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Oncolytic virotherapy represents a promising approach in cancer immunotherapy. The primary delivery method for oncolytic viruses (OVs) is intratumoral injection, which apparently limits their clinical application. For patients with advanced cancer with disseminated metastasis, systemic administration is considered the optimal approach. However, the direct delivery of naked viruses through intravenous injection presents challenges, including rapid clearance by the immune system, inadequate accumulation in tumors, and significant side effects. Consequently, the development of drug delivery strategies has led to the emergence of various bio-materials serving as viral vectors, thereby improving the anti-tumor efficacy of oncolytic virotherapy. This review provides an overview of innovative strategies for delivering OVs, with a focus on nanoparticle-based or cell-based delivery systems. Recent pre-clinical and clinical studies are examined to highlight the enhanced efficacy of systemic delivery using these novel platforms. In addition, prevalent challenges in current research are briefly discussed, and potential solutions are proposed.

Summary: Mitochondria play important roles in angiogenesis. However, the mechanisms remain elusive. In this study, we found that mitochondrial ubiquinol-cytochrome c reductase complex assembly factor 3 (UQCC3) is a key regulator of angiogenesis. TALEN-mediated knockout of Uqcc3 in mice caused embryonic lethality at 9.5–10.5 days postcoitum, and vessel density was dramatically reduced. Similarly, knockout of uqcc3 in zebrafish induced lethality post-fertilization and impaired vascular development. Knockout of UQCC3 resulted in slower tumor growth and angiogenesis. Mechanistically, UQCC3 was upregulated under hypoxia, promoted reactive oxygen species (ROS) generation, enhanced HIF-1α stability and increased VEGF expression. Finally, higher expression of UQCC3 was associated with poor prognosis in multiple types tumors, implying a role for UQCC3 in tumor progression. In conclusion, our findings highlight the important contribution of UQCC3 to angiogenesis under both physiological and pathological conditions, indicating the potential of UQCC3 as a therapeutic target for cancer.

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.

Malignant ascites frequently occur in patients with advanced ovarian cancer at initial diagnosis, and in almost all cases of relapse, they are closely related to poor prognosis, chemoresistance, and metastasis. To date, effective management strategies have been limited. In this study, we aimed to investigate the effects of oncolytic adenovirus (OV) on malignant ascites in a mouse model of advanced ovarian cancer. The results suggested that OV conferred an effective ability to reduce ascites development and prolong overall survival. Further analysis of the ascitic immune microenvironment revealed that OV treatment promoted T cell infiltration, activation, and differentiation into the effector phenotype; reprogrammed macrophages toward the M1-like phenotype; and increased the ratios of both CD8+ T cells to CD4+ T cells and M1 to M2 macrophages. However, immunosuppressive factors such as PD-1, LAG-3, and Tregs emerged after treatment. Combination therapy including OV, CSF-1R inhibitor PLX3397, and anti-PD-1 remarkably delayed the progression of ascites, and combination therapy induced a greater extent of T cell infiltration, proliferation, and activation. This study provides experimental and theoretical evidence for oncolytic virus-based treatment of malignant ascites, which may further contribute to advanced ovarian cancer therapy.

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.

Therapeutic antibodies targeting vascular endothelial growth factor (VEGF) have become a critical regimen for tumor therapy, but the efficacy of monotherapy is usually limited by drug resistance and multiple angiogenic mechanisms. Complement proteins are becoming potential candidates for cancer-targeted therapy based on their role in promoting cancer progression and angiogenesis. However, the antitumor abilities of simultaneous VEGF and complement blockade were unknown. We generated a humanized soluble VEGFR-Fc fusion protein (VID) binding VEGFA/PIGF and a CR1-Fc fusion protein (CID) targeting C3b/C4b. Both VID and CID had good affinities to their ligands and showed effective bioactivities. In vitro, angiogenesis effects induced by VEGF and hemolysis induced by complement were inhibited by VID and CID, respectively. Further, VID and CID confer a synergetic therapeutic effect in a colitis-associated colorectal cancer (CAC) model and an orthotopic 4T1 breast cancer model. Mechanically, combination therapy inhibited tumor angiogenesis, cell proliferation, and MDSC infiltration in the tumor microenvironment and promoted tumor cell apoptosis. Our study offers a novel therapeutic strategy for anti-VEGF-resistant tumors and chronic-inflammation-associated tumors. Keywords: VEGFR-Fc fusion protein, CR1-Fc fusion protein, combination therapy, angiogenesis, MDSCs