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Mucopolysaccharidosis type I-Hurler (MPS I-H) is a neurodegenerative lysosomal storage disorder (LSD) caused by inherited defects of the α-L-iduronidase (IDUA) gene. Current treatments are ineffective for treating central nervous system (CNS) manifestations because lysosomal enzymes do not effectively cross the blood-brain barrier (BBB). To enable BBB transport of the enzyme, we engineered a modified IDUA protein by adding a brain-targeting peptide from melanotransferrin. We demonstrated that fusion of melanotransferrin peptide (MTfp) at the N terminus of human IDUA (hIDUA) was enzymatically active and could efficiently cross the BBB in vitro. Then, liver-directed gene therapy using the adeno-associated virus 8 (AAV8) vector, which encoded the modified hIDUA cDNA driven by a liver-specific expression cassette was evaluated in an adult MPS I-H mouse model. The results showed that intravenous (i.v.) infusion of AAV8 resulted in sustained supraphysiological levels of IDUA activity and normalized glycosaminoglycan (GAG) accumulation in peripheral tissues. Addition of MTfp to the hIDUA N terminus allowed efficient BBB transcytosis and IDUA activity restoration in the brain, resulting in significant improvements in brain pathology and neurobehavioral deficits. Our results provide a novel strategy to develop minimally invasive therapies for treatment of MPS I-H and other neurodegenerative LSDs.

Inhibition of vascular endothelial growth factor (VEGF) is the standard therapy for neovascular age-related macular degeneration (nAMD). However, anti-VEGF agents used in the clinic require repeated injections, causing adverse effects. Gene therapy could provide sustained anti-VEGF levels after a single injection, thereby drastically decreasing the treatment burden and improving visual outcomes. In this study, we developed a novel VEGF Trap, nVEGFi, containing domains 1 and 2 of VEGFR1 and domain 3 of VEGFR2 fused to the Fc portion of human IgG. The nVEGFi had a higher expression level than aflibercept under the same expression cassettes of adeno-associated virus (AAV)8 in vitro and in vivo. nVEGFi was found to be noninferior to aflibercept in binding and blocking VEGF in vitro. AAV8-mediated expression of nVEGFi was maintained for at least 12 weeks by subretinal delivery in C57BL/6J mice. In a mouse laser-induced choroidal neovascularization (CNV) model, 4 × 108 genome copies of AAV8-nVEGFi exhibited a significantly increased reduction in the CNV area compared with AAV8-aflibercept (78.1% vs. 63.9%, p < 0.05), while causing no structural or functional changes to the retina. In conclusion, this preclinical study showed that subretinal injection of AAV8-nVEGFi was long lasting, well tolerated, and effective for nAMD treatment, supporting future translation to the clinic.

Adeno-associated virus (AAV)-mediated delivery of the clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9) has shown promising results in preclinical models. However, the long-term expression of Cas9 mediated by AAV in the post-mitotic cells raises concerns with specificity and immunogenicity. Thus, it would be advantageous to limit the duration of Cas9 expression following delivery. In this study, we have engineered an all-in-one self-cleavage AAV-CRISPR-Cas9 system to restrict the expression of Cas9 nuclease, which consists of a Cas9 nuclease from Staphylococcus aureus (SaCas9), a chimeric single guide RNA (sgRNA) molecule targeting PCSK9, and flanking sites targeted by this sgRNA. The self-cleavage system generated a negative feedback loop where Cas9 cut both the target genomic locus and the AAV vector, thus self-limiting the expression of Cas9. We demonstrated that this system could reduce ∼60% expression of SaCas9 protein and had a 20-fold reduction in off-target activity at 24 weeks post-vector administration in vivo. Moreover, the on-target editing efficacy was not compromised and resulted in a stable reduction in circulating PCSK9 and serum cholesterol. The inclusion of this self-cleavage system in gene-editing approaches could increase the safety profile of AAV-delivered genome-editing nucleases and thereby promote its clinical transformation.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 loaded by vectors could induce high rates of specific site genome editing and correct disease-causing mutations. However, most monogenic genetic diseases such as hemophilia are caused by different mutations dispersed in one gene, instead of an accordant mutation. Vectors developed for correcting specific mutations may not be suited to different mutations at other positions. Site-specific gene addition provides an ideal solution for long-term, stable gene therapy. We have demonstrated SaCas9-mediated homology-directed factor IX (FIX) in situ targeting for sustained treatment of hemophilia B. In this study, we tested a more efficient dual adeno-associated virus (AAV) strategy with lower vector dose for liver-directed genome editing that enables CRISPR-Cas9-mediated site-specific integration of therapeutic transgene within the albumin gene, and we aimed to develop a more universal gene-targeting approach. We successfully achieved coagulation function in newborn and adult hemophilia B mice by a single injection of dual AAV vectors. FIX levels in treated mice persisted even after a two-thirds partial hepatectomy, indicating stable gene integration. Our results suggest that this CRISPR-Cas9-mediated site-specific gene integration in hepatocytes could transform into a common clinical therapeutic method for hemophilia B and other genetic diseases.