Multiplex gene editing reduces oxalate production in primary hyperoxaluria type 1.

Targeting key enzymes that generate oxalate precursors or substrates is an alternative strategy to eliminate primary hyperoxaluria type I (PH1), the most common and life-threatening type of primary hyperoxaluria. The compact Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) from the Prevotella and Francisella 1 (Cpf1) protein simplifies multiplex gene editing and allows for all-in-one adeno-associated virus (AAV) delivery. We hypothesized that the multiplex capabilities of the Cpf1 system could help minimize oxalate formation in PH1 by simultaneously targeting the hepatic hydroxyacid oxidase 1 ( Hao1) and lactate dehydrogenase A ( Ldha) genes. Study cohorts included treated PH1 rats ( Agxt Q84X rats injected with AAV-AsCpf1 at 7 days of age), phosphate-buffered saline (PBS)-injected PH1 rats, untreated PH1 rats, and age-matched wild-type (WT) rats. The most efficient and specific CRISPR RNA (crRNA) pairs targeting the rat Hao1 and Ldha genes were initially screened ex vivo. In vivo experiments demonstrated efficient genome editing of the Hao1 and Ldha genes, primarily resulting in small deletions. This resulted in decreased transcription and translational expression of Hao1 and Ldha. Treatment significantly reduced urine oxalate levels, reduced kidney damage, and alleviated nephrocalcinosis in rats with PH1. No liver toxicity, ex-liver genome editing, or obvious off-target effects were detected. We demonstrated the AAV-AsCpf1 system can target multiple genes and rescue the pathogenic phenotype in PH1, serving as a proof-of-concept for the development of multiplex genome editing-based gene therapy.

[Relationship between anogenital distance and cryptorchidism in human newborns].

OBJECTIVE: To explore the relation of the anogenital distance (AGD) with cryptorchidism in male newborns. METHODS: This study included 350 male infants delivered in two community hospitals between September 2013 and September 2014. Within 24 hours after birth, a pediatric surgeon measured the AGD of the neonates and determined whether they had cryptorchidism. According to the testicular position, we divided the undescended testes into three types: upper scrotal, inguinal, and non-palpable. RESULTS: Totally 39 cases of cryptorchidism were found in the 350 newborns. The AGD of the cryptorchidism infants was significantly shorter than that of the normal neonates ([2.01 ± 0.22] vs [2.35 ± 0.19] cm, P < 0.01), and statistically significant differences remained even when preterm and low birth-weight infants were excluded ([2.32 ± 0.14] vs [2.06 ± 0.19] cm; (2.37 ± 0.17) cm vs (2.12 ± 0.12) cm, all P < 0.01). The newborns with higher-position cryptorchidism had a shorter AGD, though with no significant difference (F = 0.434, P > 0.05). No significant differences were observed in the AGD between unilateral and bilateral cryptorchidism ([1.96 ± 0.13] vs [2.02 ± 0.17] cm, P > 0.05). CONCLUSION: Shorter AGD is associated with a higher incidence of cryptorchidism in male newborns. AGD could serve as a potential biomarker for disruption of androgen action during the male programming window period.

[Staged hypospadias surgery for adult males].

OBJECTIVE: To explore the necessity of staged hypospadias surgery for adult men in order to improve the success rate of operation. METHODS: We retrospectively analyzed 52 cases of hypospadias treated in our department from January 2004 to January 2012. The patients were adult males at the mean age of 22 years and all had a history of urethroplasty, with curvature of the penis and scar tissues on the penile skin. We removed the scarred fibrous tissues on the ventral cavernosa and cut off the urethral plate following foreskin-degloving. For those still with penile curvature, we straightened the penis by plication of the dorsal tunica albuginea, with the length of the anterior urethral defect > 50% of that of the penis after penis-straightening. The patients were assigned to group 1 (n = 20) to receive stage-I foreskin vascular pedicle flap urethroplasty and group 2 (n = 32) to undergo foreskin-shaping at the ventral aspect of the penis following penile straightening to prepare the urethra plate for stage-II Duplay urethroplasty after 6-12 months. RESULTS: The success rates of urethroplasty were 25 and 56.3% in groups 1 and 2, respectively, with the post-urethroplasty incidence rates of urinary fistula of 50 and 21.9%, urethral stricture of 15 and 9.4%, local wound infection of 30 and 25%, and urethral rupture of 20 and 12.5%. There were statistically significant differences between the two groups in the incidence of urinary fistula and the success rate of urethroplasty, but not in urethral stricture, local wound infection and urethral rupture. CONCLUSION: For adult hypospadias patients with a history of urethroplasty, especially those with obvious penile curvature, long urethral defect and insufficient foreskin, staged hypospadias surgery is preferable, which can dramatically increase the success rate of second-stage urethroplasty.

