Crucial roles of the BRCA1-BARD1 E3 ubiquitin ligase activity in homology-directed DNA repair.

The tumor-suppressor breast cancer 1 (BRCA1) in complex with BRCA1-associated really interesting new gene (RING) domain 1 (BARD1) is a RING-type ubiquitin E3 ligase that modifies nucleosomal histone and other substrates. The importance of BRCA1-BARD1 E3 activity in tumor suppression remains highly controversial, mainly stemming from studying mutant ligase-deficient BRCA1-BARD1 species that we show here still retain significant ligase activity. Using full-length BRCA1-BARD1, we establish robust BRCA1-BARD1-mediated ubiquitylation with specificity, uncover multiple modes of activity modulation, and construct a truly ligase-null variant and a variant specifically impaired in targeting nucleosomal histones. Cells expressing either of these BRCA1-BARD1 separation-of-function alleles are hypersensitive to DNA-damaging agents. Furthermore, we demonstrate that BRCA1-BARD1 ligase is not only required for DNA resection during homology-directed repair (HDR) but also contributes to later stages for HDR completion. Altogether, our findings reveal crucial, previously unrecognized roles of BRCA1-BARD1 ligase activity in genome repair via HDR, settle prior controversies regarding BRCA1-BARD1 ligase functions, and catalyze new efforts to uncover substrates related to tumor suppression.

Functional analysis and clinical classification of 462 germline BRCA2 missense variants affecting the DNA binding domain.

Variants of uncertain significance (VUSs) in BRCA2 are a common result of hereditary cancer genetic testing. While more than 4,000 unique VUSs, comprised of missense or intronic variants, have been identified in BRCA2, the few missense variants now classified clinically as pathogenic or likely pathogenic are predominantly located in the region encoding the C-terminal DNA binding domain (DBD). We report on functional evaluation of the influence of 462 BRCA2 missense variants affecting the DBD on DNA repair activity of BRCA2 using a homology-directed DNA double-strand break repair assay. Of these, 137 were functionally abnormal, 313 were functionally normal, and 12 demonstrated intermediate function. Comparisons with other functional studies of BRCA2 missense variants yielded strong correlations. Sequence-based in silico prediction models had high sensitivity, but limited specificity, relative to the homology-directed repair assay. Combining the functional results with clinical and genetic data in an American College of Medical Genetics (ACMG)/Association for Molecular Pathology (AMP)-like variant classification framework from a clinical testing laboratory, after excluding known splicing variants and functionally intermediate variants, classified 431 of 442 (97.5%) missense variants (129 as pathogenic/likely pathogenic and 302 as benign/likely benign). Functionally abnormal variants classified as pathogenic by ACMG/AMP rules were associated with a slightly lower risk of breast cancer (odds ratio [OR] 5.15, 95% confidence interval [CI] 3.43-7.83) than BRCA2 DBD protein truncating variants (OR 8.56, 95% CI 6.03-12.36). Overall, functional studies of BRCA2 variants using validated assays substantially improved the variant classification yield from ACMG/AMP models and are expected to improve clinical management of many individuals found to harbor germline BRCA2 missense VUS.

Fast, precise and cloning-free knock-in of reporter sequences in vivo with high efficiency.

Targeted knock-in of fluorescent reporters enables powerful gene and protein analyses in a physiological context. However, precise integration of long sequences remains challenging in vivo. Here, we demonstrate cloning-free and precise reporter knock-in into zebrafish genes, using PCR-generated templates for homology-directed repair with short homology arms (PCR tagging). Our novel knock-in reporter lines of vesicle-associated membrane protein (vamp) zebrafish homologues reveal subcellular complexity in this protein family. Our approach enables fast and efficient reporter integration in the zebrafish genome (in 10-40% of injected embryos) and rapid generation of stable germline-transmitting lines.

Endogenous tagging of multiple cellular components in the sea anemone Nematostella vectensis.

