Purpose Plasma globotriaosylsphingosine (lyso-Gb3) is a promising secondary screening biomarker for Fabry disease. Here, we examined its applicability as a primary screening biomarker for classic and late-onset Fabry disease in males and females. Methods Between 1 July 2014 and 31 December 2015, we screened 2,360 patients (1,324 males) referred from 169 Japanese specialty clinics (cardiology, nephrology, neurology, and pediatrics), based on clinical symptoms suggestive of Fabry disease. We used the plasma lyso-Gb3 concentration, α-galactosidase A (α-Gal A) activity, and analysis of the α-Gal A gene (GLA) for primary and secondary screens, respectively. Results Of 8 males with elevated lyso-Gb3 levels (≥2.0 ng ml^–1) and low α-Gal A activity (≤4.0 nmol h^–1 ml^–1), 7 presented a GLA mutation (2 classic and 5 late-onset). Of 15 females with elevated lyso-Gb3, 7 displayed low α-Gal A activity (5 with GLA mutations; 4 classic and 1 late-onset) and 8 exhibited normal α-Gal A activity (1 with a classic GLA mutation and 3 with genetic variants of uncertain significance). Conclusion Plasma lyso-Gb3 is a potential primary screening biomarker for classic and late-onset Fabry disease probands.

Kidney-targeted gene transfer is expected to revolutionize the treatment of renal diseases. Previous gene transfer methods using nonviral vectors administered via renal arterial, pelvic, or ureteric routes into the glomerulus, tubules, or interstitial fibroblasts have resulted in low-level expression for <1 month. The peritubular capillaries (PTC) network is one of the main targets of kidney transplant rejection and of progressive tubulointerstitial fibrosis, which typifies all progressive renal diseases. To access the PTC, we retrogradely injected a lacZ expression plasmid in Ringer's solution into the renal vein of rats. We detected lacZ expression exclusively in the interstitial fibroblasts near the PTC of the injected kidney by immunoelectron microscopic analysis. Nephrotoxicity attributable to gene transfer was not apparent. We then used a rat erythropoietin (Epo) expression plasmid vector, pCAGGS-Epo, in a reporter assay. We obtained maximal Epo expression when the DNA solution was injected within 5 sec, and with a volume of 1.0 ml. We observed a dose-response relationship between serum Epo levels and the amount of injected DNA up to 100 μg. We detected the transgene-derived Epo mRNA by reverse transcription polymerase chain reaction only in the kidneys injected with pCAGGS-Epo. After an injection of 100 μg of pCAGGS-Epo, the serum Epo levels peaked at 208.3 ± 71.8 mU/ml at week 5, and gradually decreased to 116.2 ± 38.7 mU/ml at week 24. A similar pattern was obtained using smaller doses of plasmid, 2 μg or 30 μg of pCAGGS-Epo. Transgene-derived Epo secretion resulted in significant erythropoiesis. This novel technique is simple and safe, allowing high-level and long-term stable gene expression specific to the fibroblasts near the PTC, and should have therapeutic value for future applications in humans.

It has been demonstrated that gene transfer by in vivo electroporation of mouse muscle increases the level of gene expression by more than 100-fold over simple plasmid DNA injection. We tested continuous rat erythropoietin (Epo) delivery by this method in normal rats, using plasmid DNA expressing rat Epo (pCAGGSEpo) as the vector. A pair of electrodes was inserted into the thigh muscles of rat hind limbs and 100 mug of pCAGGS-Epo was injected between the electrodes. Eight 100-V, 50-msec electric pulses were delivered through the electrodes. Each rat was injected with a total of 400 mug of pCAGGS-Epo, which was delivered to the medial and lateral sides of each thigh. The presence of vector-derived Epo mRNA at the DNA injection site was confirmed by RT-PCR. The serum Epo levels peaked at 122.2 ± 33.0 mU/ml on day 7 and gradually decreased to 35.9 ± 18.2 mU/ml on day 32. The hematocrit levels increased continuously, from the preinjection level of 49.5 ± 1.1 to 67.8 ± 2.2% on day 32 (p<0.001). In pCAGGS-Epo treated rats, endogenous Epo secretion was downregulated on day 32. In a control experiment, intramuscular injection of pCAGGS-Epo without subsequent electroporation did not significantly enhance the serum Epo levels. These results demonstrate that muscle-targeted pCAGGS-Epo transfer by in vivo electroporation is a useful procedure for the continuous delivery of Epo.

