Activating human genes with zinc finger proteins, transcription activator-like effectors and CRISPR/Cas9 for gene therapy and regenerative medicine

Figures & data

Figure 1. Technologies for engineering programmable DNA-binding proteins, including (A) zinc finger proteins, (B) TALEs and (C) CRISPR/Cas9. (Top) Representative crystal structures of a (A) zinc finger protein (PDB 1P47) or (B) TALE (PDB 3UGM) fused to the p65 transcriptional activation domain (PDB 2RAM) or (C) Cas9 (green) bound to a gRNA (blue) and the corresponding DNA target site (brown) (PDB 4OO8). (Bottom) Zinc finger proteins and TALEs recognize their target sequences through non-covalent protein-DNA interactions with 3 bp or 1 bp per repeat domain, respectively. The CRISPR system consists of a complex of the gRNA and the Cas9 protein that recognizes its genomic target sequence by complementary base pairing of the gRNA (spheres) to the chromosomal DNA.

Table 1. Examples of therapeutic applications of engineered transcriptional activators.

	Application	Gene	Disease	Platform	Representative publication
Gene therapy	Compensating for genetic defect	ϒ-Globin (HBG1/2)	Sickle cell disease	Zinc finger protein	Graslund et al. [12]
	Compensating for genetic defect	Utrophin (UTRN)	Duchenne muscular dystrophy	Zinc finger protein	Di Certo et al. [13]
	Compensating for genetic defect	Frataxin (FXN)	Friedreich’s ataxia	TALE	Chapdelaine et al. [14]
	Suppressing angiogenesis	PEDF (SERPINF1)	Choroidal neovascularization	Zinc finger protein	Yokoi et al. [17]
	Inactivating oncogenes	Bax (BAX)	Cancer	Zinc finger protein	Falke et al. [16]
	Activating silenced tumor supressors	Maspin (SERPINB5)	Cancer	Zinc finger protein	Beltran et al. [15]
Regenerative medicine	Tissue regeneration	VEGF-A (VEGFA)	Amyotrophic lateral sclerosis, diabetic wounds and others	Zinc finger protein	Rebar et al. [18]
	Genetic reprogramming	Oct-4 (POU5F1)	Patient-specific cell therapy	Zinc finger protein, TALE	Bultmann et al. [19] and Gao et al. [20]

PEDF: Pigment endothelium-derived factor; TALE: Transcription activator-like effector.

Graslund T, Li X, Magnenat L, et al. Exploring strategies for the design of artificial transcription factors: targeting sites proximal to known regulatory regions for the induction of gamma-globin expression and the treatment of sickle cell disease. J Biol Chem 2005;280:3707-14

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Di Certo MG, Corbi N, Strimpakos G, et al. The artificial gene Jazz, a transcriptional regulator of utrophin, corrects the dystrophic pathology in mdx mice. Hum Mol Genet 2010;19:752-60

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Chapdelaine P, Coulombe Z, Chikh A, et al. A potential new therapeutic approach for Friedreich ataxia: induction of Frataxin expression with TALE proteins. Mol Ther Nucleic Acids 2013;2:e119

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Yokoi K, Zhang HS, Kachi S, et al. Gene transfer of an engineered zinc finger protein enhances the anti-angiogenic defense system. Mol Ther 2007;15:1917-23

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Falke D, Fisher M, Ye D, Juliano RL. Design of artificial transcription factors to selectively regulate the pro-apoptotic bax gene. Nucleic Acids Res 2003;31:e10

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Beltran A, Parikh S, Liu Y, et al. Re-activation of a dormant tumor suppressor gene maspin by designed transcription factors. Oncogene 2007;26:2791-8

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Rebar EJ, Huang Y, Hickey R, et al. Induction of angiogenesis in a mouse model using engineered transcription factors. Nat Med 2002;8:1427-32

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Bultmann S, Morbitzer R, Schmidt CS, et al. Targeted transcriptional activation of silent oct4 pluripotency gene by combining designer TALEs and inhibition of epigenetic modifiers. Nucleic Acids Res 2012;40:5368-77

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Gao X, Yang J, Tsang JC, et al. Reprogramming to pluripotency using designer TALE transcription factors targeting enhancers. Stem Cell Reports 2013;1:183-97

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