In vivo base editing rescues primary hyperoxaluria type 1 in rats.

Primary hyperoxaluria type 1 (PH1) is a childhood-onset autosomal recessive disease, characterized by nephrocalcinosis, multiple recurrent urinary calcium oxalate stones, and a high risk of progressive kidney damage. PH1 is caused by inherent genetic defects of the alanine glyoxylate aminotransferase (AGXT) gene. The in vivo repair of disease-causing genes was exceedingly inefficient before the invention of base editors which can efficiently introduce precisely targeted base alterations without double-strand DNA breaks. Adenine base editor (ABE) can precisely convert A·T to G·C with the assistance of specific guide RNA. Here, we demonstrated that systemic delivery of dual adeno-associated virus encoding a split-ABE8e could artificially repair 13% of the pathogenic allele in AgxtQ84X rats, a model of PH1, alleviating the disease phenotype. Specifically, ABE treatment partially restored the expression of alanine-glyoxylate-aminotransferase (AGT), reduced endogenous oxalate synthesis and alleviated calcium oxalate crystal deposition. Western blot and immunohistochemistry confirmed that ABE8e treatment restored AGT protein expression in hepatocytes. Moreover, the precise editing efficiency in the liver remained stable six months after treatment. Thus, our findings provided a prospect of in vivo base editing as a personalized and precise medicine for PH1 by directly correcting the mutant Agxt gene.

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.

Ureterocele with duplex collecting systems and febrile urinary tract infection risk.

PURPOSE: Ureterocele has been hypothesized to be the risk factor for febrile urinary tract infections (F-UTIs) in patients with duplex collecting systems, but this has not been proved, and our goal was to assess the relation between ureterocele with duplex collecting systems and F-UTIs. METHODS: We included individual-participant data from patients seen for complicated duplex collecting systems from 2010 to 2020 retrospectively followed. Those with using continuous low-dose antibiotic prophylaxis and incompletely duplicated systems were removed from the study. The participants were divided into two cohorts according to patients with or without ureterocele. The primary endpoint of this study was recurrent F-UTIs. RESULTS: We analyzed medical reports of 300 patients, of which 75% were female. Among the 300 patients, F-UTIs developed in 111/159 (69.8%) patients in the ureterocele group and in 69/141 (48.9%) patients in the no-ureterocele group. Univariate analysis found no discernible difference except in grade of hydronephrosis between ureterocele group and no-ureterocele group. Moreover, Cox proportional regression analysis revealed that patients of duplex system ureterocele might be intrinsically more prone to develop F-UTIs (adjusted hazard ratio 1.894; 95% CI 1.412-2.542; p  <  0.001). CONCLUSION: Among participants with duplex systems, the risk of recurrent F-UTIs in patients with ureterocele was higher than patients without it, and mini-invasive surgical correction should be considered at young age to reduce F-UTIs.

Lipidomics characterization of the lipid metabolism profiles in a cystinuria rat model: Precalculus damage in the kidney of cystinuria.

Cystinuria is a genetic disorder of cystine transport, including defective protein b0,+AT (encoded by SLC7A9), and/or rBAT (encoded by SLC3A1). Patients present hyperexcretion of cystine in the urine, recurrent cystine lithiasis, and progressive decline in kidney function. Moreover, heterodimer transport is defective. To date, little omics data are accessible regarding this metabolic disease caused by membrane proteins. Since membrane function is closely related to changes in the lipidome, we decided to explore the changes in kidney tissue of a self-established cystinuria rat model by performing lipidomic analysis by LC-MS/MS. Our results demonstrated that Slc7a9 deficiency changed the lipid profile of the renal cortex and induced vital modifications in the lipidome, including major alterations in ChE, LPA, and PA. Among those alterations, this lipidomic study highlights the lipid changes that participate in inflammatory responses during cystinuria. As a result, lipid research, perhaps has great potential, for it may lead to the identification of novel therapeutic targets for the prevention and treatment of cystinuria.