The cnidarian Nematostella vectensis has developed into a powerful model system to study the mechanisms underlying animal development, regeneration, and evolution. However, despite the significant progress in the molecular and genetic approaches in this sea anemone, endogenous protein tagging is still challenging. Here, we report a robust method for knock in for Nematostella using CRISPR/Cas9. As an outcome, we generate endogenously tagged proteins that label core molecular components of several cellular apparatus, including the nuclear envelope, cytoskeleton, cell adhesion, endoplasmic reticulum, cell trafficking, and extracellular matrix. Using live imaging, we monitor the dynamics of vesicular trafficking and endoplasmic reticulum in embryos, as well as cell contractility during the peristaltic wave of a primary polyp. This advancement in gene editing expands the molecular tool kit of Nematostella and enables experimental avenues to interrogate the cell biology of cnidarians.

Structures of partition protein ParA with nonspecific DNA and ParB effector reveal molecular insights into principles governing Walker-box DNA segregation.

Walker-box partition systems are ubiquitous in nature and mediate the segregation of bacterial and archaeal DNA. Well-studied plasmid Walker-box partition modules require ParA, centromere-DNA, and a centromere-binding protein, ParB. In these systems, ParA-ATP binds nucleoid DNA and uses it as a substratum to deliver ParB-attached cargo DNA, and ParB drives ParA dynamics, allowing ParA progression along the nucleoid. How ParA-ATP binds nonspecific DNA and is regulated by ParB is unclear. Also under debate is whether ParA polymerizes on DNA to mediate segregation. Here we describe structures of key ParA segregation complexes. The ParA-ß,γ-imidoadenosine 5'-triphosphate (AMPPNP)-DNA structure revealed no polymers. Instead, ParA-AMPPNP dimerization creates a multifaceted DNA-binding surface, allowing it to preferentially bind high-density DNA regions (HDRs). DNA-bound ParA-AMPPNP adopts a dimer conformation distinct from the ATP sandwich dimer, optimized for DNA association. Our ParA-AMPPNP-ParB structure reveals that ParB binds at the ParA dimer interface, stabilizing the ATPase-competent ATP sandwich dimer, ultimately driving ParA DNA dissociation. Thus, the data indicate how harnessing a conformationally adaptive dimer can drive large-scale cargo movement without the requirement for polymers and suggest a segregation mechanism by which ParA-ATP dimers equilibrate to HDRs shown to be localized near cell poles of dividing chromosomes, thus mediating equipartition of attached ParB-DNA substrates.

CRISPR-Cas9 delivery strategies and applications: Review and update.

Nowadays, a significant part of the investigations carried out in the medical field belong to cancer treatment. Generally, conventional cancer treatments, including chemotherapy, radiotherapy, and surgery, which have been used for a long time, are not sufficient, especially in malignant cancers. Because genetic mutations cause cancers, researchers are trying to treat these diseases using genetic engineering tools. One of them is clustered regularly interspaced short palindromic repeats (CRISPR), a powerful tool in genetic engineering in the last decade. CRISPR, which forms the CRISPR-Cas structure with its endonuclease protein, Cas, is known as a part of the immune system (adaptive immunity) in bacteria and archaea. Among the types of Cas proteins, Cas9 endonuclease has been used in many scientific studies due to its high accuracy and efficiency. This review reviews the CRISPR system, focusing on the history, classification, delivery methods, applications, new generations, and challenges of CRISPR-Cas9 technology.

HDR syndrome: Large cohort and systematic review.

HDR syndrome is a rare disease characterized by hypoparathyroidism, deafness, and renal dysplasia. An autosomal dominant disease caused by heterozygous pathogenic GATA3 variants, the penetrance of each associated condition is variable. Literature reviews have provided some answers, but many questions remain, in particular what the relationship is between genotype and phenotype. The current study examines 28 patients with HDR syndrome combined with an exhaustive review of the literature. Some conditions such as hearing loss are almost always present, while others described as rare initially, do not seem to be so rare after all (genital malformations and basal ganglia calcifications). By modeling pathogenic GATA3 variants found in HDR syndrome, we found that missense variations appear to always be located in the same area (close to the two Zinc Finger domain). We describe new pathogenic GATA3 variants, of which some seem to always be associated with certain conditions. Many audiograms were studied to establish a typical audiometric profile associated with a phenotype in HDR. As mentioned in the literature, hearing function should always be assessed as early as possible and follow up of patients with HDR syndrome should include monitoring of parathyroid function and vesicoureteral reflux in order to prevent complications.

