintain normal morphology, karyotype as well as high expression of pluripotent markers. In comparison with previous studies that employing costly ETV2 modRNA to improve differentiation from ESCs into endothelial cells (ECs), the tet-on system in Dox-ETV2-H9 cells could precisely manipulate ETV2 expression, which enables ECs differentiation process more efficient and controllable upon addition of doxycycline.

Editorial: Bioengineered gene and cell therapy for treating cardiovascular diseases.

Perspectives of genetic management strategy for inherited cardiovascular diseases in China.

Generation of a homozygous TAZ knockout hESCs line by CRISPR/Cas9 system.

Tafazzin (TAZ), a mitochondrial transacylase located on chromosome X, is required for the production of the mitochondrial phospholipid cardiolipin. Mutations occurring in the TAZ gene will lead to Barth syndrome, an X-linked recessive disease generally presenting as cardiomyopathy affecting males. Disease modeling strategies based on pluripotent stem cells (PSCs) provide an unprecedented and powerful platform to study Barth Syndrome. However, current studies were mostly based on male PSCs, the results and conclusions of which neglected the potential distinctions existing in disease phenotypes and mechanisms between gender. In this study, based on the H9 cell line (Female), we generated a homozygous TAZ knockout (TAZ-KO) human embryonic stem cell (hESC) line by employing CRISPR/Cas9 genome editing tools. This female TAZ-KO cell line, with normal karyotype, robust pluripotency and remarkably reduced TAZ expression, would be a useful tool for further deeply studying the pathogenesis of Barth syndrome cardiomyopathy in females.

Generation of a NIS-EGFP-Fluc triple-reporter human embryonic stem cell line by PiggyBac transposon system.

One major challenge in stem cell therapy is to longitudinally track cell fate after cells transplantation. Molecular Imaging approaches enabling noninvasive long-term monitoring the transplanted cells are imperative for assessment of the safety and efficiency. Here, we used PiggyBac technology to insert triple reporter genes: NIS, EGFP and Firefly luciferase into a human embryonic stem cell line (hESCs, H9) and obtained a reporter hESCs line (NIS-EGFP-Fluc H9). The triple-reporters allows the transplanted NIS-EGFP-Fluc H9 cells and their derivates to be fluorescence, bioluminescence and even PET/SPECT imaged. This triple-reporter hESCs line provides a valuable imaging platform for cell-based therapeutics clinical translation.

Efficient Correction of a Hypertrophic Cardiomyopathy Mutation by ABEmax-NG.

[Figure: see text].

In Vivo Base Editing of Scn5a Rescues Type 3 Long QT Syndrome in Mice.

BACKGROUND:Pathogenic variants in SCN5A can result in long QT syndrome type 3, a life-threatening genetic disease. Adenine base editors can convert targeted A T base pairs to G C base pairs, offering a promising tool to correct pathogenic variants.METHODS:We generated a long QT syndrome type 3 mouse model by introducing the T1307M pathogenic variant into the Scn5a gene. The adenine base editor was split into 2 smaller parts and delivered into the heart by adeno-associated virus serotype 9 (AAV9-ABEmax) to correct the T1307M pathogenic variant.RESULTS:Both homozygous and heterozygous T1307M mice showed significant QT prolongation. Carbachol administration induced Torsades de Pointes or ventricular tachycardia for homozygous T1307M mice (20%) but not for heterozygous or wild-type mice. A single intraperitoneal injection of AAV9-ABEmax at postnatal day 14 resulted in up to 99.20% Scn5a transcripts corrected in T1307M mice. Scn5a mRNA correction rate >60% eliminated QT prolongation; Scn5a mRNA correction rate <60% alleviated QT prolongation. Partial Scn5a correction resulted in cardiomyocytes heterogeneity, which did not induce severe arrhythmias. We did not detect off-target DNA or RNA editing events in ABEmax-treated mouse hearts.CONCLUSIONS:These findings show that in vivo AAV9-ABEmax editing can correct the variant Scn5a allele, effectively ameliorating arrhythmia phenotypes. Our results offer a proof of concept for the treatment of hereditary arrhythmias.

Generation of a doxycycline-inducible ETV2 expression cell line using PiggyBac transposase system.

E26 transformation-specific variant 2 (ETV2), as a member of Ets family of transcription factors, plays an important role in embryonic vasculogenesis, angiogenesis and hematopoiesis. Here, based on H9 cell line and PiggyBac technology, we generated a doxycycline-inducible ETV2 (Dox-ETV2-H9) embryonic stem cell (ESC) line. The Dox-ETV2-H9 cells maintain normal morphology, karyotype as well as high expression of pluripotent markers. In comparison with previous studies that employing costly ETV2 modRNA to improve differentiation from ESCs into endothelial cells (ECs), the tet-on system in Dox-ETV2-H9 cells could precisely manipulate ETV2 expression, which enables ECs differentiation process more efficient and controllable upon addition of doxycycline.

Editorial: Bioengineered gene and cell therapy for treating cardiovascular diseases.

Perspectives of genetic management strategy for inherited cardiovascular diseases in China.

Generation of a homozygous TAZ knockout hESCs line by CRISPR/Cas9 system.

Tafazzin (TAZ), a mitochondrial transacylase located on chromosome X, is required for the production of the mitochondrial phospholipid cardiolipin. Mutations occurring in the TAZ gene will lead to Barth syndrome, an X-linked recessive disease generally presenting as cardiomyopathy affecting males. Disease modeling strategies based on pluripotent stem cells (PSCs) provide an unprecedented and powerful platform to study Barth Syndrome. However, current studies were mostly based on male PSCs, the results and conclusions of which neglected the potential distinctions existing in disease phenotypes and mechanisms between gender. In this study, based on the H9 cell line (Female), we generated a homozygous TAZ knockout (TAZ-KO) human embryonic stem cell (hESC) line by employing CRISPR/Cas9 genome editing tools. This female TAZ-KO cell line, with normal karyotype, robust pluripotency and remarkably reduced TAZ expression, would be a useful tool for further deeply studying the pathogenesis of Barth syndrome cardiomyopathy in females.

Generation of a NIS-EGFP-Fluc triple-reporter human embryonic stem cell line by PiggyBac transposon system.

One major challenge in stem cell therapy is to longitudinally track cell fate after cells transplantation. Molecular Imaging approaches enabling noninvasive long-term monitoring the transplanted cells are imperative for assessment of the safety and efficiency. Here, we used PiggyBac technology to insert triple reporter genes: NIS, EGFP and Firefly luciferase into a human embryonic stem cell line (hESCs, H9) and obtained a reporter hESCs line (NIS-EGFP-Fluc H9). The triple-reporters allows the transplanted NIS-EGFP-Fluc H9 cells and their derivates to be fluorescence, bioluminescence and even PET/SPECT imaged. This triple-reporter hESCs line provides a valuable imaging platform for cell-based therapeutics clinical translation.

Efficient Correction of a Hypertrophic Cardiomyopathy Mutation by ABEmax-NG.

[Figure: see text].