Efficient expansion and CRISPR-Cas9-mediated gene correction of patient-derived hepatocytes for treatment of inherited liver diseases.

Cell-based ex vivo gene therapy in solid organs, especially the liver, has proven technically challenging. Here, we report a feasible strategy for the clinical application of hepatocyte therapy. We first generated high-quality autologous hepatocytes through the large-scale expansion of patient-derived hepatocytes. Moreover, the proliferating patient-derived hepatocytes, together with the AAV2.7m8 variant identified through screening, enabled CRISPR-Cas9-mediated targeted integration efficiently, achieving functional correction of pathogenic mutations in FAH or OTC. Importantly, these edited hepatocytes repopulated the injured mouse liver at high repopulation levels and underwent maturation, successfully treating mice with tyrosinemia following transplantation. Our study combines ex vivo large-scale cell expansion and gene editing in patient-derived transplantable hepatocytes, which holds potential for treating human liver diseases.

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.

Adenine transversion editors enable precise, efficient A•T-to-C•G base editing in mammalian cells and embryos.

Base editors have substantial promise in basic research and as therapeutic agents for the correction of pathogenic mutations. The development of adenine transversion editors has posed a particular challenge. Here we report a class of base editors that enable efficient adenine transversion, including precise A•T-to-C•G editing. We found that a fusion of mouse alkyladenine DNA glycosylase (mAAG) with nickase Cas9 and deaminase TadA-8e catalyzed adenosine transversion in specific sequence contexts. Laboratory evolution of mAAG significantly increased A-to-C/T conversion efficiency up to 73% and expanded the targeting scope. Further engineering yielded adenine-to-cytosine base editors (ACBEs), including a high-accuracy ACBE-Q variant, that precisely install A-to-C transversions with minimal Cas9-independent off-targeting effects. ACBEs mediated high-efficiency installation or correction of five pathogenic mutations in mouse embryos and human cell lines. Founder mice showed 44-56% average A-to-C edits and allelic frequencies of up to 100%. Adenosine transversion editors substantially expand the capabilities and possible applications of base editing technology.

Feedback activation of EGFR/wild-type RAS signaling axis limits KRASG12D inhibitor efficacy in KRASG12D-mutated colorectal cancer.

Colorectal cancer (CRC), which shows a high degree of heterogeneity, is the third most deadly cancer worldwide. Mutational activation of KRASG12D occurs in approximately 10-12% of CRC cases, but the susceptibility of KRASG12D-mutated CRC to the recently discovered KRASG12D inhibitor MRTX1133 has not been fully defined. Here, we report that MRTX1133 treatment caused reversible growth arrest in KRASG12D-mutated CRC cells, accompanied by partial reactivation of RAS effector signaling. Through a drug-anchored synthetic lethality screen, we discovered that epidermal growth factor receptor (EGFR) inhibition was synthetic lethal with MRTX1133. Mechanistically, MRTX1133 treatment downregulated the expression of ERBB receptor feedback inhibitor 1 (ERRFI1), a crucial negative regulator of EGFR, thereby causing EGFR feedback activation. Notably, wild-type isoforms of RAS, including H-RAS and N-RAS, but not oncogenic K-RAS, mediated signaling downstream of activated EGFR, leading to RAS effector signaling rebound and reduced MRTX1133 efficacy. Blockade of activated EGFR with clinically used antibodies or kinase inhibitors suppressed the EGFR/wild-type RAS signaling axis, sensitized MRTX1133 monotherapy, and caused the regression of KRASG12D-mutant CRC organoids and cell line-derived xenografts. Overall, this study uncovers feedback activation of EGFR as a prominent molecular event that restricts KRASG12D inhibitor efficacy and establishes a potential combination therapy consisting of KRASG12D and EGFR inhibitors for patients with KRASG12D-mutated CRC.

Exosomes Derived From Kartogenin-Preconditioned Mesenchymal Stem Cells Promote Cartilage Formation and Collagen Maturation for Enthesis Regeneration in a Rat Model of Chronic Rotator Cuff Tear.

