Engineering IscB to develop highly efficient miniature editing tools in mammalian cells and embryos.

The IscB proteins, as the ancestors of Cas9 endonuclease, hold great promise due to their small size and potential for diverse genome editing. However, their activity in mammalian cells is unsatisfactory. By introducing three residual substitutions in IscB, we observed an average 7.5-fold increase in activity. Through fusing a sequence-non-specific DNA-binding protein domain, the eIscB-D variant achieved higher editing efficiency, with a maximum of 91.3%. Moreover, engineered ωRNA was generated with a 20% reduction in length and slightly increased efficiency. The engineered eIscB-D/eωRNA system showed an average 20.2-fold increase in activity compared with the original IscB. Furthermore, we successfully adapted eIscB-D for highly efficient cytosine and adenine base editing. Notably, eIscB-D is highly active in mouse cell lines and embryos, enabling the efficient generation of disease models through mRNA/ωRNA injection. Our study suggests that these miniature genome-editing tools have great potential for diverse applications.

Engineering APOBEC3A deaminase for highly accurate and efficient base editing.

Cytosine base editors (CBEs) are effective tools for introducing C-to-T base conversions, but their clinical applications are limited by off-target and bystander effects. Through structure-guided engineering of human APOBEC3A (A3A) deaminase, we developed highly accurate A3A-CBE (haA3A-CBE) variants that efficiently generate C-to-T conversion with a narrow editing window and near-background level of DNA and RNA off-target activity, irrespective of methylation status and sequence context. The engineered deaminase domains are compatible with PAM-relaxed SpCas9-NG variant, enabling accurate correction of pathogenic mutations in homopolymeric cytosine sites through flexible positioning of the single-guide RNAs. Dual adeno-associated virus delivery of one haA3A-CBE variant to a mouse model of tyrosinemia induced up to 58.1% editing in liver tissues with minimal bystander editing, which was further reduced through single dose of lipid nanoparticle-based messenger RNA delivery of haA3A-CBEs. These results highlight the tremendous promise of haA3A-CBEs for precise genome editing to treat human diseases.

Engineering a precise adenine base editor with minimal bystander editing.

Adenine base editors (ABEs) catalyze A-to-G transitions showing broad applications, but their bystander mutations and off-target editing effects raise safety concerns. Through structure-guided engineering, we found ABE8e with an N108Q mutation reduced both adenine and cytosine bystander editing, and introduction of an additional L145T mutation (ABE9), further refined the editing window to 1-2 nucleotides with eliminated cytosine editing. Importantly, ABE9 induced very minimal RNA and undetectable Cas9-independent DNA off-target effects, which mainly installed desired single A-to-G conversion in mouse and rat embryos to efficiently generate disease models. Moreover, ABE9 accurately edited the A5 position of the protospacer sequence in pathogenic homopolymeric adenosine sites (up to 342.5-fold precision over ABE8e) and was further confirmed through a library of guide RNA-target sequence pairs. Owing to the minimized editing window, ABE9 could further broaden the targeting scope for precise correction of pathogenic single-nucleotide variants when fused to Cas9 variants with expanded protospacer adjacent motif compatibility. bpNLS, bipartite nuclear localization signals.

Epigenetic inactivation of ERF reactivates γ-globin expression in ß-thalassemia.