Differences in renal cortex transcriptional profiling of wild-type and novel type B cystinuria model rats.

Cystinuria is a genetic disorder of cystine transport that accounts for 1-2% of all cases of renal lithiasis. It is characterized by hyperexcretion of cystine in urine and recurrent cystine lithiasis. Defective transport of cystine into epithelial cells of renal tubules occurs because of mutations of the transport heterodimer, including protein b0,+AT (encoded by SLC7A9) and rBAT (encoded by SLC3A1) linked through a covalent disulfide bond. Study generated a novel type B cystinuria rat model by artificially deleting 7 bp of Slc7a9 gene exon 3 using the CRISPR-Cas9 system, and those Slc7a9-deficient rats were proved to be similar with cystinuria in terms of genome, transcriptome, translation, and biologic phenotypes with no off-target editing. Subsequent comparisons of renal histopathology indicated model rats gained typical secondary changes as medullary fibrosis with no stone formation. A total of 689 DEGs (383 upregulated and 306 downregulated) were differentially expressed in the renal cortex of cystinuria rats. In accordance with the functional annotation of DEGs, the potential role of glutathione metabolism processes in the kidney of cystinuria rat model was proposed, and KEGG analysis results showed that knock-out of Slc7a9 gene triggered more biological changes which has not been studied. In short, for the first time, a rat model and its transcriptional database that mimics the pathogenesis and clinical consequences of human type B cystinuria were generated.

A comparison of the clinical characteristics of pediatric urolithiasis patients with positive and negative molecular diagnoses.

PURPOSE: To compare the clinical characteristics of pediatric urolithiasis patients with positive and negative molecular diagnoses. METHODS: The clinical characteristics corresponding to pediatric urolithiasis patients that had undergone exome sequencing at our hospital between January 2016 and May 2021 were collected. Genetic analysis results were used to separate patients into positive and negative molecular diagnosis groups. Multivariate logistic regression analyses adjusted for visiting age, sex, ethnicity, province, and body mass index were used to compare differences in medical history, diagnostic imaging findings, and renal function between individuals with and without molecular diagnoses. RESULTS: In total, 194 patients with pediatric urolithiasis of unknown etiology underwent exome sequencing and were included in the present study, of whom 63 obtained urolithiasis-related molecular diagnoses. Relative to cases without a molecular diagnosis, those with a positive molecular diagnosis were more likely to be associated with a positive family history (OR 2.84, 95% CI 1.29-6.29, p = 0.008), consanguineous parents (OR 24.7, 95% CI 1.34-454, p = 0.002), early onset (OR 1.26, 95% CI 1.09-1.45, p < 0.001), nephrocalcinosis (OR 10.6, 95% CI 3.06-36.6, p < 0.001), cast stone (OR 18.9, 95% CI 4.40-81.1, p < 0.001), multiple stones (OR 13.9, 95% CI 6.39-30.2, p < 0.001), bilateral stones (OR 7.04, 95% CI 3.47-14.2, p < 0.001), a lower estimated glomerular filtration rate (OR 1.17, 95% CI 1.07-1.28, p < 0.001), and chronic kidney disease (OR 26.9, 95% CI 1.42-526, p < 0.001). CONCLUSION: A positive family history, consanguineous parents, early onset, nephrocalcinosis, severe stone burden, and impaired renal function are signals of concern that are suggestive of inherited urolithiasis.

Extended genetic analysis of exome sequencing for primary hyperoxaluria in pediatric urolithiasis patients with hyperoxaluria.

