Table 4.

Pros and cons of gene therapy methods for sickle cell disease.

Gene Addition	Mechanism	Pros	Cons
Lentiviral vector gene addition	Lentiviral vector encoding of either a human γ-globin gene or a normal or modified β-globin gene designed for anti-sickling activity Lentiviral vector encoding a short hairpin RNA molecule for posttranscriptional silencing of BCL11A	Stable integration into the host genome for long-term expression No immunogenicity Transduce non-dividing HSCs with high efficiency Can accommodate large transgenes	Semi-random integration leading to potential off-target effects or insertional mutagenesis
Gene Editing	Mechanism	Pros	Cons
Nuclease editing (CRISPR/Cas9, ZFN)	NHEJ: HbF induction via disruption of BCL11A erythroid enhancer HbF induction via disruption of BCL11A binding at the gamma globin promoter	Non-integrating Tools are transient High editing efficiency In use in multiple clinical trials	Requires DSB (genotoxicity) Potential off-target editing Induce a p53 response
	HDR: Direct correction of the sickle mutation	Non-integrating Tools are transient High editing efficiency Direct conversion	Requires DSB (genotoxicity) Potential off-target editing Induce a p53 response Requires donor template Lower editing efficiency
Base editing	Direct conversion of the sickle mutation to create Makassar mutation HbF induction by disruption of non-coding regions (BCL11A, gamma globin promoter) or generation of de novo activators (gamma globin promoter)	No DSB Limited insertion/deletions Single or multiplex genome engineering	Potential off-target editing, unwanted bystander editing, or spurious deamination

Legend: CRISPR, clustered regularly interspaced short palindromic repeats; DSB, double-stranded breaks; HSC, hematopoietic stem cell; NHEJ, non-homologous end joining; ZFN, zinc finger nuclease.