The fetal-to-adult hemoglobin switch is regulated in a developmental stage-specific manner and reactivation of fetal hemoglobin (HbF) has therapeutic implications for treatment of ß-thalassemia and sickle cell anemia, two major global health problems. Although significant progress has been made in our understanding of the molecular mechanism of the fetal-to-adult hemoglobin switch, the mechanism of epigenetic regulation of HbF silencing remains to be fully defined. Here, we performed whole-genome bisulfite sequencing and RNA sequencing analysis of the bone marrow-derived GYPA+ erythroid cells from ß-thalassemia-affected individuals with widely varying levels of HbF groups (HbF ≥ 95th percentile or HbF ≤ 5th percentile) to screen epigenetic modulators of HbF and phenotypic diversity of ß-thalassemia. We identified an ETS2 repressor factor encoded by ERF, whose promoter hypermethylation and mRNA downregulation are associated with high HbF levels in ß-thalassemia. We further observed that hypermethylation of the ERF promoter mediated by enrichment of DNMT3A leads to demethylation of γ-globin genes and attenuation of binding of ERF on the HBG promoter and eventually re-activation of HbF in ß-thalassemia. We demonstrated that ERF depletion markedly increased HbF production in human CD34+ erythroid progenitor cells, HUDEP-2 cell lines, and transplanted NCG-Kit-V831M mice. ERF represses γ-globin expression by directly binding to two consensus motifs regulating γ-globin gene expression. Importantly, ERF depletion did not affect maturation of erythroid cells. Identification of alterations in DNA methylation of ERF as a modulator of HbF synthesis opens up therapeutic targets for ß-hemoglobinopathies.

Sacubitril/valsartan inhibits the proliferation of vascular smooth muscle cells through notch signaling and ERK1/2 pathway.

AIMS: To explore the role and mechanism of Notch signaling and ERK1/2 pathway in the inhibitory effect of sacubitril/valsartan on the proliferation of vascular smooth muscle cells (VSMCs). MAIN METHODS: Human aortic vascular smooth muscle cells (HA-VSMCs) were cultured in vitro. The proliferating VSMCs were divided into three groups as control group, Ang II group and Ang II + sacubitril/valsartan group. Cell proliferation and migration were detected by CCK8 and scratch test respectively. The mRNA and protein expression of PCNA, MMP-9, Notch1 and Jagged-1 were detected by qRT-PCR and Western blot respectively. The p-ERK1/2 expression was detected by Western blot. KEY FINDINGS: Compared with the control group, proliferation and migration of VSMCs and the expression of PCNA, MMP-9, Notch1, Jagged-1 and p-ERK1/2 was increased in Ang II group. Sacubitril/valsartan significantly reduced the proliferation and migration. Additionally, pretreatment with sacubitril/valsartan reduced the PCNA, MMP-9, Notch1, Jagged-1 and p-ERK1/2 expression.

Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies.

Editing efficiency is pivotal for the efficacies of CRISPR-based gene therapies. We found that fusing an HMG-D domain to the N terminus of SpCas9 (named efficiency-enhanced Cas9 [eeCas9]) significantly increased editing efficiency by 1.4-fold on average. The HMG-D domain also enhanced the activities of non-NGG PAM Cas9 variants, high-fidelity Cas9 variants, smaller Cas9 orthologs, Cas9-based epigenetic regulators, and base editors in cell lines. Furthermore, we discovered that eeCas9 exhibits comparable off-targeting effects with Cas9, and its specificity could be increased through ribonucleoprotein delivery or using hairpin single-guide RNAs and high-fidelity Cas9s. The entire eeCas9 could be packaged into an adeno-associated virus vector and exhibited a 1.7- to 2.6-fold increase in editing efficiency targeting the Pcsk9 gene in mice, leading to a greater reduction of serum cholesterol levels. Moreover, the efficiency of eeA3A-BE3 also surpasses that of A3A-BE3 in targeting the promoter region of γ-globin genes or BCL11A enhancer in human hematopoietic stem cells to reactivate γ-globin expression for the treatment of ß-hemoglobinopathy. Together, eeCas9 and its derivatives are promising editing tools that exhibit higher activity and therapeutic efficacy for both in vivo and ex vivo therapeutics.

A natural DNMT1 mutation elevates the fetal hemoglobin level via epigenetic derepression of the γ-globin gene in ß-thalassemia.