Radiotherapy in localized prostate cancer: a multicenter analysis evaluating tumor control and late toxicity after brachytherapy and external beam radiotherapy in 1293 patients.

BACKGROUND AND PURPOSE: Comparing oncological outcomes and toxicity after primary treatment of localized prostate cancer using HDR- or LDR-mono-brachytherapy (BT), or conventionally (CF) or moderately hypofractionated (HF) external beam radiotherapy. MATERIALS AND METHODS: Retrospectively, patients with low- (LR) or favorable intermediate-risk (IR) prostate cancer treated between 03/2000 and 09/2022 in two centers were included. Treatment was performed using either CF with total doses between 74 and 78 Gy, HF with 2.4-2.6 Gy per fraction in 30 fractions, or LDR- or HDR-BT. Biochemical control (BC) according to the Phoenix criteria, and late gastrointestinal (GI), and genitourinary (GU) toxicity according to RTOG/EORTC criteria were assessed. RESULTS: We identified 1293 patients, 697 with LR and 596 with IR prostate cancer. Of these, 470, 182, 480, and 161 were treated with CF, HF, LDR-BT, and HDR-BT, respectively. For BC, we did not find a significant difference between treatments in LR and IR (p = 0.31 and 0.72). The 5­year BC for LR was between 93 and 95% for all treatment types. For IR, BC was between 88% in the CF and 94% in the HF group. For CF and HF, maximum GI and GU toxicity grade ≥ 2 was between 22 and 27%. For LDR-BT, we observed 67% grade ≥ 2 GU toxicity. Maximum GI grade ≥ 2 toxicity was 9%. For HDR-BT, we observed 1% GI grade ≥ 2 toxicity and 19% GU grade ≥ 2 toxicity. CONCLUSION: All types of therapy were effective and well received. HDR-BT caused the least late toxicities, especially GI.

Application of CRISPR-Cas9 technology in diabetes research.

Diabetes is a chronic disorder with rapidly increasing prevalence that is a major global issue of our current era. There are two major types of diabetes. Polygenic forms of diabetes include type 1 diabetes (T1D) and type 2 diabetes (T2D) and its monogenic forms are maturity-onset diabetes of the young (MODY) and neonatal diabetes mellitus (NDM). There are no permanent therapeutic approaches for diabetes and current therapies rely on regular administration of various drugs or insulin injection. Recently, gene editing strategies have offered new promise for treating genetic disorders. Targeted genome editing is a fast-growing technology, recruiting programmable nucleases to specifically modify target genomic sequences. These targeted nucleases generate double-strand breaks at target regions in the genome, which induce cellular repair pathways including non-homologous end joining (NHEJ) and homology-directed repair (HDR). Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a novel gene-editing system, permitting precise genome modification. CRISPR/Cas9 has great potential for various applications in diabetic research such as gene screening, generation of diabetic animal models and treatment. In this article, gene-editing strategies are summarized with a focus on the CRISPR/Cas9 approach in diabetes research.

Dose and fractionation regimen for brachytherapy boost in cervical cancer in the US.

