Background: We recently reported an innovative gene therapy approach for GSD III using a recombinant adeno-associated virus serotype 9 vector (AAV9-Dual-Pull) expressing a bacterial debranching enzyme (pullulanase) driven by a tandem dual promoter that consists of an immunotolerizing liver-specific promoter (LSP) and the ubiquitous CMV enhance/chicken β -actin (CB) promoter. In this follow-up study, we evaluated the long-term efficacy of this gene therapy in GSD IIIa mice. Methods: Three-month-old GSD IIIa mice were intravenously injected with AAV9-LSP-Pull or AAV9-Dual-Pull at the same dose (2.5 × 10 13 vg/kg). Tissues were collected after 9 months for AAV genome quantification, pullulanase expression determination, and glycogen content measurement. Liver and muscle enzymes in plasma and disease biomarker in urine were analyzed at multiple time points to examine the correction of liver and muscle damage. Behavioral tests were performed during the course of AAV treatment to evaluate the improvement of muscle function. Results: The AAV-Dual-Pull treatment led to persistent pullulanase expression and effective glycogen reduction in the liver, heart, and skeletal muscle, accompanied by the reversal of liver fibrosis, decrease of plasma enzyme activities, and long-term improvement of muscle function. The AAV-LSP-Pull treatment showed a better therapeutic efficacy in the liver but had no effect on the cardiac and skeletal muscles. Conclusion: Our results demonstrated the long-term efficacy and safety of systemic AAV9-Dual-Pull delivery in GSD IIIa mice. Future studies will test this gene therapy approach in GSD IIIa dogs prior to the clinical translation to GSD III patients.
Cellular immunotherapy strategies that harness the cytotoxic properties of T and NK cells by releasing granzyme B have emerged as a crucial approach in cancer therapy for both hematological and solid tumors. However, the effective release and antitumor activities of granzyme B are significantly influenced by protease inhibitors including serpin B9. Gene delivery mechanisms that can counteract these limitations of cell‐based immunotherapies are highly desired. In this regard, genetic modification of adeno‐associated virus (AAV) capsids as vehicles for targeted delivery of therapeutic genes is an emerging area of research in the field of gene therapy of both monogenic and polygenic diseases. The insertion of antigen‐specific antibody fragments and peptides into capsid sequences has been shown to redirect capsid tropism from native antigens to specific targets. Herein, we report on genetically engineered AAV2 capsid modified by inserting two antibody fragments: single‐chain variable and single‐domain antibodies and GE11 peptide, all specific for targeting epidermal growth factor receptor, at the N terminus of VP2. We observed an inverse correlation between the size of the inserted anti‐EGFR moiety, capsid structure conservation, and vector yield. After demonstrating the proof of concept by confirming antigen‐dependent transduction of cancer cells using AAV‐GFP vectors, we further demonstrated that targeted AAV vectors encoding a protease‐insensitive granzyme B mutant DNA cargo selectively eliminated cancer cells expressing serpin B9 in comparison to vectors encoding wild‐type granzyme B in vitro. Our findings suggest that the challenges posed by intracellular expression of serpin B9 to antitumor T(NK) cell–dependent immunotherapies can be curtailed by utilizing the R201K granzyme mutant in immune‐based therapy development.