DNA methyltransferase 1 (DNMT1) is a major epigenetic regulator of the formation of large macromolecular complexes that repress human γ-globin expression by maintaining DNA methylation. However, very little is known about the association of DNMT1 variants with ß-thalassemia phenotypes. We systematically investigated associations between variants in DNMT1 and phenotypes in 1142 ß-thalassemia subjects and identified a novel missense mutation (c.2633G>A, S878F) in the DNMT1 bromo-adjacent homology-1 (BAH1) domain. We functionally characterized this mutation in CD34+ cells from patients and engineered HuDEP-2 mutant cells. Our results demonstrate that DNMT1 phosphorylation is abrogated by substituting serine with phenylalanine at position 878, resulting in lower stability and catalytic activity loss. S878F mutation also attenuated DNMT1 interactions with BCL11A, GATA1, and HDAC1/2, and reduced recruitment of DNMT1 to the γ-globin (HBG) promoters, leading to epigenetic derepression of γ-globin expression. By analyzing the F-cell pattern, we demonstrated that the effect of DNMT1 mutation on increased fetal hemoglobin (HbF) is heterocellular. Furthermore, introduction of S878F mutation into erythroid cells by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) recapitulated γ-globin reactivation. Thus, the natural S878F DNMT1 mutation is a novel modulator of HbF synthesis and represents a potential new therapeutic target for ß-hemoglobinopathies.

Efficient photoactivatable Dre recombinase for cell type-specific spatiotemporal control of genome engineering in the mouse.

Precise genetic engineering in specific cell types within an intact organism is intriguing yet challenging, especially in a spatiotemporal manner without the interference caused by chemical inducers. Here we engineered a photoactivatable Dre recombinase based on the identification of an optimal split site and demonstrated that it efficiently regulated transgene expression in mouse tissues spatiotemporally upon blue light illumination. Moreover, through a double-floxed inverted open reading frame strategy, we developed a Cre-activated light-inducible Dre (CALID) system. Taking advantage of well-defined cell-type-specific promoters or a well-established Cre transgenic mouse strain, we demonstrated that the CALID system was able to activate endogenous reporter expression for either bulk or sparse labeling of CaMKIIα-positive excitatory neurons and parvalbumin interneurons in the brain. This flexible and tunable system could be a powerful tool for the dissection and modulation of developmental and genetic complexity in a wide range of biological systems.

Arthroscopic Dynamic Anterior Stabilization Using Either Long Head of the Biceps or Conjoined Tendon Transfer for Anterior Shoulder Instability Results in a Similarly Low Recurrence Rate.

PURPOSE: To compare the clinical outcomes of arthroscopic dynamic anterior stabilization (DAS) between transferring the long head of the biceps (DAS-LHB) and the conjoined tendon (DAS-CT) for anterior shoulder instability with <15% glenoid bone loss. METHODS: From January 2016 to May 2019, a total of 63 patients who underwent DAS for recurrent anterior shoulder dislocation with <15% glenoid bone loss were included, comprising 33 patients in DAS-LHB group and 30 patients in DAS-CT group. Clinical outcomes were assessed preoperatively and at a minimum 3-year follow-up, including patient-reported outcomes, range of motion, and return to sports (RTS). Postoperative recurrent instability (including dislocation, subluxation, and subjective instability with a positive apprehension test), revisions and complications also were recorded. RESULTS: No significant demographic characteristics difference was detected between the DAS-LHB (26.3 ± 7.9 years) and DAS-CT groups (26.0 ± 6.7 years). At the latest follow-up, there were no significant differences between the 2 groups in functional scores: Oxford Shoulder Instability Score (14.8 ± 2.8 vs 15.2 ± 3.6), Rowe score (95.9 ± 6.5 vs 93.2 ± 10.2), visual analog scale for pain (0.8 ± 1.2 vs 0.7 ± 1.7), and American Shoulder and Elbow Surgeons (95 ± 8.8 vs 95.2 ± 9.1) (all P > .218). No significant difference was detected between groups in the rates of RTS (90.1% vs 86.7%, P = .700) and RTS at previous level (78.7% vs 73.3%, P = .258), respectively. No recurrent dislocation occurred in either group. One patient felt occasional subluxation in the DAS-LHB group, and one was positive for the apprehension test in each group. One patient presented with postoperative shoulder stiffness and underwent a secondary arthroscopic debridement in the DAS-CT group. CONCLUSIONS: Comparable rates of recurrence, complication, return to sports, and subjective shoulder function were observed between DAS-LHB and DAS-CT groups. LEVEL OF EVIDENCE: Level III, retrospective comparative therapeutic trial.