PURPOSE: Curative-intent radiotherapy for locally advanced and select early stage cervical cancer in the US includes external beam radiotherapy (EBRT) with brachytherapy. Although there are guidelines for brachytherapy dose and fractionation regimens, there are limited data on practice patterns. This study aims to evaluate the contemporary utilization of cervical cancer brachytherapy in the US and its association with patient demographics and facility characteristics. METHODS: We retrospectively analyzed clinical covariates of cervical cancer patients diagnosed and treated in 2018-2020 with curative-intent radiotherapy from the 2020 National Cancer Database. Associations between patient and institutional factors with the number of brachytherapy fractions were identified with logistic regression. Factors with association (p < 0.10) were then included in a multivariable logistic regression model. All tests were two-sided with significance <0.05 unless specified otherwise. RESULTS: Among the eligible 2517 patients, 97.3% received HDR or LDR and is further analyzed. More patients received HDR than LDR brachytherapy (98.9% vs 1.1%) and intracavitary than interstitial brachytherapy (86.4% vs 13.6%). The most common number of HDR fractions prescribed were 5 (51.0%), 4 (32.9%), and 3 (8.6%). After adjusting for the other variables in the model, ethnicity, private insurance status, overall insurance status, and facility type were the only factors that were significantly associated with the number of brachytherapy factions (p < 0.0001, p = 0.028, p = 0.001, and p < 0.0001, respectively, n = 2184). CONCLUSIONS: In the US, various HDR brachytherapy regimens are utilized depending on patient and institutional factors. Future research may optimize cervical cancer brachytherapy by correlating specific dose and fractionation regimens with patient outcomes.

Dual-locus, dual-HDR editing permits efficient generation of antigen-specific regulatory T cells with robust suppressive activity.

Adoptive regulatory T (Treg) cell therapy is predicted to modulate immune tolerance in autoimmune diseases, including type 1 diabetes (T1D). However, the requirement for antigen (ag) specificity to optimally orchestrate tissue-specific, Treg cell-mediated tolerance limits effective clinical application. To address this challenge, we present a single-step, combinatorial gene editing strategy utilizing dual-locus, dual-homology-directed repair (HDR) to generate and specifically expand ag-specific engineered Treg (EngTreg) cells derived from donor CD4+ T cells. Concurrent delivery of CRISPR nucleases and recombinant (r)AAV homology donor templates targeting FOXP3 and TRAC was used to achieve three parallel goals: enforced, stable expression of FOXP3; replacement of the endogenous T cell receptor (TCR) with an islet-specific TCR; and selective enrichment of dual-edited cells. Each HDR donor template contained an alternative component of a heterodimeric chemically inducible signaling complex (CISC), designed to activate interleukin-2 (IL-2) signaling in response to rapamycin, promoting expansion of only dual-edited EngTreg cells. Using this approach, we generated purified, islet-specific EngTreg cells that mediated robust direct and bystander suppression of effector T (Teff) cells recognizing the same or a different islet antigen peptide, respectively. This platform is broadly adaptable for use with alternative TCRs or other targeting moieties for application in tissue-specific autoimmune or inflammatory diseases.

An optimized SpCas9 high-fidelity variant for direct protein delivery.

Electroporation of the Cas9 ribonucleoprotein (RNP) complex offers the advantage of preventing off-target cleavages and potential immune responses produced by long-term expression of the nuclease. Nevertheless, the majority of engineered high-fidelity Streptococcus pyogenes Cas9 (SpCas9) variants are less active than the wild-type enzyme and are not compatible with RNP delivery. Building on our previous studies on evoCas9, we developed a high-fidelity SpCas9 variant suitable for RNP delivery. The editing efficacy and precision of the recombinant high-fidelity Cas9 (rCas9HF), characterized by the K526D substitution, was compared with the R691A mutant (HiFi Cas9), which is currently the only available high-fidelity Cas9 that can be used as an RNP. The comparative analysis was extended to gene substitution experiments where the two high fidelities were used in combination with a DNA donor template, generating different ratios of non-homologous end joining (NHEJ) versus homology-directed repair (HDR) for precise editing. The analyses revealed a heterogeneous efficacy and precision indicating different targeting capabilities between the two variants throughout the genome. The development of rCas9HF, characterized by an editing profile diverse from the currently used HiFi Cas9 in RNP electroporation, increases the genome editing solutions for the highest precision and efficient applications.

A promoterless AAV6.2FF-based lung gene editing platform for the correction of surfactant protein B deficiency.