Investigations conducted in mice and humans have reported that the nature and the amount of lipids in plasma can predict the likelihood of cardiovascular disease (CVD) development. Although niacin has a history as treatment for dyslipidemia, only a handful of clinical trials have investigated its efficiency in the prevention of the morbidity and mortality associated with CVDs. Therefore, the purpose of this study was to assess the impact of a niacin formulation on gene expression and plasma lipids using 16 vervet monkeys (8 controls and 8 experimental). The control group was given a maintenance diet only, while the experimental group’s diet was supplemented with niacin (100 mg/kg) for a period of 3 months, followed by 4‐week washout. The investigated plasma lipids were total cholesterol, low‐density lipoprotein cholesterol (LDL‐C), high‐density lipoprotein cholesterol (HDL‐C), and triglycerides. Gene expression of proprotein convertase subtilisin/kexin type 9 ( PCSK9 ), cholesterol ester transfer protein ( CETP ), low‐density lipoprotein receptor ( LDLR ), apolipoprotein B‐100 ( APOB-100 ) and sterol regulatory element–binding protein‐2 ( SREBP-2 ), which are involved in the reverse cholesterol transport (RCT) pathway, was also determined. Niacin administration resulted in statistically significant changes for total cholesterol and HDL‐C, with the changes also significantly higher in females compared to males in the niacin‐treated group. Furthermore, gene expression analysis revealed significant decrease in CETP during niacin treatment. The downregulation suggested that the vervet monkey model supports the HDL‐C hypothesis. Future studies aimed at supporting these findings are directed towards exploring the epigenetic biomarkers influencing the RCT pathway to combat CVDs.
Background:We recently reported an innovative gene therapy approach for GSD III using a recombinant adeno-associated virus serotype 9 vector (AAV9-Dual-Pull) expressing a bacterial debranching enzyme (pullulanase) driven by a tandem dual promoter that consists of an immunotolerizing liver-specific promoter (LSP) and the ubiquitous CMV enhance/chicken β-actin (CB) promoter. In this follow-up study, we evaluated the long-term efficacy of this gene therapy in GSD IIIa mice. Methods:Three-month-old GSD IIIa mice were intravenously injected with AAV9-LSP-Pull or AAV9-Dual-Pull at the same dose (2.5 × 1013 vg/kg). Tissues were collected after 9 months for AAV genome quantification, pullulanase expression determination, and glycogen content measurement. Liver and muscle enzymes in plasma and disease biomarker in urine were analyzed at multiple time points to examine the correction of liver and muscle damage. Behavioral tests were performed during the course of AAV treatment to evaluate the improvement of muscle function. Results:The AAV-Dual-Pull treatment led to persistent pullulanase expression and effective glycogen reduction in the liver, heart, and skeletal muscle, accompanied by the reversal of liver fibrosis, decrease of plasma enzyme activities, and long-term improvement of muscle function. The AAV-LSP-Pull treatment showed a better therapeutic efficacy in the liver but had no effect on the cardiac and skeletal muscles. Conclusion:Our results demonstrated the long-term efficacy and safety of systemic AAV9-Dual-Pull delivery in GSD IIIa mice. Future studies will test this gene therapy approach in GSD IIIa dogs prior to the clinical translation to GSD III patients.
Introduction. The Tay-Sachs disease is a progressive neurodegenerative disorder that is caused by a genetic mutation in the HEXA gene coding the lysosomal α-subunit of β-hexosaminidase A. Currently, there is no effective treatment for Tay-Sachs. Induction of exocytosis as a potential treatment approach is suggested to restore lysosomal enlargement in several lysosomal storage diseases. Here, we aimed to test the therapeutic potential of two small molecules, δ-tocopherol and hydroxypropyl-β-cyclodextrin, in fibroblast and neuroglia cells derived from Hexa-/-Neu3-/- mice and Tay-Sachs patients. Method. The effect of two small molecules on lysosomal enlargement and GM2 accumulation in lysosomes was examined by LysoTracker staining and immunocytochemical colocalization analysis for GM2 and LAMP1. qRT-PCR and fluorometric enzyme assay were also used to investigate the effect of combined treatment on the level of neuraminidase 1, a negative regulator of exocytosis. Results. Single treatment with δ-tocopherol (5-40 μM) and hydroxypropyl-β-cyclodextrin (10-50 μM) for 48 hours led to significant induction of lysosomal exocytosis. We demonstrated that the combined treatment with δ-tocopherol (10 μM) and hydroxypropyl-β-cyclodextrin (25 μM) resulted in a significant reduction of lysosomal GM2 and downregulation of lysosomal Neu1 expression. Conclusion. In this study, we demonstrated that inducing exocytosis by δ-tocopherol and hydroxypropyl-β-cyclodextrin might have therapeutic potential to reduce GM2 storage and pathology in Tay-Sachs cells.