BACKGROUND: Poor tendon-to-bone healing in chronic rotator cuff tears (RCTs) is related to unsatisfactory outcomes. Exosomes derived from mesenchymal stem cells reportedly enhance rotator cuff healing. However, the difficulty in producing exosomes with a stronger effect on enthesis regeneration must be resolved. PURPOSE: To study the effect of exosomes derived from kartogenin (KGN)-preconditioned human bone marrow mesenchymal stem cells (KGN-Exos) on tendon-to-bone healing in a rat model of chronic RCT. STUDY DESIGN: Controlled laboratory study. METHODS: Exosome-loaded sodium alginate hydrogel (SAH) was prepared. Moreover, exosomes were labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR) or 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (Dil) for in vivo tracking. Bilateral rotator cuff repair (RCR) was conducted in an established chronic RCT rat model. A total of 66 rats were randomized to control, untreated exosome (un-Exos), and KGN-Exos groups to receive local injections of pure SAH, un-Exos, or KGN-Exos SAH at the repaired site. The presence of DiR/Dil-labeled exosomes was assessed at 1 day and 1 week, and tendon-to-bone healing was evaluated histologically, immunohistochemically, and biomechanically at 4 and 8 weeks. RESULTS: Both un-Exos and KGN-Exos exhibited sustained release from SAH for up to 96 hours. In vivo study revealed that un-Exos and KGN-Exos were localized to the repaired site at 1 week. Moreover, the KGN-Exos group showed a higher histological score and increased glycosaminoglycan and collagen II expression at 4 and 8 weeks. In addition, more mature and better-organized collagen fibers with higher ratios of collagen I to collagen III were observed at 8 weeks in the tendon-to-bone interface compared with those in the control and un-Exos groups. Biomechanically, the KGN-Exos group had the highest failure load (28.12 ± 2.40 N) and stiffness (28.57 ± 2.49 N/mm) among the 3 groups at 8 weeks. CONCLUSION: Local injection of SAH with sustained KGN-Exos release could effectively promote cartilage formation as well as collagen maturation and organization for enthesis regeneration, contributing to enhanced biomechanical properties after RCR. CLINICAL RELEVANCE: KGN-Exos injection may be used as a cell-free therapeutic option to accelerate tendon-to-bone healing in chronic RCT.

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.

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.

Clinical and Radiological Outcomes in Patients With Anterior Shoulder Instability and Glenoid Bone Loss after Arthroscopic Free Bone Block Combined With Dynamic Anterior Stabilization.

BACKGROUND: As an alternative to the Latarjet procedure, the arthroscopic free bone block (FBB) procedure combined with dynamic anterior stabilization (DAS) has been recently proposed to provide both glenoid augmentation and a tendon sling effect for treating anterior shoulder instability (ASI) with glenoid bone loss. PURPOSE: To evaluate the clinical and radiological outcomes of FBB-DAS for ASI with glenoid bone loss. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Patients who underwent arthroscopic FBB-DAS for ASI with >15% glenoid bone loss between February 2017 and March 2020 were screened and enrolled in this study. Clinical outcome measures were assessed preoperatively and at a minimum 2-year follow-up, including recurrence, complications, shoulder functional scores, range of motion, and return to sports. Postoperative computed tomography and magnetic resonance imaging were also performed. RESULTS: Of a total of 65 patients with a mean follow-up of 46.1 ± 13.1 months, no patients experienced a recurrent dislocation or subluxation postoperatively, while 2 had a positive anterior apprehension test (3.1%). Additionally, 2 patients (3.1%) experienced complications of hematoma and shoulder stiffness, respectively. The mean visual analog scale score, American Shoulder and Elbow Surgeons score, Rowe score, and Oxford Shoulder Instability Score all improved significantly from 3.2 ± 2.4, 75.0 ± 18.9, 43.6 ± 27.3, and 33.8 ± 9.0 preoperatively to 1.3 ± 0.8, 95.1 ± 8.0, 95.5 ± 7.8, and 14.8 ± 3.5 at final follow-up, respectively (all P < .001). No difference was detected in range of motion except for 8.1° and 7.5° external rotation limitations in adduction and abduction, respectively. There were 62 patients (95.4%) who returned to sports, and 54 patients (83.1%) returned to the preinjury level. The transferred biceps tendon was intact in all 59 patients who completed radiological examination at the latest follow-up. Good bone healing was achieved in 98.3% of patients, and the glenoid bone defect decreased from 18.1% to 4.9%. Osseous and labral glenoids were significantly enlarged in width and depth on the latest magnetic resonance imaging (all P < .001). CONCLUSION: Arthroscopic FBB-DAS provided satisfactory clinical and radiological outcomes for ASI with glenoid bone loss. Despite slight external rotation restrictions, it achieved low recurrence and complication rates, excellent shoulder functional scores, a high return-to-sports rate, and favorable graft healing and remodeling.

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.

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.

Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing.