Surfactant protein B (SP-B) deficiency is a rare genetic disease that causes fatal respiratory failure within the first year of life. Currently, the only corrective treatment is lung transplantation. Here, we co-transduced the murine lung with adeno-associated virus 6.2FF (AAV6.2FF) vectors encoding a SaCas9-guide RNA nuclease or donor template to mediate insertion of promoterless reporter genes or the (murine) Sftpb gene in frame with the endogenous surfactant protein C (SP-C) gene, without disrupting SP-C expression. Intranasal administration of 3 × 1011 vg donor template and 1 × 1011 vg nuclease consistently edited approximately 6% of lung epithelial cells. Frequency of gene insertion increased in a dose-dependent manner, reaching 20%-25% editing efficiency with the highest donor template and nuclease doses tested. We next evaluated whether this promoterless gene editing platform could extend survival in the conditional SP-B knockout mouse model. Administration of 1 × 1012 vg SP-B-donor template and 5 × 1011 vg nuclease significantly extended median survival (p = 0.0034) from 5 days in the untreated off doxycycline group to 16 days in the donor AAV and nuclease group, with one gene-edited mouse living 243 days off doxycycline. This AAV6.2FF-based gene editing platform has the potential to correct SP-B deficiency, as well as other disorders of alveolar type II cells.

The effect of vaginal cylinder inhomogeneity on the HDR brachytherapy dose calculations using Monte Carlo simulations.

PURPOSE: To analytically assess the heterogeneity effect of vaginal cylinders (VC) made of high-density plastics on dose calculations, considering the prescription point (surface or 5 mm beyond the surface), and benchmark the accuracy of a commercial model-based dose calculation (MBDC) algorithm using Monte Carlo (MC) simulations. METHODS AND MATERIALS: The GEANT4 MC code was used to simulate a commercial 192 Ir HDR source and VC, with diameters ranging from 20 to 35 mm, inside a virtual water phantom. Standard plans were generated from a commercial treatment planning system [TPS-BrachyVision ACUROS (BV)] optimized for a treatment length of 5 cm through two dose calculation approaches: (1) assuming all the environment as water (i.e., Dw,w-MC & Dw,w-TG43 ) and (2) accounting for the heterogeneity of VC applicators (i.e., Dw,w-App-MC & Dw,w-App-MBDC ). The compared isodose lines, and dose & energy difference maps were extracted for analysis. In addition, the dose difference on the peripheral surface, along the applicator and at middle of treatment length, as well as apical tip was evaluated. RESULTS: The Dw,w-App-MC results indicated that the VC heterogeneity can cause a dose reduction of (up to) % 6.8 on average (for all sizes) on the peripheral surface, translating to 1 mm shrinkage of the isodose lines compared to Dw,w-MC . In addition, the results denoted that BV overestimates the dose on the peripheral surface and apical tip of about 3.7% and 17.9%, respectively, (i.e., Dw,w-App-MBDC vs Dw,w-App-MC ) when prescribing to the surface. However, the difference between the two were negligible at the prescription point when prescribing to 5 mm beyond the surface. CONCLUSION: The VCs' heterogeneity could cause dose reduction when prescribing dose to the surface of the applicator, and hence increases the level of uncertainty. Thus, reviewing the TG43 results, in addition to ACUROS, becomes prudent, when evaluating the surface coverage at the apex.

Practical experience with initial quality assurance of a high dose rate brachytherapy grid-based Boltzmann solver algorithm.

PURPOSE: The purpose of this study was to validate the use of a model-based dose calculation algorithm (MBDCA), Acuros BV, for high dose rate brachytherapy treatment planning for a community-based hospital with a Bravos afterloader. Based on published AAPM recommendations, this work details a practical approach for community-based clinics to complete initial validation of Acuros BV, in order to add a MBDCA to a TG-43 based brachytherapy treatment planning program. METHODS: Source dimensions and materials used in Acuros BV and TG-43 source models were compared to the physical source. TG-186 testing was completed with standardized test cases externally calculated with Monte Carlo compared to locally calculated with Acuros BV. Point doses calculated using TG-43 were compared to those calculated with Acuros BV in water at various dose grid settings. Secondary dose check software was used to evaluate dose distributions resembling clinical patient plans, both in water and on CT datasets representative of patient anatomy. RESULTS: The major source of discrepancy of source models was the length of modeled steel cable. TG-186 testing showed that the largest differences between Monte Carlo and Acuros BV dose distributions were located along the source axis for cases calculated in water, as well as located in regions of high dose gradients and within the applicator for the case calculated with a generic shielded applicator. An audit of point doses calculated with both TG-43 and Acuros BV in water found that dose grid settings significantly affected agreement. Secondary dose check software indicated that Acuros BV functioned satisfactorily, and a 5% threshold was adopted for secondary dose checks on gynecologic plans. CONCLUSION: This validation process indicated that Acuros BV met expected standards and affirmed its suitability for integration into this clinical practice's brachytherapy treatment planning.