PURPOSE: Next generation sequencing-based exome sequencing can be used to identify genetic abnormalities in patients believed to be suffering from primary hyperoxaluria. We outline our efforts to improve the diagnostic capacity of exome sequencing for these patients. METHODS: We conducted a retrospective analysis of exome sequencing data from 77 pediatric urolithiasis patients with hyperoxaluria of unknown origin. Canonical exome sequencing analysis was performed to identify pathogenic variants in three known primary hyperoxaluria-related genes (AGXT, GRHPR, HOGA1) as per the guidelines of the American College of Medical Genetics. Then, extended exome sequencing analyses of 5'-untranslated region, non-canonical splicing site and copy number variant were performed on patients with negative diagnostic results in these three genes. RESULTS: Canonical exome sequencing analyses led to the diagnosis of primary hyperoxaluria in 20/77 (26%) patients, including eight, four, and eight patients diagnosed with type 1, 2 and 3 primary hyperoxaluria, respectively. Non-canonical splicing site analyses discovered a pathogenic variant in the HOGA1 gene, which led to the diagnosis of six additional patients with type 3 primary hyperoxaluria, while copy number variant analyses from exome sequencing data identified a 1.8 kb copy number loss that impacted the AGXT gene, resulting in the diagnosis of an additional type 1 primary hyperoxaluria case. CONCLUSIONS: Extended non-canonical splicing site and copy number variant analyses improve the diagnostic yield of canonical exome sequencing analysis for primary hyperoxaluria from 26% (20/77) to 35% (27/77) in 77 pediatric urolithiasis patients with hyperoxaluria.

Enhanced genome editing to ameliorate a genetic metabolic liver disease through co-delivery of adeno-associated virus receptor.

Genome editing through adeno-associated viral (AAV) vectors is a promising gene therapy strategy for various diseases, especially genetic disorders. However, homologous recombination (HR) efficiency is extremely low in adult animal models. We assumed that increasing AAV transduction efficiency could increase genome editing activity, especially HR efficiency, for in vivo gene therapy. Firstly, a mouse phenylketonuria (PKU) model carrying a pathogenic R408W mutation in phenylalanine hydroxylase (Pah) was generated. Through co-delivery of the general AAV receptor (AAVR), we found that AAVR could dramatically increase AAV transduction efficiency in vitro and in vivo. Furthermore, co-delivery of SaCas9/sgRNA/donor templates with AAVR via AAV8 vectors increased indel rate over 2-fold and HR rate over 15-fold for the correction of the single mutation in PahR408W mice. Moreover, AAVR co-injection successfully increased the site-specific insertion rate of a 1.4 kb Pah cDNA by 11-fold, bringing the HR rate up to 7.3% without detectable global off-target effects. Insertion of Pah cDNA significantly decreased the Phe level and ameliorated PKU symptoms. This study demonstrates a novel strategy to dramatically increase AAV transduction which substantially enhanced in vivo genome editing efficiency in adult animal models, showing clinical potential for both conventional and genome editing-based gene therapy.

Generation and characterization of a novel rat model of primary hyperoxaluria type 1 with a nonsense mutation in alanine-glyoxylate aminotransferase gene.