Cytosine base editors (CBEs) efficiently generate precise C·G-to-T·A base conversions, but the activation-induced cytidine deaminase/apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (AID/APOBEC) protein family deaminase component induces considerable off-target effects and indels. To explore unnatural cytosine deaminases, we repurpose the adenine deaminase TadA-8e for cytosine conversion. The introduction of an N46L variant in TadA-8e eliminates its adenine deaminase activity and results in a TadA-8e-derived C-to-G base editor (Td-CGBE) capable of highly efficient and precise C·G-to-G·C editing. Through fusion with uracil glycosylase inhibitors and further introduction of additional variants, a series of Td-CBEs was obtained either with a high activity similar to that of BE4max or with higher precision compared to other reported accurate CBEs. Td-CGBE/Td-CBEs show very low indel effects and a background level of Cas9-dependent or Cas9-independent DNA/RNA off-target editing. Moreover, Td-CGBE/Td-CBEs are more efficient in generating accurate edits in homopolymeric cytosine sites in cells or mouse embryos, suggesting their accuracy and safety for gene therapy and other applications.

CRISPR-Cas9-mediated gene editing of the BCL11A enhancer for pediatric ß0/ß0 transfusion-dependent ß-thalassemia.

Gene editing to disrupt the GATA1-binding site at the +58 BCL11A erythroid enhancer could induce γ-globin expression, which is a promising therapeutic strategy to alleviate ß-hemoglobinopathy caused by HBB gene mutation. In the present study, we report the preliminary results of an ongoing phase 1/2 trial (NCT04211480) evaluating safety and efficacy of gene editing therapy in children with blood transfusion-dependent ß-thalassemia (TDT). We transplanted BCL11A enhancer-edited, autologous, hematopoietic stem and progenitor cells into two children, one carrying the ß0/ß0 genotype, classified as the most severe type of TDT. Primary endpoints included engraftment, overall survival and incidence of adverse events (AEs). Both patients were clinically well with multilineage engraftment, and all AEs to date were considered unrelated to gene editing and resolved after treatment. Secondary endpoints included achieving transfusion independence, editing rate in bone marrow cells and change in hemoglobin (Hb) concentration. Both patients achieved transfusion independence for >18 months after treatment, and their Hb increased from 8.2 and 10.8 g dl-1 at screening to 15.0 and 14.0 g dl-1 at the last visit, respectively, with 85.46% and 89.48% editing persistence in bone marrow cells. Exploratory analysis of single-cell transcriptome and indel patterns in edited peripheral blood mononuclear cells showed no notable side effects of the therapy.

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.

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.

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.

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.

Gain-of-function variants in SYK cause immune dysregulation and systemic inflammation in humans and mice.

Spleen tyrosine kinase (SYK) is a critical immune signaling molecule and therapeutic target. We identified damaging monoallelic SYK variants in six patients with immune deficiency, multi-organ inflammatory disease such as colitis, arthritis and dermatitis, and diffuse large B cell lymphomas. The SYK variants increased phosphorylation and enhanced downstream signaling, indicating gain of function. A knock-in (SYK-Ser544Tyr) mouse model of a patient variant (p.Ser550Tyr) recapitulated aspects of the human disease that could be partially treated with a SYK inhibitor or transplantation of bone marrow from wild-type mice. Our studies demonstrate that SYK gain-of-function variants result in a potentially treatable form of inflammatory disease.

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.

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.

Tissue-Engineered Decellularized Allografts for Anterior Cruciate Ligament Reconstruction.

Anterior cruciate ligament (ACL) reconstruction with allografts is limited by high immunogenicity, poor cellularization, and delayed tendon-bone healing. Decellularized tendons (DAs) have been used as bioscaffolds to reconstruct ligaments with variable success. In the study, four kinds of decellularized allogeneic hamstring tendons were prepared and their microstructure and cytocompatibility were examined in vitro. The results showed that decellularized allografts neutralized by 5% calcium bicarbonate had typical reticular and porous microstructures with optical cytocompatibility. Tissue-engineering decellularized allografts (TEDAs) were prepared with the selected decellularized allografts and tendon stem/progenitor cells and used for ACL reconstruction in a rabbit model. Histological staining showed that the TEDAs promoted cellular infiltration and new vessel formation significantly and improved tendon-bone healing moderately compared to decellularized allografts. Better macroscopic scores and biomechanical results were observed in TEDA groups, but there were no significant differences between DA and TEDA groups at months 1, 2, and 3 postoperatively. Immunohistochemical data showed that the tissue-engineering decellularized allografts enhanced the expression of collagen I at each timepoint and collagen III at months 1 and 2. ELISA analysis showed that the tissue-engineering decellularized allografts reduced the secretion of IgE and IL-1ß within 1 month and promoted the secretion of IL-2, IL-4, IL-10, and IL-17 after 1 month. The results showed that tissue-engineering decellularized allografts strengthened intra-articular graft remodeling significantly and provided moderate improvements in tendon-bone healing by creating more suitable immune responses than decellularized allografts. The study revealed that tissue-engineering decellularized allografts as a promising option for ACL reconstruction could achieve more favorable outcomes.