Rotator cuff tear reaching the superior half portion of the humeral head causes shoulder abduction malfunction.

PURPOSE: To examine the biomechanical properties governing posterosuperior rotator cuff (RC) tear progression and dynamic shoulder abduction function, in the absence of excess loading. METHODS: Twelve freshly frozen cadaveric shoulders were evaluated via an established dynamic shoulder abduction stimulator. The shoulder abduction functions were primarily evaluated using subacromial contact pressure (SACP) during an abduction procedure, and subsequent middle deltoid force (MDF) under 5 conditions: (1) intact, (2) anterior 1/3 posterosuperior rotator cuff (PSRC) tear, (3) anterior 2/3 PSRC tear, (4) entire PSRC tear, and (5) global RC tear (tear involving the entire superior RC). RESULTS: No obvious differences were observed in the peak MDF required for abduction, and in the peak SACP among the four PSRC tear statuses (49.8 ± 9.2 N, 0.39 ± 0.05 mPa [1/3 PSRC tear]; 49.3 ± 6.8 N, 0.40 ± 0.06 mPa [2/3 PSRC tear]; 51.6 ± 7.0 N, 0.44 ± 0.08 mPa [entire PSRC tear]), as well as intact statuses (48.3 ± 9.8 N, 0.40 ± 0.05 mPa). However, significant elevations in the peak MDF and peak SACP levels were observed among the four PSRC tear statuses and global RC tear (68.1 ± 9.3 N; 4.12 ± 1.50 mPa, P < 0.01). CONCLUSION: In the absence of excess loading, the biomechanical function of the shoulder was not impaired by a simple PSRC tear. However, once the tear size reached the half superior portion of the humeral head, the humeral head migrated to the surface of the subacromion, and this action markedly decreased shoulder abduction function.

Generation of genetically modified rat models via the CRISPR/Cas9 technology.

The RNA-guided CRISPR/Cas9 genomic editing system consists of a single guide RNA (sgRNA) and a Cas9 nuclease. The two components form a complex in cells and target the genomic loci complementary to the sgRNA. The Cas9 nuclease cleaves the target site creating a double stranded DNA break (DSB). In mammalian cells, DSBs are often repaired via error prone non-homologous end joining (NHEJ) or via homology directed repair (HDR) with the presence of donor DNA templates. Micro-injection of the CRISPR/Cas9 system into the rat embryos enables generation of genetically modified rat models. Here, we describe a detailed protocol for creating gene knockout or knockin rat models via the CRISPR/Cas9 technology.

Dynamic Double-Sling Augmentation Prevents Anteroinferior Translation for Recurrent Anteroinferior Shoulder Dislocation With 20% Glenoid Bone Loss: A Cadaveric Biomechanical Study.

PURPOSE: To biomechanically compare the dynamic double-sling with single-sling augmentation using the conjoined tendon (CT) with 20% of an anteroinferior glenoid bone defect under the high loads in shoulders. METHODS: With the shoulder in 60° of glenohumeral abduction and 60° of external rotation, the 12 shoulders stability was tested sequentially in 5 conditions: intact, 20% glenoid bone loss, Bankart repair, single-sling augmentation with the CT, and double-sling augmentation with both the CT and long head of the biceps tendon (LHBT). The anteroinferior humeral head (HH) translation force of 20N, 30N, 40N, 50N, or 60N was applied to determine the shoulder stability in each condition. RESULTS: The total HH translation over 8.77 mm represented the anteroinferior shoulder instability (95% confidence interval of bone defect: 7.76-8.77 mm). A significant increase in anteroinferior HH translation was demonstrated after the creation of 20% glenoid bone defect under the 20N translational force (10.52 ± 0.71 mm). Structural failure after the Bankart repair and the single-sling augmentation under the 30N (9.84 ± 1.25 mm) and 40N (9.59 ± 0.66 mm) translational forces, respectively, were observed. The double-sling augmentation effectively prevented the anteroinferior HH translation under the translational force of less than 40N, and only half of the augmentation structure (8.25 ± 1.66 mm) had failed under the 50N translational forces. CONCLUSION: In the absence of any Hill-Sachs lesion and when tested at 60° abduction and external rotation in shoulders with 20% glenoid bone defects, at time-zero, the double-sling augmentation strategy could effectively prevent anteroinferior translation when compared with the Bankart repair or the single-sling augmentation technique under all magnitudes of the translational force in biomechanical simulation. Nevertheless, none of the constructs restored the HH translation to the normal intact state. CLINICAL RELEVANCE: Double-sling augmentation technique may represent a reliable option for preventing anteroinferior translation.

