TALENs can be easily assembled in arbitrarily large arrays to bind the sequence of interest, but their intrinsic repetitiveness and large size impair efficient cloning and limit delivery by viral vectors
TALENs can be easily assembled in arbitrarily large arrays to bind the sequence of interest, but their intrinsic repetitiveness and large size impair efficient cloning and limit delivery by viral vectors. confine the manipulation to a defined cell subset, thus diminishing Hh-Ag1.5 the risk of off-target effects and bystander toxicity spillover. Rabbit polyclonal to PCDHGB4 gene therapy based on semi-randomly integrating retro- (RV) or lenti-viral (LV) vectors has demonstrated an outstanding potential for the treatment of several inherited and acquired hematological diseases (Ghosh et al., 2015; Naldini, 2019). To this goal, autologous HSPCs are harvested, transduced by viral vectors and ultimately infused into the patient. A conditioning regimen is usually administered prior to infusion to deplete host cells and maximize engraftment of the engineered product (Bernardo and Aiuti, 2016). The discovery and repurposing of programmable molecules, such as nucleases, base editors and prime editors have opened the door to Hh-Ag1.5 targeted genome editing, i.e., site-specific nucleotide(s) deletion, insertion and substitution, or integration of a therapeutic transgene cassette at a pre-determined genomic locus (Doudna, 2020). These new technologies may be exploited to deliver a wide spectrum of genetic manipulations, Hh-Ag1.5 with potential applications for several hematological diseases. Indeed, targeted genome editing by programmable nucleases has already entered the clinic and is currently being tested with encouraging results (Xu et al., 2019; Frangoul et al., 2021). While blossoming, gene editing is still in its infancy, and both knowledge and technological gaps await to be filled to broaden its clinical applicability. Furthermore, safety and efficacy, both in the short and long term, are still unknown. In this Review, we highlight the therapeutic potential and the current challenges toward clinical translation of targeted genome editing by programmable nucleases in human HSPCs for the treatment Hh-Ag1.5 of blood diseases. Programmable Nucleases for Targeted Genome Editing Programmable nucleases are chimeric molecules composed by (i) a protein- or an RNA-based DNA binding structure, which dictates nuclease specificity, and (ii) an effector domain with catalytic nuclease activity, which induces a DNA double strand break (DSB) nearby or within the binding site. Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas systems are the most exploited nuclease platforms for targeted genome editing (Carroll, 2014). ZFNs are composed by an array of three to six zinc-finger (ZF) DNA binding domains, linked by a flexible peptide linker to a non-specific FokI cleavage domain. Each ZF domain is composed by 30 amino acids and recognizes nucleotide triplets in the major groove of the DNA double helix; in total each ZFN recognizes 9C18 nucleotides (Gaj et al., 2013). Sequence and structure of the aforementioned flexible peptide linker is fundamental to achieve efficient cleavage and targeting specificity (Handel and Cathomen, 2011). Mechanistically, a pair of ZFN monomers must bind the DNA, typically in a head-to-head configuration, by associating with DNA strands of opposite polarity and leaving a 5C7 bp gap. This leads to dimerization of the two FokI domains that catalyze the DNA DSB (Urnov et al., 2010). TALENs consist of a DNA-binding domain composed by modular TALE repeats, fused with a FokI nuclease domain. Each TALE repeat is composed by 33C35 amino acids and recognizes a single nucleotide; specificity is determined by two hypervariable residues, known as Repeated Variable Diresidues (RVDs) (Gaj et al., 2013). Indeed, TALE repeats can be assembled together in a rather straightforward way to pair the desired DNA sequence, nucleotide by nucleotide. As for ZFNs, a pair of TALEN monomers is necessary to introduce a DSB. Finally, CRISPR/Cas is an RNA-based DNA targeting-system found in bacteria as an acquired immune system against transmissible genetic elements, such as viruses and plasmids (Barrangou et al., 2007; Brouns et al., 2008; Garneau et al., 2010). (Sp) Cas9 protein (SpCas9) (Nozawa et al., 2011), which belongs to type II family of CRISPR/Cas systems, is the most widely used platform for CRISPR-based targeted genome editing. Mechanistically, the CRISPR/Cas9 system is composed by a single-stranded guide RNA Hh-Ag1.5 (sgRNA) and the Cas9 endonuclease, which is the enzyme required to mediate target DNA cleavage. The sgRNA contains a unique 20 base-pair sequence which complements the target DNA site, and can be easily customized to bind the desired genomic sequence by Watson-Crick base-pairing (Jinek et al., 2012). The presence of a protospacer adjacent motif (PAM), immediately downstream the target DNA site, is necessary to efficiently bind and cut the DNA, e.g. 5-NGG-3 for SpCas9, although some cleavage activity has also been observed with the 5-NAG-3 motif (Hsu et al., 2013; Sternberg et al., 2014). All these platforms have intrinsic advantages and disadvantages (Gaj et al., 2013). TALENs can be easily assembled in arbitrarily large arrays to bind the sequence of interest,.