A deep learning-based 3D Prompt-nnUnet model for automatic segmentation in brachytherapy of postoperative endometrial carcinoma.

PURPOSE: To create and evaluate a three-dimensional (3D) Prompt-nnUnet module that utilizes the prompts-based model combined with 3D nnUnet for producing the rapid and consistent autosegmentation of high-risk clinical target volume (HR CTV) and organ at risk (OAR) in high-dose-rate brachytherapy (HDR BT) for patients with postoperative endometrial carcinoma (EC). METHODS AND MATERIALS: On two experimental batches, a total of 321 computed tomography (CT) scans were obtained for HR CTV segmentation from 321 patients with EC, and 125 CT scans for OARs segmentation from 125 patients. The numbers of training/validation/test were 257/32/32 and 87/13/25 for HR CTV and OARs respectively. A novel comparison of the deep learning neural network 3D Prompt-nnUnet and 3D nnUnet was applied for HR CTV and OARs segmentation. Three-fold cross validation and several quantitative metrics were employed, including Dice similarity coefficient (DSC), Hausdorff distance (HD), 95th percentile of Hausdorff distance (HD95%), and intersection over union (IoU). RESULTS: The Prompt-nnUnet included two forms of parameters Predict-Prompt (PP) and Label-Prompt (LP), with the LP performing most similarly to the experienced radiation oncologist and outperforming the less experienced ones. During the testing phase, the mean DSC values for the LP were 0.96 ± 0.02, 0.91 ± 0.02, and 0.83 ± 0.07 for HR CTV, rectum and urethra, respectively. The mean HD values (mm) were 2.73 ± 0.95, 8.18 ± 4.84, and 2.11 ± 0.50, respectively. The mean HD95% values (mm) were 1.66 ± 1.11, 3.07 ± 0.94, and 1.35 ± 0.55, respectively. The mean IoUs were 0.92 ± 0.04, 0.84 ± 0.03, and 0.71 ± 0.09, respectively. A delineation time < 2.35 s per structure in the new model was observed, which was available to save clinician time. CONCLUSION: The Prompt-nnUnet architecture, particularly the LP, was highly consistent with ground truth (GT) in HR CTV or OAR autosegmentation, reducing interobserver variability and shortening treatment time.

Characterisation of a Silicon Photomultiplier Based Oncological Brachytherapy Fibre Dosimeter.

Source localisation and real-time dose verification are at the forefront of medical research in brachytherapy, an oncological radiotherapy procedure based on radioactive sources implanted in the patient body. The ORIGIN project aims to respond to this medical community's need by targeting the development of a multi-point dose mapping system based on fibre sensors integrating a small volume of scintillating material into the tip and interfaced with silicon photomultipliers operated in counting mode. In this paper, a novel method for the selection of the optimal silicon photomultipliers to be used is presented, as well as a laboratory characterisation based on dosimetric figures of merit. More specifically, a technique exploiting the optical cross-talk to maintain the detector linearity in high-rate conditions is demonstrated. Lastly, it is shown that the ORIGIN system complies with the TG43-U1 protocol in high and low dose rate pre-clinical trials with actual brachytherapy sources, an essential requirement for assessing the proposed system as a dosimeter and comparing the performance of the system prototype against the ORIGIN project specifications.

Multi-Frame Content-Aware Mapping Network for Standard-Dynamic-Range to High-Dynamic-Range Television Artifact Removal.

