DNA Repair Genes as Drug Candidates for Early Breast Cancer Onset in Latin America: A Systematic Review

The prevalence of breast cancer in young women (YWBC) has increased alarmingly. Significant efforts are being made to elucidate the biological mechanisms concerning the development, prognosis, and pathological response in early-onset breast cancer (BC) patients. Dysfunctional DNA repair proteins are implied in BC predisposition, progression, and therapy response, underscoring the need for further analyses on DNA repair genes. Public databases of large patient datasets such as METABRIC, TCGA, COSMIC, and cancer cell lines allow the identification of variants in DNA repair genes and possible precision drug candidates. This study aimed at identifying variants and drug candidates that may benefit Latin American (LA) YWBC. We analyzed pathogenic variants in 90 genes involved in DNA repair in public BC datasets from METABRIC, TCGA, COSMIC, CCLE, and COSMIC Cell Lines Project. Results showed that reported DNA repair germline variants in the LA dataset are underrepresented in large databases, in contrast to other populations. Additionally, only six gene repair variants in women under 50 years old from the study population were reported in BC cell lines. Therefore, there is a need for new approaches to study DNA repair variants reported in young women from LA.

Iron-Rich Conditions Induce OmpA and Virulence Changes of Acinetobacter baumannii

Background: Iron ions affect the expression of outer membrane protein A (OmpA), a major pathogenic protein in Acinetobacter baumannii.Objective: To analyze the effect of iron ions on the expression of the OmpA protein of A. baumannii and explore its association with the virulence of OmpA.Methods: Site-directed mutagenesis was used to construct ompA gene deletion strains and gene repair strains. The OmpA protein expression of A. baumannii under culture with different contents of iron ions was detected. The virulence of A. baumannii with different OmpA protein expression levels were evaluated in macrophages and mice.Results: OmpA protein levels of the three strains were enhanced under iron-rich conditions. They were reduced in the presence of the iron-chelating agent 2,2′-bipyridine. A. baumannii wild type and + ompA had a remarkable toxic effect on RAW246.7 macrophages (P < 0.05). In contrast, the ΔompA had a significantly reduced toxic effect on RAW246.7 macrophages (P < 0.05). The levels of the inflammatory factors IL-1β, IL-6, IL-8, and TNFα in the mice spleen were significantly increased in the + ompA strain treatment group compared with the ΔompA strain group (all P < 0.05). In addition, the levels were higher in the presence of iron ions than in the presence of the chelating agent.Conclusion: Iron-rich conditions increase the OmpA protein expression of A. baumannii. Strains with high OmpA protein expression were more invasive, which may be a key determinant of A. baumannii infection and pathogenicity. Iron control strategies might be used for the management of A. baumannii.

Genome Evolution of Filamentous Cyanobacterium Nostoc Species: From Facultative Symbiosis to Free Living

In contrast to obligate bacteria, facultative symbiotic bacteria are mainly characterized by genome enlargement. However, the underlying relationship of this feature with adaptations to various habitats remains unclear. In this study, we used the global genome data of Nostoc strains, including 10 novel genomes sequenced in this study and 26 genomes available from public databases, and analyzed their evolutionary history. The evolutionary boundary of the real clade of Nostoc species was identified and was found to be consistent with the results of polyphasic taxonomy. The initial ancestral species of Nostoc was demonstrated to be consistent with a facultative symbiotic population. Further analyses revealed that Nostoc strains tended to shift from facultative symbiosis to a free-living one, along with an increase in genome sizes during the dispersal of each exterior branch. Intracellular symbiosis was proved to be essentially related to Nostoc evolution, and the adaptation of its members to free-living environments was coupled with a large preference for gene acquisition involved in gene repair and recombination. These findings provided unique evidence of genomic mechanisms by which homologous microbes adapt to distinct life manners and external environments.

Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas

Programmable site-specific nucleases, such as the CRISPR/Cas9 ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas (Chlamydomonas reinhardtii). However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous double-stranded DNA (dsDNA) encoding a selectable marker gene. Repair of the double-stranded DNA breaks induced by the ribonucleoproteins is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. Here, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker) - in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precise edits in the homolog of bacterial ftsY or the WD and TetratriCopeptide repeats protein 1 (WDTC1) genes in ∼1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonas acetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways or structures.

CRISPR/Cas9-Mediated in vivo Genetic Correction in a Mouse Model of Hemophilia A

Hemophilia A (HA), a common bleeding disorder caused by a deficiency of coagulation factor VIII (FVIII), has long been considered an attractive target for gene therapy studies. However, full-length F8 cDNA cannot be packaged efficiently by adeno-associated virus (AAV) vectors. As the second most prevalent mutation causing severe HA, F8 intron 1 inversion (Inv1) is caused by an intrachromosomal recombination, leaving the majority of F8 (exons 2–26) untranscribed. In theory, the truncated gene could be rescued by integrating a promoter and the coding sequence of exon 1. To test this strategy in vivo, we generated an HA mouse model by deleting the promoter region and exon 1 of F8. Donor DNA and CRISPR/SaCas9 were packaged into AAV vectors and injected into HA mice intravenously. After treatment, F8 expression was restored and activated partial thromboplastin time (aPTT) was shortened. We also compared two liver-specific promoters and two types of integrating donor vectors. When an active promoter was used, all of the treated mice survived the tail-clip challenge. This is the first report of an in vivo gene repair strategy with the potential to treat a recurrent mutation in HA patients.