Primary hyperoxaluria type 1 (PH1) is a severe inherited disorder caused by a genetic defect in alanine-glyoxylate aminotransferase (AGXT), which results in recurrent urolithiasis and renal failure. Animal models that precisely reflect human PH1 phenotypes are lacking. We aimed to develop a novel PH1 rat model and study the mechanisms involved in PH1 deterioration. One cell stage Sprague-Dawley embryos were injected with the CRISPR/Cas9 system to introduce a Q84X mutation in Agxt. Liver tissues were harvested to determine Agxt expression. Urine oxalate, crystals, and electrolyte levels in AgxtQ84X and wild-type (WT) littermates were evaluated. Kidney tissues were used for Pizzolato staining and kidney injury evaluation. Data showed that Agxt mRNA and protein were absent in AgxtQ84X rats. At 4 and 24 wk, AgxtQ84X rats displayed 2.1- and 2.9-fold higher urinary oxalate levels, respectively, compared with WT littermates. As a result, calcium oxalate (CaOx) crystals in urine were revealed in all AgxtQ84X rats but in none of the WT rats. We also observed bladder stones in 36.4% of AgxtQ84X rats, of which 44.4% had renal CaOx deposition. Moreover, the elevated serum urea and creatinine levels indicated the impaired renal function in AgxtQ84X rats. Further investigation revealed significantly increased expression of inflammation-, necroptosis-, and fibrosis-related genes in the kidneys of AgxtQ84X rats with spontaneous renal CaOx deposition, indicating that these pathways are involved in PH1 deterioration. Collectively, these results suggest that this rat model has broad applicability in mechanistic studies and innovative therapeutics development for PH1 and other kidney stone diseases.NEW & NOTEWORTHY Primary hyperoxaluria type 1 is a severe inherited disorder that results in recurrent urolithiasis and renal failure. We generated an alanine-glyoxylate aminotransferase (Agxt)Q84X nonsense mutant rat model that displayed an early onset of hyperoxaluria, spontaneous renal CaOx precipitation, bladder stone, and kidney injuries. Our results suggest an interaction of renal CaOx crystals with the activation of inflammation-, fibrosis-, and necroptosis-related pathways. In all, the AgxtQ84X rat strain has broad applicability in mechanistic studies and the development of innovative therapeutics.

Integration of exome sequencing and metabolic evaluation for the diagnosis of children with urolithiasis.

PURPOSE: To investigate the prevalence of inherited causes in an early onset urolithiasis cohort and each metabolic subgroup. METHODS: A retrospective analysis of both metabolic and genomic data was performed for the first 105 pediatric urolithiasis patients who underwent exome sequencing at our hospital from February 2016 to October 2018. Measurements included the diagnostic yield of exome sequencing in the entire cohort and each metabolic subgroup (hyperoxaluria, hypocitraturia, hypercalciuria, hyperuricosuria and cystine stone subgroups). The conformity between molecular diagnoses and metabolic evaluation was also evaluated. RESULTS: The present study involved a cohort of 105 pediatric patients with urolithiasis, from which diagnostic variants were identified in 38 patients (36%), including 27 primary hyperoxaluria and 11 cystinuria. In the metabolic subgroup analyses, 41% hyperoxaluria cases were primary hyperoxaluria caused by monogenic defects, and 100% of the causes of cystine stones could be explained by monogenic defects. However, no appropriate inherited causes were identified for hypocitraturia, hypercalciuria, or hyperuricosuria in the cohort. A high conformity (100%) was obtained between the molecular diagnoses and metabolic evaluation. CONCLUSION: Exome sequencing in a cohort of 105 pediatric patients with urolithiasis yielded a genetic diagnosis in 36% of cases and the molecular diagnostic yield varies substantially across different metabolic abnormalities.

Knockdown of lactate dehydrogenase by adeno-associated virus-delivered CRISPR/Cas9 system alleviates primary hyperoxaluria type 1.

BACKGROUND: Primary hyperoxaluria type 1 (PH1) is a rare genetic disorder caused by endogenous overproduction of hepatic oxalate, leading to hyperoxaluria, recurrent calcium oxalate kidney stones, and end-stage renal disease. Lactate dehydrogenase (LDH) is an ideal target for diminishing oxalate production as it is responsible for glyoxylate to oxalate conversion in the liver, the last step of oxalate metabolism. Here, we investigated the therapeutic efficacy and potential side effects of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology to ameliorate PH1 via specifically disrupting the hepatic LDH. METHODS: Pheochromocytoma (PC12) cells were used to assess the efficacy of cleavage of single-guide RNAs in vitro. PH1 neonatal rats were injected with a single administration of adeno-associated virus to deliver the CRISPR/Cas9 system that targeted LDH. Three weeks after injection, a liver biopsy was performed to detect LDH expression, liver injury, and liver metabolomics. Urinary oxalate was regularly monitored, and renal calcium oxalate deposition was evaluated after 4 weeks of 0.5% ethylene glycol challenge. After 6 months of treatment, animals were euthanized, and ex-liver organs were harvested for toxicity analysis. RESULTS: The Ldha gene was specifically knocked out in 20% of the liver cells of PH1 rats in the treatment group, leading to a 50% lower LDH expression than that in the control group. Compared to the control groups, urinary oxalate levels were significantly decreased, and renal calcium oxalate precipitation was largely mitigated in the treatment group throughout the entire 6-month study period. While no CRISPR/Cas9-associated off-target edits or hepatotoxicity were detected, we observed mild metabolic changes in the liver tricarboxylic acid (TCA) and glycolysis pathways. CONCLUSIONS: CRISPR/Cas9-mediated LDH disruption may represent an applicable new strategy for alleviating PH1 for its long-lasting effect and low editorial efficiency requirements.

