7. Conclusions

Skipping exon 6 alone or exon 5 alone would be degraded by NMD, and those transcripts

The exercise to identify an optimal inhibitor of TGF-β1 involved screening multiple gene targets and dozens of PMO inhibitors. Qualitative differences between splice-altering strategies and translation inhibitors involve the preservation of feedback inhibition of the promoter. Translation inhibitors and splice-altering targets that induce a nonsense-mediated decay (NMD) prevent the synthesis of the negative feedback, resulting in compensatory transcription followed by rebound translation of TGF-β1. By contrast, skipping of exons 5 and 6 leads to translation products with altered function but includes the LAP portion of the translated product, resulting in a prolonged inhibition of TGF-β1. Transient inhibition of TGF-β1 is desired [48], so the optimal approach favors the AUG and NMD PMO over exon skipping and ligand-neutralizing antibodies. Translation inhibition is preferred over NMD because

The Centers for Disease Control and Prevention report that 4.2 million (28.5%) of US diabetics aged ≥40 years have diabetic retinopathy (DR) or damage to the small blood vessels in the retina that may result in loss of vision [49]. The direct costs for DR in the US were over \$4.5 billion, and the indirect economic impact was an additional \$5 billion. Retinopathy occurs in almost all patients with type 1 diabetes and 75% of patients with type 2 diabetes within 15 years of the manifestation of diabetes [50]. Over 12,000 diabetic patients become blind each year due to ocular complications [51]. Current therapy addresses the end stages of DR including laser photocoagulation, intravitreal antivascular endothelial growth factor (VEGF) agents such as Bevacizumab and Aflibercept, intravitreal corticosteroids such as Triamcinolone, and vitreoretinal surgery. CD34+ stem cells from diabetic patients cannot generate endothelial cells to repair the vasculature, instead generating more inflammatory monocytes [52]. The CD34+ stem cell therapy described here exploits the ability of these cells to differentiate into a wide variety of cell types to stimulate both vascular and neural regeneration to treat early stages of DR. CD34+ cells are capable of homing to vascular lesions in the eye, mediating vascular repair [53]. The use of autologous CD34+ cells eliminates the significant complication of transplant rejection. However, diabetic CD34+ cells are dysfunctional, contributing to the diabetic complication of DR [54]. While CD34+ cells from healthy subjects could repair retinal capillaries in streptozotocin-induced diabetic mice, spontaneously diabetic obese BBZDR/Wor rats and neonatal mouse oxygen-induced retinopathy animal models CD34+ cells from diabetic mice could not [55]. The approach described here restores function to dysfunctional diabetic CD34+ cells. TGF-β1 is overexpressed and may cause dysfunction in diabetic CD34+ cells, and correction of this overexpression can restore the regenerative ability of those cells in diabetics. TGF-β1 is the major regulator of the balance between CD34+ proliferation, differentiation, and quiescence. Transient inhibition of TGF-β1 with an optimal PMO (1) activates human CD34+ proliferation, whereas ID11 antibody does not, (2) enhances CXCR4 cell surface expression and effective stem

would not be observed. Skipping exons 5 and 6 will also remain in frame.

80 In Vivo and Ex Vivo Gene Therapy for Inherited and Non-Inherited Disorders

NMD responses may be less reliable.

6. Stem cell therapy for diabetic retinopathy

Damaged retinal vessels are repaired by HSC in individuals throughout their life. Diabetic HSC function is impaired, leading to the development of numerous clinically important conditions including diabetic retinopathy. Selective ex vivo manipulation of TGF-β1 in diabetic HSC represents a therapeutic approach to maintain, enhance, and restore vascular viability in the retina. The PMO offers transcript selective binding and transient interference with translation of TGF-β1. The PMO offers a feasible technology in which they enter HSC, can inhibit autocrine TGF-β1 signaling in HSC, and have an excellent safety profile. We presented the process of selecting TGF-β1 as an optimal transcript and the optimal PMO sequence targeting TGF-β1 mRNA. Our studies identified a transient interference with the translation of TGF-β1 in diabetic CD34+ HSC with an antisense PMO that will (1) upregulate the expression of CXCR4, enabling stem cell homing and adhesion to sites of vascular injury in the retina, (2) stimulation of nitric oxide production, enabling stem cell mobility, and (3) the release of cell cycle checkpoints, enabling stem cell proliferation and differentiation required for the repair of vascular lesions. The manipulated stem cell treatment strategy is making the transition from discovery to preparation for clinical evaluation.