Concurrent Scoliosis and Dentofacial Anomaly: A Case Report

The relationship between scoliosis, dentofacial anomaly, and malocclusion is poorly understood. We report a novel and complex pediatric case of concurrent juvenile scoliosis, dentofacial anomaly, and malocclusion, successfully treated and managed by an interdisciplinary hospital team over a 15-year period. The degree of severity of the scoliosis and dentofacial anomaly necessitates surgical intervention. Successful orthopedic surgical procedures have improved the patient’s quality of life. Future surgical correction of the dentofacial anomaly and malocclusion is necessary to improve the patient’s condition from psychosocial, aesthetic, and functional standpoints. The patient’s condition is characterized by multiple congenital abnormalities, developmental delay, seizure disorder, juvenile scoliosis, and dentofacial anomaly with malocclusion. Intriguingly, a unifying diagnosis for the patient’s condition could not be confirmed despite the indication of a syndromic cause. The collection of characteristics is suggestive of the chromosome 22q11.2 deletion syndromes (including velocardiofacial syndrome [VCFS] or DiGeorge syndrome) as possible genetic causes. Clinical genetics testing was unable to establish a diagnosis of chromosome 22q11.2 deletion, VCFS or DiGeorge syndrome. Further investigation of the genotype-phenotype relationships of scoliosis, dentofacial anomaly, and malocclusion is required to improve medical knowledge, diagnostic capability, and patient care, specifically relating to cases of undiagnosed diseases. Future research utilizing next-generation sequencing techniques is necessary to aim for precise genetic diagnosis, including knowledge of the underlying cellular and molecular mechanisms, for the development of the potential of therapeutic approaches targeting gene repair.

Sickle Cell Disease Hematopoietic Stem Cell gene therapy with globin gene addition is promising

Autologous transplantation of gene-modified HSCs might be used to treat Sickle Cell Disease (SCD) once and for all. Hematopoietic Stem Cell (HSC) gene therapy with lentiviral-globin gene addition was optimized by HSC collection, vector constructs, lentiviral transduction, and conditioning in the current gene therapy experiment for SCD, resulting in higher gene marking and phenotypic correction. Further advancements over the next decade should allow for a widely approved gene-addition therapy. Long-term engraftment is crucial for gene-corrected CD34+ HSCs, which might be addressed in the coming years, and gene repair of the SCD mutation in the-globin gene can be achieved in vitro using genome editing in CD34+ cells. Because of breakthroughs in efficacy, safety, and delivery strategies, in vivo gene addition and gene correction in BM HSCs is advancing. Overall, further research is needed, but HSC-targeted gene addition/gene editing therapy is a promising SCD therapy with curative potential that might be widely available soon.

Early Disruption of Photoreceptor Cell Architecture and Loss of Vision in a Humanized Pig Model of Usher Syndrome

Usher syndrome (USH) is the most common form of monogenic deaf-blindness. Loss of vision is untreatable and, so far, there are no suitable animal models for testing therapeutic strategies. By introducing a human mutation into the harmonin-encoding USH1C gene in pigs, we generated the first translational animal model for USH type 1 with characteristic hearing defect, vestibular dysfunction and visual impairment. Changes in photoreceptor architecture, quantitative motion analysis and electroretinography were characteristics of the reduced retinal virtue in USH1C pigs. Primary cells from those animals and USH1C patients showed significantly elongated primary cilia, compared to wild-type, confirming the nature of USH as a true and general ciliopathy and proving the therapeutic capacity of gene supplementation and gene repair approaches.

Prediction of sgRNA Off-Target Activity in CRISPR/Cas9 Gene Editing Using Graph Convolution Network

CRISPR/Cas9 is a powerful genome-editing technology that has been widely applied in targeted gene repair and gene expression regulation. One of the main challenges for the CRISPR/Cas9 system is the occurrence of unexpected cleavage at some sites (off-targets) and predicting them is necessary due to its relevance in gene editing research. Very few deep learning models have been developed so far to predict the off-target propensity of single guide RNA (sgRNA) at specific DNA fragments by using artificial feature extract operations and machine learning techniques; however, this is a convoluted process that is difficult to understand and implement for researchers. In this research work, we introduce a novel graph-based approach to predict off-target efficacy of sgRNA in the CRISPR/Cas9 system that is easy to understand and replicate for researchers. This is achieved by creating a graph with sequences as nodes and by using a link prediction method to predict the presence of links between sgRNA and off-target inducing target DNA sequences. Features for the sequences are extracted from within the sequences. We used HEK293 and K562 t datasets in our experiments. GCN predicted the off-target gene knockouts (using link prediction) by predicting the links between sgRNA and off-target sequences with an auROC value of 0.987.

Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas

Programmable site-specific nucleases, such as the CRISPR/Cas9 ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas. However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous double-stranded DNA (dsDNA) encoding a selectable marker gene. Repair of the double-stranded DNA breaks induced by the ribonucleoproteins is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. In this study, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker) - in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precisely, scarless edited FTSY or WDTC1 genes in ~1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonas acetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways or structures.