Corrigendum: The Application of External Ureteral Catheters in Children With Acute Kidney Injury Caused by Ceftriaxone-Induced Urolithiasis.

[This corrects the article DOI: 10.3389/fped.2020.00200.].

Quantitative Proteome of Infant Stenotic Ureters Reveals Extracellular Matrix Organization and Oxidative Stress Dysregulation Underlying Ureteropelvic Junction Obstruction.

PURPOSE: Ureteropelvic junction obstruction (UPJO) is the most frequent cause of congenital hydronephrosis in child. To better investigate the molecular mechanisms of this pathological process, the stenotic ureter proteome of UPJO in infants is compared with their own normal pre-stenotic segments. EXPERIMENTAL DESIGN: Data independent acquisition-based proteomics are performed to compare proteome between pre-stenotic and stenotic ureter from nine UPJO infants. Gene ontology analysis, hierarchical cluster analysis, and network interaction are performed to characterize biological functions of significantly altered proteins. Selected significantly altered proteins are validated by western blot on another three UPJO infants. RESULTS: 15 proteins are up-regulated and 33 proteins are down-regulated during stenotic pathology. Significantly altered proteins are involved in decreased extracellular matrix and cytoskeleton organization, increased regulation of oxidative activity, and altered inflammatory associated exocytosis. Significant expression of biglycan, fibulin-1, myosin-10, cytochrome b5 are validated providing possible mechanism in UPJO which could be associated impaired smooth muscle cell, epithelial integrity, and increased oxidative stress. CONCLUSIONS AND CLINICAL RELEVANCE: This study provides molecular evidence of dysregulated extracellular matrix organization, impaired smooth muscle cell, and oxidative stress during UPJO pathology, indicating that biglycan, fibulin-1, myosin-10, cytochrome b5 might reflect the pathology of UPJO.

Dual base editor catalyzes both cytosine and adenine base conversions in human cells.

Although base editors are useful tools for precise genome editing, current base editors can only convert either adenines or cytosines. We developed a dual adenine and cytosine base editor (A&C-BEmax) by fusing both deaminases with a Cas9 nickase to achieve C-to-T and A-to-G conversions at the same target site. Compared to single base editors, A&C-BEmax's activity on adenines is slightly reduced, whereas activity on cytosines is higher and RNA off-target activity is substantially decreased.

Urethral Triplication With Diverticulum Malformation: A Case Report and Literature Review.

A 3-year-old boy presented to our pediatric urology with a history of urine flow under the scrotum when voiding in a squatting position but not when standing. And the ventral side of the front penis became enlarged during urination and dribbled afterward. Physical examination revealed the boy had 2 urethras opening at the tip of glans, and another accessory urethra opening at perineum. Rigid cystoscopy and voiding cystourethrography confirmed it to be a urethral triplication malformation. This condition, the combination of urethral diverticulum and urethral triplication, consisting of urethro-perineum fistula, has not been previously reported.

CRISPR/Cas9-mediated metabolic pathway reprogramming in a novel humanized rat model ameliorates primary hyperoxaluria type 1.