Tissue engineering of bladder using vascular endothelial growth factor gene-modified endothelial progenitor cells.

PURPOSE: This study assessed the use of vascular endothelial growth factor (VEGF) gene-modified endothelial progenitor cells (EPCs) seeded onto bladder acellular matrix grafts (BAMGs), to enhance the blood supply in tissue-engineered bladders in a porcine model. METHODS: Autologous porcine peripheral EPCs were isolated, cultured, expanded, characterized, and modified with the VEGF gene using an adenovirus vector. The expression of VEGF was examined using reverse transcriptase polymerase chain reaction (RT-PCR) and an enzyme-linked immunosorbent assay (ELISA). VEGF gene modified EPCs were seeded onto BAMG and cultured for 3 days before implantation into pigs for bladder tissue engineering. A partial bladder cystectomy was performed in 12 pigs. The experimental group (6 pigs) received VEGF gene-modified EPC-seeded BAMG. The control group (6 pigs) received BAMG without seeded EPCs. The resulting tissue-engineered bladders were subject to a general and histological analysis. Microvessel density (MVD) was assessed using immunohistochemistry. RESULTS: The ex vivo transfection efficiency of EPCs was greater than 60%-70% when concentrated adenovirus was used. The genetically modified cells expressed both VEGF and green fluorescent protein (GFP). Scanning electron microscopy (SEM) and Masson's trichrome staining of cross sections of the cultured cells seeded to BAMG showed cell attachment and proliferation on the surface of the BAMG. Histological examination revealed bladder regeneration in a time-dependent fashion. Significant increases in MVD were observed in the experimental group, in comparison with the control group. CONCLUSIONS: VEGF-modified EPCs significantly enhanced neovascularization, compared with BAMG alone. These results indicate that EPCs, combined with VEGF gene therapy, may be a suitable approach for increasing blood supply in the tissue engineering of bladders. Thus, a useful strategy to achieve a tissue-engineered bladder is indicated.

[Amniotic fluid: a novel source for tissue engineering].

OBJECTIVE: To investigate the possibility of using amniotic fluid cells as seed cells for tissue engineering. METHODS: Amniotic fluid was obtained by ultrasound-guided amniocentesis performed on pregnant women with a gestational age ranging from 16(th) approximately 23(rd) weeks. The cells isolated from the amniotic fluid were cultured in F10 culture fluid with 10% FBS. Immunocytochemistry was used to examine the standard intermediate filaments. After 3 passages of subculture, the cells were harvested and seeded onto PGA polymer scaffold. The cellular morphology, structure and adhesion with scaffold were evaluated by contrast microscope and scanning electronic microscope. RESULTS: The amniocytes expanded rapidly in culture media. Immunocytochemistry revealed positive signals for vimentin, smooth muscle action (SMA), and pan cytokeratin, and negative signals for desmin. Amniocytes-polymer complex analysis showed confluent cells firmly attached to the scaffold, with no evidence of cell death. CONCLUSION: The expansion potential of amniotic fluid cells is active. They express the characteristics of mesenchymal cells. The cells on PGA polymer scaffold can grow rapidly and maintain its morphological property. So the amniotic fluid may be a practical cell source for tissue engineering.

[Eosinophilic cystitis in children:a report of six cases and review of literature].

OBJECTIVE: Eosinophilic cystitis is a very rare tumor-like inflammatory disease especially in children. It can involve any portion of the bladder and interfere with its function. Here we present 6 pediatric cases and review the literature. METHODS: Six children, with mean age of 8.3 years, were referred to our clinic. Their major complaints included voiding urgency, frequency, dysuria, enuresis, intermittent hematuria and abdominal pain. Ultrasound, CT scan and voiding cystourethrogram (VCUG) showed diffuse thickening of bladder wall and a filling-defect lesion. They all suggested the possibility of bladder tumor. The final diagnosis of eosinophilic cystitis was made by cystoscopic tissue biopsy or open surgery. Patients underwent bladder autoaugmentation, mass resection or mere oral administration of corticosteroid and antibiotics respectively. RESULTS: 5 among 6 patients were followed up for 5 - 28 months. Their voiding complaints and image changes gradually disappeared. Up to now, there is no relapse. CONCLUSIONS: The clinical presentation of eosinophilic cystitis includes irritative voiding symptoms, hematuria and abdominal pain. Tumor-like changes could be found in the imagination examinations. A biopsy is mandatory to establish the diagnosis. Treatment in general varies from the removal of the antigenic stimulus, if identifiable, to administration of corticosteroid and antibiotics. When patients fail to respond to the medication treatment, the opening operation could be required.