Transcutaneous in vivo electroporation is expected to be an effective gene-transfer method for promoting bone regeneration using the BMP-2 plasmid vector. To promote enhanced osteoinduction using this method, we simultaneously transferred cDNAs for BMP-2 and BMP-7, as inserts in the non-viral vector pCAGGS. First, an in vitro study was carried out to confirm the expression of BMP-2 and BMP-7 following the double-gene transfer. Next, the individual BMP-2 and BMP-7 plasmids or both together were injected into rat calf muscles, and transcutaneous electroporation was applied 8 times at 100 V, 50 msec. In the culture system, the simultaneous transfer of the BMP-2 and BMP-7 genes led to a much higher ALP activity in C2C12 cells than did the transfer of either gene alone. In vivo, ten days after the treatment, soft X-ray analysis showed that muscles that received both pCAGGS-BMP-2 and pCAGGS-BMP-7 had better-defined opacities than those receiving a single gene. Histological examination showed advanced ossification in calf muscles that received the double-gene transfer. BMP-4 mRNA was also expressed, and RT-PCR showed that its level increased for 3 days in a time-dependent manner in the double-gene transfer group. Immunohistochemistry confirmed that BMP-4-expressing cells resided in the matrix between muscle fibers. The simultaneous transfer of BMP-2 and BMP-7 genes using in vivo electroporation induces more rapid bone formation than the transfer of either gene alone, and the increased expression of endogenous BMP-4 suggests that the rapid ossification is related to the induction of BMP-4.

Background: We previously demonstrated that erythropoietin (Epo) expression increases in five-sixths nephrectomized rats, after muscle-targeted gene transfer by in vivo electroporation, using plasmid DNA expressing rat Epo (pCAGGS-Epo). Here, we apply this method to a rat model with severe anemia associated with chronic renal failure; these rats have hematocrit levels in the 30–35% range, similar to those in humans with end-stage renal disease. Methods: Wistar rats were treated to produce adenine-induced uremia. The uremic rats were then treated with muscle-targeted gene transfer using pCAGGS-Epo. Some uremic rats died from chronic renal failure; one of these was dissected, and the kidneys were histologically examined. For the remaining rats, we measured body weight and blood pressure, and obtained blood samples regularly. Results: The uremic rats showed severe anemia, with hematocrit levels at 32.6 ± 3.3%. Epo-gene transfer increased Epo expression and serum Epo levels, and also increased the hematocrit levels to 64.5 ± 4.8%. The dose of pCAGGS-Epo used in this study did not induce severe hypertension. Conclusions: Continuous Epo-gene expression improves the anemia associated with chronic renal failure, and without severe side effects. Our results support the potential use of gene electrotransfer for human gene therapy applications.

Summary: Background and objectives: Previous reports of Fabry disease screening in dialysis patients indicate thatα-galactosidase A activity alone cannot specifically and reliably identify appropriate candidates for genetic testing; a marker for secondary screening is required. Elevated plasma globotriaosylsphingosine is reported to be a hallmark of classic Fabry disease. The purpose of this study was to examine the usefulness of globotriaosylsphingosine as a secondary screening target for Fabry disease.Design, setting, participants, & measurements: This study screened 1453 patients, comprising 50% of the male dialysis patients in Niigata Prefecture between July 1, 2010 and July 31, 2011. Screening for Fabry disease was performed by measuring the plasmaα-galactosidase A enzyme activity and the globotriaosylsphingosine concentration, by high-performance liquid chromatography. Genetic testing and genetic counseling were provided.Results: A low level of plasmaα-galactosidase A activity (≤4.0 nmol/h per milliliter) was observed in 47 patients (3.2%). Of these, 3 (0.2%) had detectable globotriaosylsphingosine levels. These patients all hadα-galactosidase Agene mutations: one was p.Y173X and two were the nonpathogenic p.E66Q. The patient with p.Y173X started enzyme replacement therapy. Subsequent screening of his family identified the same mutation in his elder sister and her children. Genetic testing for 33 of the other 44 patients detected 7 patients with p.E66Q. Thus, the plasma lyso-Gb3 screen identified Fabry disease with high sensitivity (100%) and specificity (94.3%).Conclusions: Plasma globotriaosylsphingosine is a promising secondary screening target that was effective for selecting candidates for genetic counseling and testing and for uncovering unrecognized Fabry disease cases.