Bankart Repair With Transferred Long Head of the Biceps Provides Better Biomechanical Effect Than Conjoined Tendon Transfer in Anterior Shoulder Instability With 20% Glenoid Defect.

PURPOSE: To examine the biomechanical differences between labral repair with transferred conjoined tendon and transferred long head of the biceps tendon (LHBT) for anterior shoulder instability with 20% bone loss. METHODS: Twelve cadaveric shoulders were tested in sequent 5 conditions: intact, 20% glenoid defect, Bankart repair, Bankart repair with transferred conjoined tendon (dynamic conjoined tendon sling, DCS), and with transferred LHBT (dynamic LHBT sling, DLS) at 60° of glenohumeral abduction and 60° of external rotation. The physiological glenohumeral joint load was created by forces applied to the rotator cuff, conjoined tendon, and LHBT. The glenohumeral compression force and range of motion were recorded before anteroinferior force application. The anterior, inferior, and total translations were measured with 20, 30, 40, and 50 N of anteroinferior force, respectively. RESULTS: Anteroinferior glenoid defect led to significant increase of humerus translation and decrease of glenohumeral compression force. DLS provided better resistance effect in both anterior-posterior and superior-inferior directions than DCS under high loading condition (40 N, P =.03; 50 N, P <.01). Both DCS and DLS procedures could further restore glenohumeral compression force with Bankart repair (Bankart repair: 32.1 ± 4.0 N; DCS: 36.7 ± 3.2 N, P < .01; DLS: 35.8 ± 3.6 N, P =.03). No range of motion restrictions were observed relative to the normal shoulder. CONCLUSIONS: Both the DLS and DCS techniques could reduce the anterior-inferior translation and partially restore the glenohumeral stability in anterior shoulder instability with 20% anteroinferior glenoid defect compared with Bankart repair. Under greater loading conditions, DLS provides better stability than DCS. CLINICAL RELEVANCE: Shoulder stability can be restored by DLS and DCS with low load. With greater shoulder stability requirements, DLS might be a better option than DCS for anterior shoulder instability with 20% bone loss.

GATA zinc finger domain-containing protein 2A (GATAD2A) deficiency reactivates fetal haemoglobin in patients with ß-thalassaemia through impaired formation of methyl-binding domain protein 2 (MBD2)-containing nucleosome remodelling and deacetylation (NuRD) complex.

Reactivation of fetal haemoglobin (HbF) expression is an effective way to treat ß-thalassaemia and sickle cell anaemia. In the present study, we identified a novel GATA zinc finger domain-containing protein 2A (GATAD2A) mutation, which contributed to the elevation of HbF and ameliorated clinical severity in a patient with ß-thalassaemia, by targeted next-generation sequencing. Knockout of GATAD2A led to a significant induction of HbF in both human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) and human cluster of differentiation (CD)34+ cells with a detectable impact on erythroid differentiation. Furthermore, heterozygous knockout of GATAD2A impaired recruitment of chromodomain helicase DNA-binding protein 4 (CHD4) to the methyl-binding domain protein 2 (MBD2)-containing nucleosome remodelling and deacetylation (NuRD) complex. Our present data suggest that mutations causing the haploinsufficiency of GATAD2A might contribute to amelioration of clinical severity in patients with ß-thalassaemia.

A novel series of benzothiazepine derivatives as tubulin polymerization inhibitors with anti-tumor potency.