Recently, advancements in image sensor technology have paved the way for the proliferation of high-dynamic-range television (HDRTV). Consequently, there has been a surge in demand for the conversion of standard-dynamic-range television (SDRTV) to HDRTV, especially due to the dearth of native HDRTV content. However, since SDRTV often comes with video encoding artifacts, SDRTV to HDRTV conversion often amplifies these encoding artifacts, thereby reducing the visual quality of the output video. To solve this problem, this paper proposes a multi-frame content-aware mapping network (MCMN), aiming to improve the performance of conversion from low-quality SDRTV to high-quality HDRTV. Specifically, we utilize the temporal spatial characteristics of videos to design a content-aware temporal spatial alignment module for the initial alignment of video features. In the feature prior extraction stage, we innovatively propose a hybrid prior extraction module, including cross-temporal priors, local spatial priors, and global spatial prior extraction. Finally, we design a temporal spatial transformation module to generate an improved tone mapping result. From time to space, from local to global, our method makes full use of multi-frame information to perform inverse tone mapping of single-frame images, while it is also able to better repair coding artifacts.

Post-operative KEloids iRradiation (POKER): does the surgery/high-dose interventional radiotherapy association make a winning hand?

PURPOSE: To report the results involving post-operative interventional radiotherapy (POIRT) in a homogenous cohort of patients affected by keloid and treated at a single institution with the same fractionation schedule. PATIENTS AND METHODS: Inclusion criteria were: surgery with a histopathological diagnosis of keloid, subsequent high-dose rate interventional radiotherapy (HDR-IRT)-12 Gy in 4 fractions (3 Gy/fr) twice a day-and follow-up period ≥ 24 months. RESULTS: One-hundred and two patients and a total of 135 keloids were eligible for the analyses. Median follow-up was 64 [IQR: 25-103] months. Thirty-six (26.7%) recurrences were observed, 12-months and 36-months cumulative incidence of recurrence were 20.7% (95% CI 12.2-28.5) and 23.8% (95% CI 14.9-31.7) respectively. History of spontaneous keloids (HR = 7.00, 95% CI 2.79-17.6, p < 0.001), spontaneous cheloid as keloid cause (HR = 6.97, 95% CI 2.05-23.7, p = 0.002) and sternal (HR = 10.6, 95% CI 3.08-36.8, p < 0.001), ear (HR = 6.03, 95% CI 1.71-21.3, p = 0.005) or limb (HR = 18.8, 95% CI 5.14-68.7, p < 0.001) keloid sites were significantly associated to a higher risk of recurrence. CONCLUSIONS: The findings support the use of surgery and POIRT as an effective strategy for controlling keloid relapses. Further studies should focus on determining the optimal Biologically Effective Dose and on establishing a scoring system for patient selection.

Current Strategies for Increasing Knock-In Efficiency in CRISPR/Cas9-Based Approaches.

Since its discovery in 2012, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system has supposed a promising panorama for developing novel and highly precise genome editing-based gene therapy (GT) alternatives, leading to overcoming the challenges associated with classical GT. Classical GT aims to deliver transgenes to the cells via their random integration in the genome or episomal persistence into the nucleus through lentivirus (LV) or adeno-associated virus (AAV), respectively. Although high transgene expression efficiency is achieved by using either LV or AAV, their nature can result in severe side effects in humans. For instance, an LV (NCT03852498)- and AAV9 (NCT05514249)-based GT clinical trials for treating X-linked adrenoleukodystrophy and Duchenne Muscular Dystrophy showed the development of myelodysplastic syndrome and patient's death, respectively. In contrast with classical GT, the CRISPR/Cas9-based genome editing requires the homologous direct repair (HDR) machinery of the cells for inserting the transgene in specific regions of the genome. This sophisticated and well-regulated process is limited in the cell cycle of mammalian cells, and in turn, the nonhomologous end-joining (NHEJ) predominates. Consequently, seeking approaches to increase HDR efficiency over NHEJ is crucial. This manuscript comprehensively reviews the current alternatives for improving the HDR for CRISPR/Cas9-based GTs.