Production of therapeutic iduronate‐2‐sulfatase enzyme with a novel single‐stranded RNA virus vector

Abstract

The Sendai virus vector has received a lot of attention due to its broad tropism for mammalian cells. As a result of efforts for genetic studies based on a mutant virus, we can now express more than 10 genes of up to 13.5 kilo nucleotides in a single vector with high protein expression efficiency. To prove this benefit, we examined the efficacy of the novel ribonucleic acid (RNA) virus vector harboring the human iduronate‐2‐sulfatase (IDS) gene with 1,653 base pairs, a causative gene for mucopolysaccharidosis type II, also known as a disorder of lysosomal storage disorders. As expected, this novel RNA vector with the human IDS gene exhibited its marked expression as determined by the expression of enhanced green fluorescent protein and IDS enzyme activity. While these cells exhibited a normal growth rate, the BHK‐21 transformant cells stably expressing the human IDS gene persistently generated an active human IDS enzyme extracellularly. The human IDS protein produced failed to be incorporated into the lysosome when cells were pretreated with mannose‐6‐phosphate, demonstrating that this human IDS enzyme has potential for therapeutic use by cross‐correction. These results suggest that our novel RNA vector may be applicable for further clinical settings.

Enhanced osteoblastic differentiation of parietal bone in a novel murine model of mucopolysaccharidosis type II

“…We established modified cell lines derived from baby hamster kidney cells (BHK-21 cells) that consistently produce human IDS. In the culture supernatant, we measured IDS activity at 2 μmol/h/L [ 33 ]. We then examined whether supplementing this exogenous IDS enzyme could correct the Ids-deficient phenotypes.…”

“…We established modified cell lines derived from baby hamster kidney cells (BHK-21 cells) that consistently produce human IDS. In the culture supernatant, we measured IDS activity to be 2 μmol/h/L [ 33 ]. Separately, Ids -deficient parietal bone-derived osteoblasts were cultured and differentiated in a 96-well plate at 37 °C under 5% CO2, following the procedure described above.…”

Current Understanding on the Genetic Basis of Key Metabolic Disorders: A Review

“…Enzyme replacement therapy (ERT) is one of the options for patients diagnosed with the phenotype [ 87 ]; however, the inability of the intravenously administered enzymes to overcome the blood brain barrier renders ERT ineffective against progressive neurodegeneration and severe symptoms in the Central Nervous System as reported in patients with neuronopathic MPS [ 88 ]. Recent advances in the development of RNA technology have led to novel approaches such as the experimental production of human iduronate-2-sulfatase (IDS) using a Sendai virus vector [ 89 ].…”

An engineered ligand-responsive Csy4 endoribonuclease controls transgene expression from Sendai virus vectors

“…These features are especially suited for cell reprogramming by transcription factors, such as generation of induced pluripotent stem cells (iPSCs), for which durable expression of multiple factors at high levels is imperative [ 22 ]. Thus, SeVdp vectors are increasingly utilized as a powerful tool for research in gene therapy, regenerative medicine, and biopharmaceutical development [ 23 – 25 ]. However, because SeVdp vectors exist as a single-stranded RNA in cells, no reliable system is available yet to adjust expression of the transgene from SeVdp in a time- or dose-dependent manner.…”