Table1.

Advantages and limitations of novel approaches to gene therapy

Technology	Basic concept	Major advantages	Major limitations	Technical aspects that need to be improved prior to clinical implementation
Homing Endonucleases (HEs)	Use of highly specific engineered endonuclease to induce a DSB in a desired locus and promote homologous recombination with a repair matrix.	Gene correction within the endogenous locus, thus maintaining proper regulation of gene expression.	The low number of HEs available limit applicability of this strategy for gene correction. Lack of animal models for pre- clinical trials.	Low efficiency Off-target cleavage Inefficient intranuclear delivery of HE and repair matrix.
Zinc Finger Nucleases (ZFNs)	Use of an artificial fusion protein that combines a nuclease with a Zinc-finger protein to induce site- specific DSBs and promote homologous recombination with a repair matrix.	Gene correction within the natural locus allows for maintenance of endogenous regulation	Need for a specific system for each gene and sometimes for different mutations with in the same gene. Lack of animal models for pre- clinical trials.	Low efficiency Off target cleavage Inefficient intranuclear delivery of HE and repair matrix.
Safe Harbor targeting	Targeting of specific and safe (ie, far from oncogenes) loci in the genome to introduce a normal copy of the desired gene.	Safe and efficient insertion of a normal copy of the desired gene under any desired promoter. One system will work for various defects and diseases.	Insertion of an extra copy of the desired gene. Does not utilize the natural regulatory elements in the endogenous locus. Significant limitation for use in dominant negative defects and leaky phenotypes	Inefficient intranuclear delivery of HE and repair matrix.
Transposons and transposases	Use of transposase protein that targets any DNA cargo sequence flanked by ITR sequences for random integration of a normal copy of the desired gene	Efficient delivery and insertion of large sequences (up to 11 Kb). One system will work for various defects and diseases.	Random integration may lead to insertional mutagenesis and/or intragenic disruption. Does not utilize the natural regulatory elements in the endogenous locus. Significant limitation for use in dominant negative defects and leaky phenotypes	Inefficient intranuclear delivery of HE and repair matrix.

DSB: double-strand DNA break; ITR: inverted terminal repeats