Engineering of Human Pluripotent Stem Cells by AAV-mediated Gene Targeting

Abstract

Precise genetic manipulation of human pluripotent stem cells will be required to realize their scientific and therapeutic potential. Here, we show that adeno-associated virus (AAV) gene targeting vectors can be used to genetically engineer human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Different types of sequence-specific changes, including the creation and correction of mutations, were introduced into the human HPRT1 and HMGA1 genes (HPRT1 mutations being responsible for Lesch-Nyhan syndrome). Gene targeting occurred at high frequencies in both ESCs and iPSCs, with over 1% of all colony-forming units (CFUs) undergoing targeting in some experiments. AAV vectors could also be used to target genes in human fibroblasts that were subsequently used to derive iPSCs. Accurate and efficient targeting took place with minimal or no cytotoxicity, and most of the gene-targeted stem cells produced were euploid and pluripotent.

The Nontoxic Cell Cycle Modulator Indirubin Augments Transduction of Adeno-Associated Viral Vectors and Zinc-Finger Nuclease-Mediated Gene Targeting

“…In particular, incubation of cells with indirubin-3¢-monoxime, a well-tolerated CDK inhibitor with minimal toxicity in normal cells, augmented the genetargeting frequency up to 10-fold to correct a mutated reporter gene in up to 34% of infected human cells. Given the recent success in AAV-mediated gene targeting in other multipotent (Chamberlain et al, 2004;Jang et al, 2011) or pluripotent stem cells (Mitsui et al, 2009;Khan et al, 2010;Asuri et al, 2012), respectively, as well as in vivo (Papadopoulos et al, 1997;Paulk et al, 2012), these findings present a promising strategy to enhance AAV/ZFN-mediated genome engineering for potential clinical applications.…”

“…Although ZFN-mediated targeted integration has been achieved in up to 40% of a mesenchymal stromal cell (MSC) population (Benabdallah et al, 2010), such high frequencies have remained unmatched in other therapeutically relevant human stem cells, including embryonic stem or induced pluripotent stem cells (Mitsui et al, 2009;Zou et al, 2009). Hence, gene targeting in human stem cells has been dependent on the co-integration of a selection marker to enrich for targeting events (Hockemeyer et al, 2009;Mitsui et al, 2009;Zou et al, 2009;Khan et al, 2010). Importantly, MSCs, which bear differentiation potential to multiple lineages, such as bone, neurons, and cardiac tissues, have been effectively transduced with AAV serotype 2 vectors (Kumar et al, 2004;Chng et al, 2007).…”

“…Meanwhile, this technology has been increasingly employed in biotechnology, and therapeutic applications are tangible, as epitomized by the introduction of targeted alterations in the genome of multi-and pluripotent human stem cells (Chamberlain et al, 2004;Hockemeyer et al, 2009;Zou et al, 2009;Benabdallah et al, 2010). Two major platforms have been described to overcome the low HR frequency in such cells: a viral vector platform based on adeno-associated virus (AAV) (Chamberlain et al, 2004;Mitsui et al, 2009;Khan et al, 2010) and DNA double-strand break (DSB)-inducing agents, such as meganucleases (Silva et al, 2011;Takeuchi et al, 2011), zinc-finger nucleases (ZFNs) (Urnov et al, 2010;Carroll, 2011;Rahman et al, 2011), and transcriptional activator-like effector (TALE) nucleases (Bogdanove and Voytas, 2011;Mussolino and Cathomen, 2012).…”

Directed evolution of novel adeno-associated viruses for therapeutic gene delivery

“…40 The most successful report of gene targeting to human pluripotent stem cells using wild-type AAV showed correct targeting of 1.3% of all colony-forming units, which corresponds to an overall gene targeting frequency for the originally infected hESCs of approximately 0.03%. 41 To build upon this result, Asuri et al 24 applied directed evolution to create an AAV variant capable of enhanced gene delivery and gene targeting in hESCs and human induced pluripotent stem cells. Selecting an AAV2 and AAV6 error-prone library, an AAV2 loopswap library, a shuffled library (containing AAV 1, 2, 4-6, 8 and 9) and a random peptide insert library for enhanced infection of hESCs yielded a simple AAV2 variant harboring a single R459G mutation.…”

Find and replace: editing human genome in pluripotent stem cells

“…A few other research groups also successfully obtained gene-modified cell lines with this conventional approach, nevertheless with extremely low efficiency (Urbach et al, 2004;Costa et al, 2007;Irion et al, 2007;Davis et al, 2008;Di Domenico et al, 2008;Bu et al, 2009;Kamei et al, 2009;Ruby and Zheng, 2009;Xue et al, 2009;Buecker et al, 2010;Fischer et al, 2010;Goulburn et al, 2011). To overcome technical limitations, novel gene editing strategies have been developed to enhance targeting efficiency in hPSCs, such as bacterial artificial chromosome (BAC) (Song et al, 2010), adenoassociated virus vector (AAV) (Khan et al, 2010;Asuri et al, 2011), zinc finger nuclease (ZFN) (Lombardo et al, 2007;Hockemeyer et al, 2009;Zou et al, 2009), transcription activator-like effecter nuclease (TALEN) (Cermak et al, 2011;Hockemeyer et al, 2011;Miller et al, 2011;Mussolino et al, 2011;Zhang et al, 2011), and helper-dependent adenoviral vector (HDAdV) (Suzuki et al, 2008;Li et al, 2011;Liu et al, 2011b). Here we focus on comparing cons and pros of the above novel techniques to explore their potential for efficient and safe gene targeting in hPSCs.…”