Sustained Knockdown of a Disease-Causing Gene in Patient-Specific Induced Pluripotent Stem Cells Using Lentiviral Vector-Based Gene Therapy

Abstract

Patient-specific induced pluripotent stem cells (iPSCs) hold great promise for studies on disease-related developmental processes and may serve as an autologous cell source for future treatment of many hereditary diseases. New genetic engineering tools such as zinc finger nucleases and transcription activator-like effector nuclease allow targeted correction of monogenetic disorders but are very cumbersome to establish. Aiming at studies on the knockdown of a disease-causing gene, lentiviral vector-mediated expression of short hairpin RNAs (shRNAs) is a valuable option, but it is limited by silencing of the knockdown construct upon epigenetic remodeling during differentiation. Here, we propose an approach for the expression of a therapeutic shRNA in disease-specific iPSCs using third-generation lentiviral vectors. Targeting severe α-1-antitrypsin (A1AT) deficiency, we overexpressed a human microRNA 30 (miR30)-styled shRNA directed against the PiZ variant of A1AT, which is known to cause chronic liver damage in affected patients. This knockdown cassette is traceable from clonal iPSC lines to differentiated hepatic progeny via an enhanced green fluorescence protein reporter expressed from the same RNA-polymerase II promoter. Importantly, the cytomegalovirus i/e enhancer chicken β actin (CAG) promoter-driven expression of this construct is sustained without transgene silencing during hepatic differentiation in vitro and in vivo. At low lentiviral copy numbers per genome we confirmed a functional relevant reduction (-66%) of intracellular PiZ protein in hepatic cells after differentiation of patient-specific iPSCs. In conclusion, we have demonstrated that lentiviral vector-mediated expression of shRNAs can be efficiently used to knock down and functionally evaluate disease-related genes in patient-specific iPSCs.

Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase

“…After successful evaluation of AAT sgRNAs we attempted correction of the human Z-AAT gene in a previously published patient-specific iPSC line (hPi) generated from an individual with severe ZZ-AAT deficiency20. This cell line is homozygous for the E342K missense point mutation and has been used for functional hepatic disease modeling studies.…”

“…In previous studies, we had compared the EF-1α short promoter (EFS), the full length EF-1α promoter and the CAG promoter for their resistance to transgene silencing in mouse iPSC20. We found that CAG was the most stable of these promoters and expression persisted even throughout differentiation in vitro and in vivo , and that expression was also stable in human iPSC even at low lentiviral vector copy numbers (VCN).…”

“…However, our data must not necessarily oppose the findings of Yusa et al ., as we have used an entirely different iPSC cell line for our approach and it is known that vast epigenetic differences can exist between various iPSC cell lines, even when they have originated from the same reprogramming attempt25. Previously, we showed complete reprogramming and full genomic integrity in the hPi cell line we have used here20, but for future more in-depth investigations it could be interesting to directly compare the epigenetic configuration as well as expression levels of epigenetic modifier enzymes such as DNMTs and TETs in the cell lines used by Yusa and colleagues with our cell line. Of note, the PGK-based donor construct published by Kosuke Yusa has also been used by others in the mean time for efficient mono-allelic and allele-specific Cas9-based gene targeting of the SERPINA1 locus26.…”

“…All lentiviral vectors were produced by transient transfection of HEK293T cells using the standard CaCl 2 method, as described previously20. 36 hours post transfection supernatants were harvested, filtrated through a 0.45 μm bottle-top filter (Sarstedt, Germany) and subjected to ultracentrifugation at 14,000 × g for 8 h. Pellets were re-suspended in ice-cold PBS, using 0.5% of the total supernatant volume (200x concentration) and frozen in 100 μl aliquots at −80 °C.…”

“…36 hours post transfection supernatants were harvested, filtrated through a 0.45 μm bottle-top filter (Sarstedt, Germany) and subjected to ultracentrifugation at 14,000 × g for 8 h. Pellets were re-suspended in ice-cold PBS, using 0.5% of the total supernatant volume (200x concentration) and frozen in 100 μl aliquots at −80 °C. Titers of lentiviral vectors CiG-MAAT-t-IP, CiG-ZAAT-t-IP, CiG-TTR-t-IP, CiG-SOD1-t-IP and Alb-Neo were determined by a dilution series transduction of HEK293T cells and subsequent multiplex qRT-PCR analysis of isolated genomic DNA for vector copy numbers using specific primer/probe sets for the WPRE element of the vectors and the genomic PTBP2 locus, as described previously20. Titers of lentiviral CGIP and PGIP vectors were determined by dilution series transduction followed by FACS analysis 72 h post transduction.…”