Primary hyperoxaluria type I is caused by mutations in the alanine glyoxylate aminotransferase gene (AGXT), leading to accumulation of glyoxylate and subsequent production of oxalate and urolithiasis. Here, we generated a novel rat model of primary hyperoxaluria type I that carries a D205N mutation in the partially humanized Agxt gene through the CRISPR/Cas9 system. The AgxtD205N mutant rats showed undetectable alanine glyoxylate aminotransferase protein expression, developed hyperoxaluria at 1 month of age and exhibited severe renal calcium oxalate deposition after ethylene glycol challenge. This suggests our novel model is more relevant to the human disease than existing animal models. To test whether this model could be used for the development of innovative therapeutics, SaCas9 targeting hydroxyacid oxidase 1, responsible for metabolizing glycolate into glyoxylate, was delivered via adeno-associated viral vectors into newborn rats with primary hyperoxaluria type 1. This approach generated nearly 30% indels in the Hao1 gene in the liver, leading to 42% lower urine oxalate levels in the treated group than in the control group and preventing the rats with primary hyperoxaluria type 1 from undergoing severe nephrocalcinosis for at least 12 months. Thus, our results demonstrate that this partially humanized AgxtD205N rat strain is a high-performing model of primary hyperoxaluria type 1 for understanding pathology, and the development of novel therapeutics, such as reprogramming of the metabolic pathway through genome editing.

The Application of External Ureteral Catheters in Children With Acute Kidney Injury Caused by Ceftriaxone-Induced Urolithiasis.

Objective: To evaluate our use of external ureteral catheters in children with acute kidney injury (AKI) resulting from ceftriaxone-induced urolithiasis. Methods: From July 2010 to June 2015, a series of 15 children, including 12 males and 3 females, were referred to our department. All of them were diagnosed of post-renal AKI and underwent emergent hospitalization. Evaluation of serum electrolytes, creatinine (Cr), blood urea nitrogen (BUN), complete blood count, and blood gas analysis were completed in each child both before they were admitted, and again after surgery. Bilateral externalized ureteral catheters were placed cystoscopically in each of these patients. The composition of collected calculi was analyzed by infrared spectrography. Results: Bilateral externalized ureteral catheters were placed successfully in all patients. There were no procedure-related complications. Two days after catheter placement, the levels of serum Cr and BUN had improved in all patients, and these levels were noted to be significantly lower than before catheterization (P < 0.001). Infrared spectrography demonstrated that the primary composition of all calculi collected was ceftriaxone. No recurrent AKI or renal deterioration was detected during the follow-up which ranged from 3 to 8 years. Conclusions: These results show that short-term external ureteral catheters can be effectively utilized in children with AKI caused by ceftriaxone-induced urolithiasis. We recommend this procedure as a viable replacement to indwelling stents in patients with bilateral ureteral stones.

Amelioration of an Inherited Metabolic Liver Disease through Creation of a De Novo Start Codon by Cytidine Base Editing.

Base editing technology efficiently generates nucleotide conversions without inducing excessive double-strand breaks (DSBs), which makes it a promising approach for genetic disease therapy. In this study, we generated a novel hereditary tyrosinemia type 1 (HT1) mouse model, which contains a start codon mutation in the fumarylacetoacetate hydrolase (Fah) gene by using an adenine base editor (ABE7.10). To investigate the feasibility of base editing for recombinant adeno-associated virus (rAAV)-mediated gene therapy, an intein-split cytosine base editor (BE4max) was developed. BE4max efficiently induced C-to-T conversion and restored the start codon to ameliorate HT1 in mice, but an undesired bystander mutation abolished the effect of on-target editing. To solve this problem, an upstream sequence was targeted to generate a de novo in-frame start codon to initiate the translation of FAH. After treatment, almost all C-to-T conversions created a start codon and restored Fah expression, which efficiently ameliorated the disease without inducing off-target mutations. Our study demonstrated that base editing-mediated creation of de novo functional elements would be an applicable new strategy for genetic disease therapy.