In this work, a series of diaryl benzo[b][1,4]thiazepine derivatives D1-D36 were synthesized and screened as tubulin polymerization inhibitors with anti-tumor potency. They were designed by introducing the seven-member ring benzothiazepine as the linker for CA-4 modification for the first time. Among them, the hit compound D8 showed potential on inhibiting the growth of several cancer cell lines (IC50 values: 1.48 µM for HeLa, 1.47 µM for MCF-7, 1.52 µM for HT29 and 1.94 µM for A549), being comparable with the positive controls Colchicine and CA-4P. The calculated IC50 value of D8 as an tubulin polymerization inhibitor was 1.20 µM. The results of the flow cytometry assay revealed that D8 could induce the mitotic catastrophe and the death of living cancer cells. D8 also indicated the anti-vascular activity. The possible binding pattern was implied by docking simulation, inferring the possibility of introducing interactions with the nearby tubulin chain. Since the novel structural trial has been conducted with preliminary discussion, this work might stimulate new ideas in further modification of tubulin-related anti-cancer agents and therapeutic approaches.

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.

Incomplete Rotator Cable Did Not Cause Rotator Cuff Dysfunction in Case of Rotator Cuff Tear: A Biomechanical Study of the Relationship Between Rotator Cable Integrity and Rotator Cuff Function.

PURPOSE: This study seeks to evaluate the biomechanical relationship between the severity of rotator cable tears and the function of the rotator cuff. METHODS: Twelve cadaveric shoulders with intact rotator cuff, existing rotator cable, and a critical shoulder angle below 35° were included. For each shoulder, a posterosuperior rotator cuff tear (PSRCT) (model 2) in the crescent area was formed. Then anterior insertion detached (model 3), anterior insertion detached together with the middle cable tear (model 4), and the whole rotator cable tear (model 5) were subsequently created. The rotator cuff that lay above the humeral head rotation center was detached as a global tear control (model 6), along with the primitive status as the intact control (model 1). Glenohumeral abduction was initiated by simulating deltoid and remaining rotator cuff force. Functioning of the remaining rotator cuff was evaluated using the middle deltoid force (MDF), as required for abduction. RESULTS: No statistically significant differences in peak MDF values were seen among the 4 PSRCT statuses (44.10 ± 7.30 N [model 2], P = .96; 45.50 ± 9.55 N [model 3], P = .86; 45.90 ± 3.53 N [model 4], P = 0.30; 44.20 ± 8.19 N [model 5], P = .80) and intact control status (39.79 ± 7.65 N [model 1]). However, significant differences in peak MDF values were found among the 4 PSRCT statuses and the global tear control status (54.53 ± 7.46 N [model 6], P < .01). CONCLUSION: The PSRCT, regardless of the severity of the rotator cable tear, does not induce functionally significant biomechanical impairment. Tear extension involving all rotator cuff tissue above the geometric rotation center of the humeral head results in obvious functional impairment. CLINICAL RELEVANCE: For PSRCT, the remaining rotator cuff tissue above the geometric rotation center may contribute to the preservation of shoulder function in RCT patients.

Structure and function of Norrin in assembly and activation of a Frizzled 4-Lrp5/6 complex.

Norrin is a cysteine-rich growth factor that is required for angiogenesis in the eye, ear, brain, and female reproductive organs. It functions as an atypical Wnt ligand by specifically binding to the Frizzled 4 (Fz4) receptor. Here we report the crystal structure of Norrin, which reveals a unique dimeric structure with each monomer adopting a conserved cystine knot fold. Functional studies demonstrate that the novel Norrin dimer interface is required for Fz4 activation. Furthermore, we demonstrate that Norrin contains separate binding sites for Fz4 and for the Wnt ligand coreceptor Lrp5 (low-density lipoprotein-related protein 5) or Lrp6. Instead of inducing Fz4 dimerization, Norrin induces the formation of a ternary complex with Fz4 and Lrp5/6 by binding to their respective extracellular domains. These results provide crucial insights into the assembly and activation of the Norrin-Fz4-Lrp5/6 signaling complex.

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.